LOOP MEDIATED ISOTHERMAL AMPLIFICATION

Loop-mediated isothermal amplification (LAMP) has been used to detect several pathogens including malaria parasites from field and clinical samples. In this protocol, the malaria LAMP technology is developed to differentiate between Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) species by targeting the dihydrofolate reductase thymidylate synthase (dhfr-ts) gene, a known target for the antifolate class of drugs such as Pyrimethamine. LAMP primer sets are designed and validated for species specific amplification. Additionally, specific probes help improve detection and visualization of the products when combined with lateral flow dipstick-based (LFD) detection. The protocols are further simplified to eliminate tedious sample preparation steps, such that crude lysis prepared simply by diluting few microliter (μL) of blood sample with distilled water is sufficient. The LAMP-LFD malaria dhfr-ts protocols are sensitive and can detect as little as 1 picogram (pg) of PfDNA and 1 nanogram (ng) of PvDNA, or a few microliters of crude lysate from infected blood samples (Yongkiettrakul et al., Parasitol Int 63: 777-784, 2014). These simplified steps not only reduce cost but also increase the potential for large application in the fields and clinical settings.

The applications of correct diagnostic approaches play a decisive role in timely containment of infectious diseases spread and mitigation of public health risks. Nevertheless, there is a need to update the diagnostics regularly to capture the new, emergent, and highly divergent viruses. Acute gastroenteritis of viral origin has been identified as a significant cause of mortality across the globe, with the more serious consequences seen at the extremes of age groups (young and elderly) and immunecompromised individuals. Therefore, significant advancements and efforts have been put in the development of enteric virus diagnostics to meet the WHO ASSURED criteria as a benchmark over the years. The Enzyme-Linked Immunosorbent (ELISA) and Polymerase Chain Reaction (PCR) are the basic assays that provided the platform for development of several efficient diagnostics such as real-time RT-PCR, loopmediated isothermal amplification (LAMP), polymerase spiral reaction (PSR), biosensors, microarrays and next generation sequencing. Herein, we describe and discuss the applications of these advanced technologies in context to enteric virus detection by delineating their features, advantages and limitations.

The rapid spread of Zika virus (ZIKV) represents a global public health problem, especially in countries that circulate several vector mosquito vectors and favorable conditions for virus transmission, such as Brazil. In these areas, improvement in mosquito control needs to be a top priority, but mosquito viral surveillance occurs inefficiently in endemic countries. Currently, the reverse transcriptase reaction followed by quantitative polymerase chain reaction (qRT-PCR) is the gold standard for molecular diagnostic of ZIKV in mosquito samples. However, the technique presents high cost and limitations for Point-of-care (POC) diagnostics, which hampers its application for a large number of samples in entomological surveillance programs in low-resource areas. In view of this scenario, there remains the need to develop new POC diagnostics. Objective: In this context, the aim of this work was to develop and validate a diagnostic platform based on the reverse transcriptase technique followed by isothermal loop-mediated amplification (RT-LAMP) for detection of ZIKV in mosquito samples. Methodology: Initially, was determined the ability of RT-LAMP to detect ZIKV in Aedes aegypti under controlled conditions, including experimentally infected female A. aegypti mosquitoes. In addition, the specificity were assayed by testing the cross-reactivity with other arboviruses currently circulating in Brazil, including ZIKV (PE243), DENV-1 (PE/97-42735), DENV-2 (PE/95-3808), DENV-3 (PE/02-95016), DENV-4 (PE/10-0081), YFV (17DD) and CHIKV (PE2016-480). To evaluate the analytical sensitivity, ZIKV strain PE243 was 10-fold serially diluted in crude lysates of uninfected A. aegypti mosquito. After dilution, samples were directly assayed by RT-LAMP without RNA isolation. Subsequently, the validation of the RT-LAMP test was performed with 60 samples of A. aegypti and Culex quinquefasciatus mosquitoes, including samples of laboratory-infected mosquitoes and naturally infected mosquitoes collected in the metropolitan region of Recife-PE. Lastly, the value per reaction was calculated based on the cost of all the reagents. Results: Regarding the results, the RT-LAMP assay was highly specific for detection of ZIKV in 20 minutes and was up to 10,000 times more sensitive than qRT-PCR for detection of ZIKV in mosquito samples. The RT-LAMP had a sensitivity of 100%, specificity of 91.18 %, and overall accuracy of 95.24%, highlighting the potential of RT-LAMP for detection of ZIKV. As for the cost of each reaction of the RT-LAMP, the value was one real (R$ 1.00). We have developed a low cost, POC diagnostic platform based on the RT-LAMP assay to detect ZIKV in mosquito samples collected at the epicenter of the Zika epidemics in Brazil. The test is a robust, fast and inexpensive tool for surveillance of ZIKV in mosquito populations and will enable developing countries to establish better viral surveillance in vectors and improve the efficacy of control programs. Our results provide a potential new straightforward and molecular diagnostic test for ZIKV in arthropod vectors.

The newly identified coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causes coronavirus disease 2019 (COVID-19) and has affected over 25 million people worldwide as of 31 August 2020. To aid in the development of diagnostic kits for rapid and sensitive detection of the virus, we evaluated a combination of polymerase chain reaction (PCR) and isothermal nucleic acid amplification techniques. Here, we compared conventional PCR and loop-mediated isothermal amplification (LAMP) methods with hybrid techniques such as polymerase chain displacement reaction (PCDR) and a newly developed PCR-LAMP method. We found that the hybrid methods demonstrated higher sensitivity and assay reaction rates than those of the classic LAMP and PCR All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

Abstract⎯The results of detection and identification of Bacillus anthracis strains in loop-mediated isothermal DNA amplification (LAMP) reaction performed under optimized conditions with original primers and thermostable DNA polymerase are presented. Reproducible LAMP-based detection of chromosomal and plasmid DNA targets specific for B. anthracis strains has been demonstrated. No cross reactions with DNA from bacterial strains of other species of the B. cereus group were detected. The development of tests for anthrax-pathogen detection based on the optimized reaction of loop isothermal DNA amplification is planned. These tests will be convenient for clinical studies and field diagnostics due to the absence of requirements for sophisticated equipment.

The reliability of loop-mediated isothermal amplification (LAMP), initially developed for the detection of human herpesvirus 7 (HHV-7), was evaluated in this study. Although a LAMP product was detected in HHV-7 DNA, neither HHV-6 nor human cytomegalovirus DNA produced a product. When agarose gel electrophoresis was used for the detection of LAMP products, the sensitivity of a 30-min HHV-7 LAMP reaction reached 250 copies/tube. The use of turbidity for the detection of the LAMP products gave a sensitivity of 500 and 250 copies/tube for 30- and 60-min reactions, respectively. Following these initial validation studies, clinical samples collected from two patients with primary HHV-7 infections were examined by HHV-7 LAMP. By use of agarose gel electrophoresis, HHV-7 LAMP products could be detected in acute-phase plasma samples but no LAMP product was detectable in convalescent-phase plasma samples from either patient. Since a turbidity assay is less sensitive than agarose gel electroph...

Background: Buruli ulcer (BU) caused by Mycobacterium ulcerans (M. ulcerans) has emerged as an important public health problem in several rural communities in sub-Saharan Africa. Early diagnosis and prompt treatment are important in preventing disfiguring complications associated with late stages of the disease progression. Presently there is no simple and rapid test that is appropriate for early diagnosis and use in the low-resource settings where M. ulcerans is most prevalent. We compared conventional and pocket warmer loop mediated isothermal amplification (LAMP) methods (using a heat block and a pocket warmer respectively as heat source for amplification reaction) for the detection of M. ulcerans in clinical specimens. The effect of purified and crude DNA preparations on the detection rate of the LAMP assays were also investigated and compared with that of IS2404 PCR, a reference assay for the detection of M. ulcerans. Thirty clinical specimens from suspected BU cases were examined by LAMP and IS2404 PCR. Principal Findings: The lower detection limit of both LAMP methods at 60uC was 300 copies of IS2404 and 30 copies of IS2404 for the conventional LAMP at 65uC. When purified DNA extracts were used, both the conventional LAMP and IS2404 PCR concordantly detected 21 positive cases, while the pocket warmer LAMP detected 19 cases. Nine of 30 samples were positive by both the LAMP assays as well as IS2404 PCR when crude extracts of clinical specimens were used. The LAMP method can be used as a simple and rapid test for the detection of M. ulcerans in clinical specimens. However, obtaining purified DNA, as well as generating isothermal conditions, remains a major challenge for the use of the LAMP method under field conditions. With further improvement in DNA extraction and amplification conditions, the pwLAMP could be used as a point of care diagnostic test for BU

The increasing incidence of infectious diseases caused by fungi in immunocompromised patients has encouraged researchers to develop rapid and accurate diagnosis methods. Identification of the causative fungal species is critical in deciding the appropriate treatment, but it is not easy to get satisfactory results due to the difficulty of fungal cultivation and morphological identification from clinical samples. In this study, we established a microarray system that can identify 42 species from 24 genera of clinically important fungal pathogens by using a chemical color reaction in the detection process. The array uses the internal transcribed spacer region of the rRNA gene for identification of fungal DNA at the species level. The specificity of this array was tested against a total of 355 target and nontarget fungal species. The fungal detection was succeeded directly from 10 3 CFU/ml for whole blood samples, and 50 fg DNA per 1 ml of serum samples indicating that the array system we established is sensitive to identify infecting fungi from clinical sample. Furthermore, we conducted isothermal amplification in place of PCR amplification and labeling. The successful identification with PCR-amplified as well as isothermally amplified target genes demonstrated that our microarray system is an efficient and robust method for identifying a variety of fungal species in a sample.

An assay to detect Strongyloides stercoralis in stool specimens was developed using the loop-mediated isothermal amplification (LAMP) method. Primers were based on the 28S ribosomal subunit gene. The reaction conditions were optimized and SYTO-82 fluorescent dye was used to allow real-time and visual detection of the product. The product identity was confirmed with restriction enzyme digestion, cloning, and sequence analysis. The assay was specific when tested against DNA from bacteria, fungi and parasites, and 30 normal stool samples. Analytical sensitivity was to 10 copies of target sequence in a plasmid and up to a 10 -2 dilution of DNA extracted from a Strongyloides ratti larva spiked into stool. Sensitivity was increased when further dilutions were made in water, indicative of reduced reaction inhibition. Twenty-seven of 28 stool samples microscopy and polymerase chain reaction positive for S. stercoralis were positive with the LAMP method. On the basis of these findings, the assay warrants further clinical validation.

Campylobacteriosis is one of the most common foodborne diseases worldwide. Two Campylobacter species -C. jejuni and C. coli in poultry and poultry products are considered to be the main source of human campylobacteriosis. Therefore, studying Campylobacter status in poultry flocks is needed to prevent transmission of disease and reduce human risk, health cost, and economic losses. In this study, we adapted and used a Loop-Mediated Isothermal Amplification (LAMP) assay for specific, sensitive, simple and cost-effective rapid detection of C. jejuni and C. coli in the poultry production chain. Amplified LAMP products were detected using a small, low-cost portable commercial blue LED transilluminator and a direct visual detection strategy was demonstrated. By using optimized conditions for amplification a limit of detection (LOD) of 50 CFU/ml was achieved for testing of C. jejuni and C. coli in spiked chicken feces without enrichment. The method took 60-70 min from receiving the samples to the final results (including 30 min for amplification). The optimized LAMP showed a relative accuracy of 98.4%, a specificity of 97.9%, and a sensitivity of 100% in comparison to real-time PCR method. Cohen's kappa index also showed an excellent agreement (0.94) between the two methods. The results showed that the method is specific, sensitive and is suitable to develop for rapid detection of Campylobacter spp. at poultry production.

A cost-effective, sensitive and speci c Chlamydia trachomatis (CT) diagnostic test is essential for resource limited countries. A loop mediated isothermal ampli cation (LAMP) assay was optimized for detection of CT ompA DNA in urine. Crude DNA extraction (method-1) from urine was by centrifugation at 14,000g for 30 minutes, heating at 95°C for ve minutes with buffer and centrifugation at 17,000g for one minute. A 25µl LAMP reaction comprising, 7µl crude extract in 20mM Tris-HCl, 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 2mM MgSO 4 , 0.1% Tween 20, six LAMP primers, 1.4mM deoxynucleoside triphosphate, 0.8M Betaine, 6mM MgSO 4 and 8U Bsm polymerase, were incubated at 56°C for 60 minutes and the colour change, when a nucleic acid gel stain was added, was noted by ultraviolet illumination. Urine from 326 sexually transmitted disease clinic attendees were tested, both by LAMP and real time PCR (rPCR). Analytical sensitivity was 0.8 copies per reaction volume. Compared to rPCR, LAMP assay had sensitivity, speci city, positive predictive and negative predictive values of 71.4%, 99.7%, 96.2%, 96.7% respectively. LAMP assay was highly speci c for CT ompA DNA in urine. The sensitivity improved further when urinary inhibitors were diluted and volume of urine for extraction was increased (method-2). LAMP-Sybr Gold assay for Chlamydia trachomatis ompA DNA was 71.4% sensitive and 99.7% speci c for urine Analytical sensitivity of LAMP-Sybr Gold assay was 8.06 x 10 -1 copies CT ompA DNA 25µl reaction volume Method-2 for preparation of crude DNA extract improved sensitivity of LAMP-Sybr Gold assay

BstLF, the large fragment of Bst DNA polymerase, is a recombinant strand-displacing DNA polymerase derived from Geobacillus stearothermophilus and serves as the primary enzyme utilized in loopmediated isothermal amplification (LAMP). Recently, the application of LAMP as a convenient and accessible diagnostic tool has gained considerable attention. However, the high cost of reagents presents a significant barrier to the development of LAMP diagnostic kits, limiting accessibility for laboratories in low-resource settings. In this study, we report a low-cost production method for BstLF utilizing more readily available alternative reagents coupled with a simplified purification process. The resulting homebrewed BstLF demonstrated comparable amplification efficiency and maintained thermal and storage stability after 10 months, aligning closely with the performance of the commercially available Bst 2.0 DNA polymerase. Furthermore, we propose this alternative production method to democratize access to LAMP-based diagnostics research. A cost analysis reveals a remarkable 94.17% reduction in the cost per reaction when using the home-brewed BstLF compared to the commercial Bst 2.0 DNA polymerase. Overall, this research introduces a streamlined and cost-effective method for the small-scale production of BstLF, thus facilitating greater access to LAMP assays in areas with limited resources.

Fire blight caused by the gram-negative bacterium Erwinia amylovora is a major disease of pome fruit like apple (Malus domestica) or pear (Pyrus communis). Detection of the pathogen is hampered by low titres usually being present during initial flower infection and the brief time frame for analysis to decide upon subsequent countermeasures. Here we describe a loop-mediated isothermal amplification of DNA (LAMP) assay for genomic DNA of Erwinia amylovora, which relies on a highly specific primer design that excludes amplification from typical DNA sources of the orchard biological system and from sample handling. The assay enables the fast detection of down to approximately 20 cfu of pure Erwinia amylovora or 100 fg genomic DNA (corresponding to approximately 25 E. amyolovora equivalents) per reaction within 45 min. Fast and reliable detection of E. amylovora in orchard samples by naked eye is achieved through a visual colour change indication with hydroxynaphthol blue. The assay avoids use of complex technical devices and is thus suitable for field testing.

The applications of correct diagnostic approaches play a decisive role in timely containment of infectious diseases spread and mitigation of public health risks. Nevertheless, there is a need to update the diagnostics regularly to capture the new, emergent, and highly divergent viruses. Acute gastroenteritis of viral origin has been identified as a significant cause of mortality across the globe, with the more serious consequences seen at the extremes of age groups (young and elderly) and immunecompromised individuals. Therefore, significant advancements and efforts have been put in the development of enteric virus diagnostics to meet the WHO ASSURED criteria as a benchmark over the years. The Enzyme-Linked Immunosorbent (ELISA) and Polymerase Chain Reaction (PCR) are the basic assays that provided the platform for development of several efficient diagnostics such as real-time RT-PCR, loopmediated isothermal amplification (LAMP), polymerase spiral reaction (PSR), biosensors, microarrays and next generation sequencing. Herein, we describe and discuss the applications of these advanced technologies in context to enteric virus detection by delineating their features, advantages and limitations.

Objective: The Polymerase Chain Reaction (PCR) provides a more sensitive, specific, and efficient approach for identifying Mycobacterium tuberculosis (Mtb) in comparison to traditional (AFB) smear and culture procedures. This research seeks to evaluate the potential benefits of replacing the nested PCR technique with the Xpert MTB/rifampicin (RIF) test for the detection of Mycobacterium tuberculosis in an area with a moderate incidence of tuberculosis (TB). Methodology: This research retrospectively evaluated the diagnostic accuracy of Xpert MTB/RIF and nested PCR for tuberculosis, examining sensitivity, specificity, and predictive values using SPSS. A cost-effectiveness study was performed. Data was collected from 350 individuals (210 men, 140 females) at Ibn Sina institutions and hospitals in Dhaka City from July 2021 to June 2023, using standardized questionnaires to obtain demographic and clinical information. Result: An examination including 350 individuals (210 men, 140 women) revealed that 28% of samples tested positive for Mycobacterium tuberculosis (Mtb), with 72.44% of the cases occurring in males. Pulmonary specimens, especially sputum, had the greatest detection rates. Molecular techniques including as GeneXpert and Nested PCR exceeded AFB smear and culture, with Nested PCR demonstrating 100% sensitivity and specificity across all specimen types. Conclusion: In conclusion, the Xpert MTB/RIF assay offers faster, more sensitive detection of rifampicin resistance than nested PCR, making it key for rapid TB diagnosis. However, nested PCR remains superior in low-bacterial-load cases. High costs and specialized equipment limit Xpert's use in resourcepoor settings, emphasizing the need for affordable, accessible diagnostics.

Coronavirus disease (COVID-19) is a respiratory syndrome that rapidly spreads in 195 countries all over the world in a very short period by Severe Acute Respiratory SARS-CoV2. In this pandemic situation, RT-LAMP is used to detect the COVID-19 because it is time-saving and cost-effective, samples are run at optimum temperature, and there is no need for well-equipped laboratories and experienced staff that is used in RT-PCR. This study reveals the screening of SARS-CoV2 by using RT-LAMP which is very specific than RT-PCR. This study was done by taking the safety approvals and taking care of bioethical values, 170 total samples of coronavirus suspected patients are used for screening the spike (S) and nsp1 protein by using RT-LAMP. Samples are taken from the swabs of the patients, then the isolation, extraction, and purification of RNA were done and the reaction was carried out on the RT-LAMP at optimum temperature and colour changes were seen from pink to yellow for the results with t...

Loop-mediated isothermal amplification (LAMP) assay was introduced in the year 2000 by Notomi, as a highly sensitive, specific and cost-effective technique for microbial identification. In contrast to the polymerase chain reaction (PCR) technology in which the reaction is carried out with a series of alternating temperature steps or cycles, isothermal amplification is carried out at a constant temperature and does not require a thermal cycler. LAMP, a simple DNA amplification technique, with its field amenable nature has been used to detect a variety of pathogens including viruses, fungi, bacteria and parasites and in most of the cases it surpasses polymerase chain reaction. In this study the authors investigated the Loop mediated isothermal amplification technique (LAMP) which is a novel nucleic acid amplification technique. They tried to apply LAMP technique to detection of mitochondrial cytochrome b gene in dorcas gazelles. They designed LAMP specific primers for targeted gene and have verified the LAMP sensitivity up to 4 particles. The authors suggested that LAMP technique could be an appropriate replacement for PCR and may be useful in low resource or field settings where conventional DNA or RNA extraction prior.

The advent of Polymerase Chain Reaction (PCR) has revolutionized the human molecular diagnostic scenario. Its ability to exponentially generate desired stretch of DNA from chromosomal or complimentary DNA has laid the foundation for sensitive and specific detection of a pathogen or an aberrant stretch of DNA. Importantly, PCR technology has helped generate diagnostic immunity from phenotypic variation of pathogenic targets. The technological evolution of conventional end-point PCR to its real time version further added clinically important features such as enhanced sensitivity and quantification of target DNA molecules. Two important chemistries became prominent in the real-time PCR domain. The intercalating dye-based chemistry comes with convenience, economy and feature such as melt curve analysis but also carries the disadvantage of overestimation of signal due to unwanted signal from primer dimers. Dual labelled probe on the other hand provides unprecedented sensitivity and specificity. Isothermal method of genome nucleic acid amplification has also evolved as a parallel and convenient method of amplification-based detection of nucleic acid targets. Tuberculosis diagnostics has immensely benefitted from these technologies. However, unmet need exists in sputum handling methods and in its adaptation for molecular diagnostics in resource limited settings. Significant dependence for diagnostic needs on the western world is a matter of concern in India. The country will benefit from rapid and indigenous research and development of molecular diagnostic solutions by way of formulating kits for rapid extraction of DNA and RNA from various clinical sources and developing detection technologies that can compete international products in its category. A solution to address sputum transportation, storage and DNA extraction that has enhanced advantages compared to current methods will also be helpful in the efforts to make India free from tuberculosis.

Background: To improve urinary tract infection detection, we evaluated the specificity and sensitivity of Loop-mediated isothermal Amplification Method (LAMP) for detection of the Eschericia coli (E. coli) in urine samples, for the first time. Methods: Primers were designed to target the malB gene of Escherichia coli. LAMP assay was performed on urine specimens collected from patients with urinary tract infection symptoms. Results: As expected, LAMP was more specific and sensitive than direct microscopic tests. LAMP assay showed the best detection limit of DNA copies with 1.02 copies. Conclusion: LAMP method offers several advantages in terms of sensitivity, rapidness and simplicity for detection of E. coli infection in urine samples. The LAMP method would be highly suitable for the early detection of the UTIs and also comfort quick diagnosis of UTI in clinical laboratories with limited equipment.

DNA-paramagnetic silica bead aggregation in a rotating magnetic field facilitates the quantification of DNA with femtogram sensitivity, but yields no sequence-specific information. Here we provide an original description of aggregation inhibition for the detection of DNA and RNA in a sequence-specific manner following loop-mediated isothermal amplification (LAMP). The fragments generated via LAMP fail to induce chaotrope-mediated bead aggregation; however, due to their ability to passivate the bead surface, they effectively inhibit bead aggregation by longer 'trigger' DNA. We demonstrate the utility of aggregation inhibition as a method for the detection of bacterial and viral pathogens with sensitivity that approaches single copies of the target. We successfully use this methodology for the detection of notable food-borne pathogens Escherichia coli O157:H7 and Salmonella enterica, as well as Rift Valley fever virus, a weaponizable virus of national security concern. We also...

Purpose Timely diagnosis of intraocular tuberculosis (IOTB) along with detection of drug resistance can save many eyes from visual impairment. With the growing incidence of IOTB and rising drug resistance, a reliable diagnostic platform for simultaneous detection of the agent and mutated gene is urgently needed. The MTBDRplus assay was evaluated directly on vitreous fluid samples for the same. In a prospective study, The MTBDRplus assay was performed on 127 vitreous fluid samples (77 'study group' comprising cases of presumed ocular tuberculosis and 50 'control group' cases of disease controls (n = 25) and non-uveitic controls (n = 25)). All samples positive by MTBDRplus assay were subjected to gene sequencing to confirm the mutations for rifampicin and isoniazid resistance. The MTBDRplus assay produced a sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 36.36%, 100%, 100%, and 50.50%, respectively, for the detection of IOTB. Among the 28 cases from study group that were positive by MTBDRplus assay, rifampicin resistance was reported in six and isoniazid resistance in two cases. On sequencing of rpoB and katG gene, one case of false rifampicin-resistant by MTBDRplus was found. The other resistant isolates showed concordant mutations between MTBDRplus assay and sequencing. Conclusion The MTBDRplus assay is an effective tool for the rapid diagnosis of IOTB along with detection of drug resistance, thereby improving the outcome in IOTB.

Spider mites are difÞcult to identify because they are very small and have a limited number of diagnostic characters. Most species of the spider mite genus Tetranychus in Japan are morphologically similar, differing only in the diameter of the aedeagal knob in males. Because this genus contains many important pests, the unambiguous identiÞcation of species is crucial for effective pest management and quarantine procedures. DNA-based methods could complement the morphological methods. We examined whether Tetranychus species in Japan could be identiÞed by DNA sequences using the internal transcribed spacer region of nuclear ribosomal DNA and the cytochrome c oxidase subunit I gene of mitochondrial DNA. We determined sequences of the 13 known Tetranychus species in Japan. We could identify 10 of the 13 species in the internal transcribed spacer tree. In the cytochrome c oxidase subunit I tree, we could identify all 13 known Tetranychus species in Japan. Although Tetranychus kanzawai Kishida and T. parakanzawai Ehara were identiÞed by DNA sequences, they were clearly separated into two monophyletic clades each, indicating that a cryptic species existed in each species.

Introduction: Tuberculosis (TB) continues to be a public health problem globally. The burden is further exacerbated in developing countries like Nigeria, by poor diagnosis, management and treatment, as well as rapid emergence of drug-resistant TB. This study was conducted to evaluate the prevalence of drug-resistant TB, determine the rpoB gene mutation patterns of Mycobacterium tuberculosis (MTB) and model the dynamics of multidrug resistant TB (MDR-TB) in Enugu, Nigeria. Methodology: A total of 868 samples, from patients accessing DOTS services in designated centres within the zone, were screened by sputum-smear microscopy, while 207 samples were screened by Nucleic Acid Amplification (Xpert® MTB/Rif) Test (NAAT). A deterministic model was formulated to study the transmission dynamics of TB and MDR-TB, using live data generated through epidemiological study. Results: The results showed TB prevalence values of 22.1% and 21.3% by sputum-smear and NAAT assays, respectively. Analysis o...

The aim of the study was to compare Polymerase Chain Reaction (PCR) and Loop Mediated Isothermal Amplification (LAMP) assay for detection of Salmonella  in Chicken Meat.  Food safety remains a major concern worldwide, with outbreaks of foodborne illness resulting in significant costs to individuals and to the food industry. The Salmonella genus is the most prevalent group of foodborne pathogens that are regularly isolated from food-producing animals and thus are one of the leading causes of food borne diseases. Out of total 40 Salmonella isolates, LAMP and PCR assay detected 36 (90%) and 32 (80%) isolates, respectively. The sensitivity of the LAMP assay was noted to be 10-fold higher than that of PCR whereas the specificity of both LAMP and PCR was same.

Introduction. Accurate detection of filarial parasites in humans and vectors is essential for the implementation and evaluation of Global and National Programs to eliminate lymphatic filariasis. Immunological methods to detect infection are available; however, cross-reactivity issues have been reported in most of them. Nucleic acid-based molecular assays offer high levels of specificity and sensitivity and can be used to detect the infections. Methods. In this study, we evaluated loop-mediated isothermal amplification (LAMP) tests to amplify Wuchereria bancrofti DNA in patients' blood. The amplicons were tested by both pH-sensitive dyes for enhanced visual detection and agarose gel electrophoresis. A closed-tube LAMP assay was also evaluated. Cohen's Kappa statistics was used for statistical analysis of the assays. 125 patients consented for blood sampling which were used for clinical analysis of LAMP assays with the PCR method used as the "gold standard." Results. The sensitivity of the evaluated Wuchereria bancrofti LAMP was 92.3%, with a specificity of 97.3% and kappa statistics value of 0.84, which is in a strong agreement. Conclusion. In this study, LAMP assays coupled with fluorescence dye detection have been found to be suitable for diagnosis and monitoring of Wuchereria bancrofti infections in the Kenyan population.

Sensitive, specific, rapid, inexpensive and easy-to-use nucleic acid tests for use at the point-of-need are critical for the emerging field of personalised medicine for which companion diagnostics are essential, as well as for application in low resource settings. Here we report on the development of a point-of-care nucleic acid lateral flow test for the direct detection of isothermally amplified DNA. The recombinase polymerase amplification method is modified slightly to use tailed primers, resulting in an amplicon with a duplex flanked by two single stranded DNA tails. This tailed amplicon facilitates detection via hybridisation to a surface immobilised oligonucleotide capture probe and a gold nanoparticle labelled reporter probe. A detection limit of 1 × 10(-11) M (190 amol), equivalent to 8.67 × 10(5) copies of DNA was achieved, with the entire assay, both amplification and detection, being completed in less than 15 minutes at a constant temperature of 37 °C. The use of the tail...

Rapid, scalable, point-of-need, COVID-19 diagnostic testing is necessary to safely re-open economies and prevent future outbreaks. We developed an assay that detects single copies of SARS-CoV-2 virus directly from saliva and swab samples in 30 min using a simple, one-step protocol that utilizes only a heat block and microcentrifuge tube prefilled with a mixture containing the necessary reagents and has a sensitivity and specificity of 97% and 100%, respectively.

To safely re-open economies and prevent future outbreaks, rapid, frequent, point-of-need, SARS-CoV-2 diagnostic testing is necessary. However, existing field-deployable COVID-19 testing methods require the use of uncomfortable swabs and trained providers in PPE, while saliva-based methods must be transported to high complexity laboratories for testing. Here, we report the development and clinical validation of High-Performance Loop-mediated isothermal Amplification (HP-LAMP), a rapid, saliva-based, SARS-CoV-2 test with a limit of detection of 1.4 copies of virus per µl of saliva and a sensitivity and specificity with clinical samples of > 96%, on par with traditional RT-PCR based methods using swabs, but can deliver results using only a single fluid transfer step and simple heat block. Testing of 120 patient samples in 40 pools comprised of 5 patient samples each with either all negative or a single positive patient sample was 100% accurate. Thus, HP-LAMP may enable rapid and acc...

Mycobacterium avium subspecies paratuberculosis (MAP), causative agent of Johne’s disease (JD) is chronic granulomatous enteritis affecting domestic and wild ruminants. Since, MAP is not killed by pasteurization, it has been isolated from commercially pasteurized milk and milk products resulting exposure of human population to this pathogen through milk. Control and eradication of JD is considered difficult because of its insidious nature and lack of early, rapid and accurate diagnostic tests. Therefore in present study, a visual loop-mediated isothermal amplification (LAMP) assay method has been developed using a total of six primers including 2 outer (F3 and B3), 2 inner (FIP and BIP) and 2 loop (LF and LB) primers specific for MAP for the first time on ‘S 5’ strain of Mycobacterium avium subsp. paratuberculosis ‘Indian Bison type’ biotype. After laboratory standardization, final optimized reaction performed at 65°C for 45 min was achieved after titration of incubation time, tempe...

Background: Until 2016, microscopy was the main tool for the early detection of pulmonary tuberculosis in Cameroon, especially in remote settings. Due to the poor sensitivity of microscopy, there was a need to implement a molecular assay in order to improve tuberculosis case detection.Intervention: In 2017, tuberculosis loop-mediated isothermal amplification (TB-LAMP), a molecular rapid diagnostic test recommended by the World Health Organization, was implemented in Cameroon as a replacement test of microscopy for initial diagnosis of pulmonary tuberculosis and also as a follow-on test to microscopy for smear-negative sputum specimens. A roll out plan for TB-LAMP implementation in Cameroon had been developed from January 2017 to April 2017, followed by initial implementation at four sites in May 2017. Additional sites were added progressively.Lessons learnt: The use of TB-LAMP as a follow-on test to microscopy for smear-negative sputum specimens helped in the detection of tuberculos...

One challenge in point-of-care diagnostics is the lack of room-temperature methods for RNA detection based on enzymatic amplification and visualization steps. Here we perform a reverse transcription ligase chain reaction using our isothermal lesion induced DNA amplification (LIDA) technique that can be tuned to operate at any desired temperature. Using RNA-triggered LIDA, we can detect as little as ~100 attomoles target RNA and can distinguish RNA target from total cellular RNA. Finally, we demonstrate that the resulting DNA amplicons can be detected colorimetrically, also at room temperature, by rapid, target-triggered disassembly of DNA-modified gold nanoparticles. This integrated amplification/detection platform requires no heating or visualization instrumentation, which is an important step towards realizing instrument-free POC testing.

Prevention of life threatening hypersensitivity reactions to carbamazepine is possible through pre-treatment screening of the associated HLA-B*15:02 risk allele. However, clinical implementation of screening is hindered by the high cost and slow turnaround of conventional HLA typing methods. We have developed an interdigitated electrode (IDE) biosensor platform utilizing loop mediated isothermal amplification (LAMP) that can rapidly detect the HLA-B*15:02 allele. DNA amplification is followed by solid-phase hybridization of LAMP amplicons to a DNA probe immobilized on the IDE sensor surface, resulting in a change in sensor impedance. The testing platform does not require DNA extraction or post-amplification staining, achieving sample-to-answer in 1 h and 20 min. The platform was tested on 27 whole blood samples (14 HLA-B*15:02 positive and 13 negative) with sensitivity of 92.9% and specificity of 84.6% when applying a cutoff of impedance change. Based on these characters the LAMP-ID...

A multitargeted loop-mediated isothermal amplification (MT-LAMP) assay targeting mpt64 (Rv1980c) and IS 6110 was designed to diagnose genitourinary tuberculosis (GUTB) cases. While assessing gel-based, hydroxynaphthol blue (HNB) and SYBR Green I MT-LAMP assays on GUTB specimens (n = 28) in a pilot study, both gel-based/SYBR Green I assays exhibited better sensitivity than HNB LAMP. Since SYBR Green MT-LAMP is easier to perform compared with a gel-based assay, a higher number of GUTB specimens (n = 55) were evaluated by SYBR Green MT-LAMP, wherein 85.5% sensitivity and 94.4% specificity (n = 36) were obtained. Moreover, the sensitivity attained by MT-LAMP was significantly higher (p < 0.05) than with multiplex-PCR ( mpt64 + IS 6110). After further validating these MT-LAMP data in different epidemiological settings, this assay may be developed as a diagnostic kit.

TÓM TẮT: Vi khuẩn Escherichia coli O157:H7 là nguyên nhân quan trọng gây nên các vụ ngộ độc thực phẩm và gây chết người trên toàn thế giới. Một kỹ thuật phát hiện nhanh và chính xác là rất quan trọng. Shiga toxin 2 (mã hóa bởi gen stx2) là một tác nhân quan trọng gây của dòng STEC nói chung và E. coli O157:H7 nói riêng, do vậy gen st2 là một trong những gen đích cho rất nhiều các kỹ thuật phát hiện sử dụng. Trong nghiên cứu này, chúng tôi đã phát triển kỹ thuật LAMP để phát hiện nhanh và chính xác chủng vi khuẩn Escherichia coli O157:H7. Kỹ thuật này có ưu điểm là phát hiện nhanh, chính xác và thiết bị đơn giản. Bộ mồi được thiết kế đặc biệt dựa trên trình tự gen stx2. Chúng tôi nhận thấy không có sự khác nhau khi sử dụng duy nhất mồi trong (BIP/FIP) hoặc cả 4 mồi. Phản ứng dương tính được quan sát bằng mắt thường khi sử dụng SYBR green hoặc dưới tia UV.

Background: The global pandemic coronavirus SARS-CoV-2 has a healthcare, social and economic burden. To limit the spread of the virus, the World Health Organization (WHO) urgently called for extensive screening of suspected individuals; thus, a quick, simple, and sensitive diagnostic assay is always in need. Methods: We applied reverse transcription-loop-mediated isothermal amplification (RT-LAMP) for the detection of SARS-CoV-2. The RT-LAMP method was optimized by evaluating two fluorescence amplification mixes and several reaction times, and results were compared to the standard real-time RT-PCR (rtRT-PCR). The assay was validated using 200 nasopharyngeal swabs collected in viral transport media (62 positive for SARS-CoV-2, and 138 negative for SARS-CoV-2 detected by the rtRT-PCR method). The samples were diluted 1:4 in diethylpyrocarbonate (DEPC)-treated water, utilized for RT-LAMP using different singleplex and multiplex sets of LAMP primers (N gene, S gene, and orf1ab gene), an...

Background: Contagious Bovine Pleuropneumonia (CBPP) is the most important chronic pulmonary disease of cattle on the African continent causing severe economic losses. The disease, caused by infection with Mycoplasma mycoides subsp. mycoides is transmitted by animal contact and develops slowly into a chronic form preventing an early clinical diagnosis. Because available vaccines confer a low protection rate and short-lived immunity, the rapid diagnosis of infected animals combined with traditional curbing measures is seen as the best way to control the disease. While traditional labour-intensive bacteriological methods for the detection of M. mycoides subsp. mycoides have been replaced by molecular genetic techniques in the last two decades, these latter approaches require well-equipped laboratories and specialized personnel for the diagnosis. This is a handicap in areas where CBPP is endemic and early diagnosis is essential. Results: We present a rapid, sensitive and specific diagnostic tool for M. mycoides subsp. mycoides detection based on isothermal loop-mediated amplification (LAMP) that is applicable to field conditions. The primer set developed is highly specific and sensitive enough to diagnose clinical cases without prior cultivation of the organism. The LAMP assay detects M. mycoides subsp. mycoides DNA directly from crude samples of pulmonary/ pleural fluids and serum/plasma within an hour using a simple dilution protocol. A photometric detection of LAMP products allows the real-time visualisation of the amplification curve and the application of a melting curve/re-association analysis presents a means of quality assurance based on the predetermined strand-inherent temperature profile supporting the diagnosis. The CBPP LAMP developed in a robust kit format can be run on a battery-driven mobile device to rapidly detect M. mycoides subsp. mycoides infections from clinical or post mortem samples. The stringent innate quality control allows a conclusive on-site diagnosis of CBPP such as during farm or slaughter house inspections.

RT-qPCR utilising upper respiratory swabs are the diagnostic gold standard for SARS-CoV-2 despite reported low sensitivity and limited scale up due to global shortages. Saliva is a non-invasive, equipment independent alternative to swabs. We collected 145 paired saliva and nasal/throat (NT) swabs at diagnosis (day 0) and repeated on day 2 and day 7 dependent on inpatient care and day 28 for study follow up. Laboratory cultured virus was used to determine the analytical sensitivity of spiked saliva and swabs containing amies preservation media. Self-collected saliva samples were found to be consistent, and in some cases superior when compared to healthcare worker collected NT swabs from COVID-19 suspected participants. We report for the first time the analytical limit of detection of 10 -2 and 10 0 pfu/ml for saliva and swabs respectively. Saliva is a easily self-collected, highly sensitive specimen for the detection of SARS-CoV-2.

RT-qPCR utilising upper respiratory swabs are the diagnostic gold standard for SARS-CoV-2 despite reported low sensitivity and limited scale up due to global shortages. Saliva is a non-invasive, equipment independent alternative to swabs.We collected 145 paired saliva and nasal/throat (NT) swabs at diagnosis (day 0) and repeated on day 2 and day 7 dependent on inpatient care and day 28 for study follow up. Laboratory cultured virus was used to determine the analytical sensitivity of spiked saliva and swabs containing amies preservation media.Self-collected saliva samples were found to be consistent, and in some cases superior when compared to healthcare worker collected NT swabs from COVID-19 suspected participants. We report for the first time the analytical limit of detection of 10−2and 100 pfu/ml for saliva and swabs respectively.Saliva is a easily self-collected, highly sensitive specimen for the detection of SARS-CoV-2.

Morphological and molecular analyses were used to identify the potato cyst-forming nematodes found in soil samples from Costa Rica. The morphometric characters analyzed in cysts and second-stage juveniles conformed to those of Globodera pallida. PCR with species-specific primers (single or multiplex reaction) produced an amplification product of the same size as the G. pallida control one. The PCR-RFLP patterns obtained from the Costa Rican samples revealed no difference among them and were identical to those of the G. pallida control. Evidence of the presence of G. rostochiensis was not found. This is the first molecular report of the presence of G. pallida in Costa Rica.

Background and aim: Diagnosis of abdominal TB is an exigent task due to variable anatomical sites and non-specific clinical manifestations that closely resemble other diseases. Most of the available diagnostic modalities yield low sensitivities and need expertise to handle the specialized equipment. Hence, there is an urgent need to develop a rapid and reliable diagnostic test, so as to reduce the unnecessary morbidity. Therefore, we designed a multi-targeted loop-mediated isothermal amplification (MT-LAMP) for diagnosing abdominal TB. Methods: We evaluated an MT-LAMP (using mpt64 and IS6110) to diagnose abdominal TB within ascitic fluids and intestinal/peritoneal biopsies and compared these results with multiplex-PCR (M-PCR) using the same targets. MT-LAMP products were analyzed by gel electrophoresis and visual detection methods, that is, hydroxy naphthol blue and SYBR Green I reaction. Results: Sensitivities of 80.9% and 84.6% were obtained in suspected (n = 42) and total abdominal TB (n = 52) cases, respectively by gel-based MT-LAMP, with 97.3% (n = 37) specificity in non-TB controls. Notably, sensitivities attained by gel-based/SYBR Green I MT-LAMP in both clinically suspected and total abdominal TB cases were significantly higher (P < 0.05) than M-PCR. Furthermore, sensitivity obtained with SYBR Green I was equivalent to that of gel-based MT-LAMP, while somewhat lesser specificity (94.6%) was attained with SYBR Green I, compared with gel-based MT-LAMP. Conclusion: Both gel-based and SYBR Green MT-LAMP exhibited equivalent sensitivities to diagnose abdominal TB. Because SYBR Green LAMP is easier to perform than a gelbased assay, we are currently focused on improving the specificity of this assay so as to develop a diagnostic kit.

Background and study aim: The use of LPA is still new in Nigeria and only available in TB reference laboratories. In this study, the performance of LPA version 2.0 was evaluated for the detection of resistant to first-line anti-TB drugs. We evaluated the performance of LPA version 2.0 for the detection of rifampicin (RIF) and isoniazid (INH) resistance. Sputum samples from 223 participants were subjected to phenotypic drug susceptibility testing (PDST) and LPA. Statistical analyses included calculation of sensitivity, specificity, positive and negative predictive values. Cross tabulation was done along the kappa test to measure the degree of agreement between PDST and LPA. P-Value > 0.05 was considered significant. The overall sensitivity and specificity of 89.6% (95% C.I 82.5-94.5%) and 65.4% (95% C.I 44.3-82.7%) for detection of RIF resistance; for INH they were 76.6 (95% C.I 67.5-84.5%) and 76.7% (95% C.I 49.5-82.6%); and for MDR-TB, they were 67.0% (95% C.I 56.4-76.5%) and 72.0% (95% C.I 57.6-83.7%). The kappa values were 0.53 (0.001), 0.38 (p = 0.000) and 0.36 (p = 0.000) for the detection of RIF, INH and MDR-TB. There was moderate agreement between PDST and LPA for detection of RIF (κ = 0.57; P = 0.0001), INH (κ = 0.44; P = 0.0001), MDR-TB (κ = 0.43; P = 0.001). The Line probe assay has good sensitivity and specificity for detecting rifampicin and isoniazid. However, the overall performance is moderate; this should be considered when interpreting the assay's results.

Recombinase polymerase amplification (RPA) is an isothermal method to amplify nucleic acid sequences without the temperature cycling that classical PCR uses. Instead of using heat to denature the DNA duplex, RPA uses recombination enzymes to swap single-stranded primers into the duplex DNA product; these are then extended using a strand-displacing polymerase to complete the cycle. Because RPA runs at low temperatures, it never forces the system to recreate base-pairs following Watson-Crick rules, and therefore it produces undesired products that impede the amplification of the desired product, complicating downstream analysis. Herein, we show that most of these undesired side products can be avoided if the primers contain components of a self-avoiding molecular recognition system (SAMRS). Given the precision that is necessary in the recombination systems for them to function biologically, it is surprising that they accept SAMRS. SAMRS-RPA is expected to be a powerful tool within the...

Sensitive precise measurements were taken (high signal-to-noise ratios, 10-Pm image resolution, 99% scan-to-scan reproducibility). These features confirmed their potential as analytical tools, are a competitive alternative to fluorescence scanners, and incorporate additional advantages, such as user-friendly interface and connectivity for telemedicine needs. The analytical performances of the integrated platform (assay and reader) in the human samples were also excellent, with a low detection limit (100 genomic DNA copies), and reproducible (<15%) and cheap assays (< 10 €/test). The correct genotyping of a genetic biomarker (singlenucleotide polymorphism located in the GRIK4 gene) was achieved as the assigned genotypes agreed with those determined by using sequencing. The portability, favourable discriminating and read-out capabilities reveal that the implementation of mass-produced low-cost devices into minimal-specialised clinical laboratories is closer to becoming a reality.

The studied challenge is the specific detection of low-abundant genomic variants that differ by a single nucleotide from the wild type. The combination of blocked recombinase polymerase amplification (RPA) and selective capture by probes immobilised on magnetic-core particles integrated into a flow system is presented. The sensing principle was demonstrated as the effective concentration-detection of the specific generated products was achieved. The analytical performance of resulting assay was successfully compared to PCR-based methods or array formats, providing faster effective detection of the selective products. As proof of concept, the single-nucleotide substitutions of the KRAS gene at codon 12 were studied in chip with parallel microchambers and permanent magnets. The blocked RPA products (generated at 37 C) from tumour biopsies (extracted DNA 4 ng) provided a specific fluorescent bead-line that depends on the present mutation. The results agree with those reported by next-generation sequencing and provide new opportunities for in vitro diagnostic and personalised medicine.

An integrated device composed of micro-reactors embedded onto compact discs is proposed for real-time targeted DNA determination. The method principle is based on in-disc loop-mediated isothermal amplification (iD-LAMP) and quantitative optical read-out by a disc drive. In the presence of a target, the turbidimetric or colorimetric properties of reaction solution change, and the transmitted intensity of the disc drive laser modifies according to reaction yield. Monitoring real-time curves allowed the quantitative determination of DNA template amounts. The best amplification/detection results were obtained with micro-reactors (2 mm diameter and 1.1 mm in depth) drilled on a digital video disc (DVD) and detection based on the colorimetric mode. As proofof-concept, the assay was applied to detect pathogenic bacteria Salmonella spp. and to identify bovine meat in food samples. Ninety-six samples were simultaneously analysed in 15 min, with high selectivity and sensitivity (5 CFU/mL and 10 µg/g for bacteria and meat, respectively). The in-disc results were comparable to those obtained by conventional LAMP or qPCR approaches. The developed device allows low sample and reagent consumption (3 µL of reaction), portability, ease-of-use, and rapid low-cost high-throughput analyses.

The genotyping of a single-nucleotide polymorphism (SNP) is addressed through methods based on loop-mediated isothermal amplification (LAMP) combined with user-friendly optical read-outs to cover the current demand for point-of-care DNA biomarker detection. The modification of primer design and reaction composition improved the assay selectivity yielding allele-specific results and reducing false-positive frequency. Furthermore, the reduced cost, ease of use and effectiveness of colorimetric detection (solution and hybridization chip formats) were availed for the image capture by a smartphone, reching high sensitivity. In order to evaluate their discriminating capacities, LAMP-based methods were applied to human samples to genotype an SNP biomarker (rs1954787) located in the GRIK4 gene and related to the treatment response to anti-depressants drugs. Sensitive (limit of detection: 100 genomic DNA copies), reproducible (<15% error), fast (around 70 min) and low-cost assays were accomplished. Patient subgroups were correctly discriminated, agreeing with reference sequencing techniques. The achieved analytical performances using the developed amplification-detection principles confirmed the approach potential for point-of-care optical DNA testing.

An integrated sensor comprising isothermal DNA amplification and in situ detection is presented. The method principle is based on recombinase polymerase amplification (RPA) and detection in the microarray format by compact disc technology as a highthroughput sensing platform. Primers were immobilised on the polycarbonate surface of digital versatile discs (DVD) and, after hemi-nested amplification, multiplexing identification of each tethered product was achieved by optical scanning with a 650 nmlaser of the DVD drive. The efficiency of one-pot hybridisation/elongation/detection depended strongly on probe density and other factors such as the concentration of the unbound primers present in solution. The optimised conditions provided equivalent amplification factors (7.310 8  8.910 8 fold) to those obtained by conventional reactions performed in vials. The proposed method was applied to Salmonella detection (generic by hns and oriC genes, and specific for subspecies I by STM4507 gene). A triplex assay was satisfactorily compared to the non-integrated protocols. Food and vaccine samples were analysed in a shorter time with less handling. The results indicate that the multiplex DVD assay is a simple, competitive, isothermal, portable system that is particularly useful for microbiological routine analysis.