LOOP MEDIATED ISOTHERMAL AMPLIFICATION

Ebola virus (EBOV) causes severe hemorrhagic fever in humans and nonhuman primates with high mortality rates. Rapid identification of the virus is required to prevent spread of the infection. In this study, we developed and evaluated a one-step simple reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for the rapid detection of Zaire ebolavirus (ZEBOV), the most virulent species of EBOV, targeting the trailer region of the viral genome. The assay could detect 20 copies of the artificial ZEBOV RNA in 26 min with a real time-monitoring detection, and also detect 10 −3 FFU of the cell-culture propagated viruses. The reaction time needed to detect 10 4 FFU of ZEBOV was only 20 min. In addition, the assay was highly specific for ZEBOV. The RT-LAMP assay developed in this study is rapid, simple, highly specific, and sensitive for the detection of ZEBOV, and so may be an effective diagnostic tool. Furthermore, as this technique does not require sophisticated instrumentation, it seems very suitable for diagnosis in the field or laboratories in Ebola outbreak areas such as Central Africa.

Caprine arthritis encephalitis virus (CAEV), of the genus Lentivirus of the Retroviridae family, causes persistent disease, which is characterized by polyarthritis and mastitis in adult goats and progressive paresis (leukoencephalomyelitis) in kids. A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of CAEV in blood samples. Species-specific primers amplifying the gag gene region in the provirus were used for the detection of CAEV. The LAMP assay result was obtained 30 min after incubation on a constant temperature at 63°C in a heat block. Resulting amplicons were visualized by addition of SYBR green dye after the reaction and checked by agarose gel electrophoresis. The sensitivity of LAMP assay was evaluated by comparing the result with the nested polymerase chain reaction. Based on the experiments, the result of the assay indicated a rapid and sensitive test for the detection of CAEV.

Lycopersicum root, an agricultural waste was used as a biosorbent for the removal of Pb(II) ions from aqueous solution. The biosorptive capacity was determined using Atomic Adsorption Spectrophotometry, (AAS). Optimum metal ion concentration (5.0 mg/l), contact time (30 minutes), contact temperature (15 o C) and pH (5.5), dependence was established with a biosorbent dose of 3.0 g. The experimental data were analyzed using Langmuir, Freundlich, Temkin Dubinin-Radushkevich (D-R) isotherm models. The Freundlich Isotherm best described the biosorption process (R 2 = 0.9477), followed by Temkin (R 2 = 0.9130), Langmuir (R 2 = 0.8860) and D-R (R 2 = 0.0075). The kinetics of the adsorption process was evaluated using pseudo-first-order, pseudo-second-order and intra-particle diffusion models. The pseudo-second-order best described the process with R 2 = 0.9950 and the qe, cal (0.0600 mg/g) matched more closely to the qe, exp (0.0822 mg/g). The thermodynamic analysis showed that the adsorption was spontaneous, feasible and exothermic in nature. Therefore, Lycopersicum root can be used as a biosorbent for aqueous removal of Pb(II) ions. To cite this article

Loop mediatedisothermalamplification(LAMP)assay,apromisingdiagnostictest,hasbeendevelopedfor
detectionofdifferentpathogensofhumanaswellasanimals.Variouspositivepointssupportitsuseasafield
level testbutthemajorproblemisproductcrosscontaminationleadingtofalsepositiveresults.Different
methodswereadoptedbyvariousresearcherstocontrolthisfalsepositiveamplificationduetocross
contaminationbutallhavetheirownadvantagesanddisadvantages.AnewclosedtubeLAMPassaybasedon
agar dyecapsulewasdevelopedinthepresentstudyandthistechniquehassomeadvantagesovertheother
closed tubetechnique.
 Agar attheconcentrationof1.5%wasusedtosandwichSYBRgreendyeIwiththeaidofintradermalsyringe.
This agardyecapsulewasplacedovertheLAMPreactionmixturebeforeitwasamplified.
 To eliminatethehazardousnatureofUltraViolet(UV)lightduringresultvisualizationofLAMPproducts,the
present studydemonstratestheuseofLightEmittingDiode(LED)lightsforresultvisualization.LAMP wascarriedoutfor Brucella speciesdetectionusingthismodifiedtechniquesyieldinggoodresults
withoutanycrosscontaminationandLEDshowedsimilarfluorescencecomparedtoUV

A loop-mediated isothermal amplification (LAMP) assay was developed for the diagnosis of Theileria lestoquardi infection. The primers were designed based on the clone-5 sequence of T. lestoquardi. The specificity and sensitivity of the assay were established. Analysis of the specificity showed that the selected LAMP primers amplified the target sequence from T. lestoquardi DNA successfully, while no amplification was seen with DNA from Theileria annulata, Theileria ovis, Babesia ovis, Anaplasma ovis, or ovine genomic DNA. The specificity of the LAMP product was further confirmed by restriction digestion and sequencing. The sensitivity of the LAMP assay was analyzed in comparison to PCR resulting in a detection limit of 10 fg/μl of plasmid DNA containing the clone-5 sequence. The suitability for utilizing the LAMP assay in the field for the diagnosis of T. lestoquardi infection was tested on 100 field samples collected in Sudan and compared with results obtained by PCR. The relative specificity and sensitivity of the established LAMP assay was determined to be 92.1% and 87.5%, respectively, indicating that it may be regarded as an alternative molecular diagnostic tool to PCR which could be used for epidemiological surveys on T. lestoquardi infection.

Objectives: Loop-mediated isothermal amplification (LAMP) is a newly developed molecular method that can be performed isothermally. We developed and evaluated a LAMP assay using novel primers to diagnose tuberculosis directly from clinical samples. Materials: Primers were designed to amplify the specific novel esat-6 gene target of Mycobacterium tuberculosis (MTB). Quantitated DNA was used to determine analytical sensitivity and specificity was evaluated by testing 29 NTM and 37 other bacterial species. After standardization, its sensitivity and specificity were evaluated on samples from 118 TB suspected and 31 non-TB patients and compared it with smear, culture and mPCR methods. Results: LAMP was able to detect 5 fg DNA (one MTB) within 21 min and found to be 10 times more sensitive than mPCR and showed 100% specificity against NTM and other bacterial species. In clinical samples, LAMP showed highest MTB detection rate (52.5%) as compared to mPCR (44%) and culture (30.5%). On culture positive and mPCR positive samples, the sensitivity of LAMP was found to be 100% (95% CI 90.2e100) and 96.1% (95% CI 86.7e99.5) respectively with 93.5% (95% CI 78.5e99.2) of overall specificity. Conclusion: LAMP was found to be more sensitive than culture and mPCR for the detection of MTB. It showed specificity comparable to mPCR but was rapid and cost effective.

Potato viruses such as Potato virus Y (PVY) cause diseases that affect potato quality and thus damage potato production worldwide. Current tests for viral infection use double-antibody sandwich enzyme linked immunosorbent assays (DAS ELISA) or reverse transcription polymerase chain reaction (RT-PCR)/real-time RT-PCR. Despite many advantages, these assays have a number of drawbacks that affect cost and time of diagnosis. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) allows fast detection of target RNA. Here, we developed a closed-tube real-time RT LAMP assay for fluorescent detection of PVY. Specific RT-LAMP primers were designed to target the conserved region of the sequence encoding the PVY coat protein. The assay was specific and facilitated sensitive PVY detection in a single tube at 65 °C. The time-to positive values depended on the PVY concentration in tested samples. The effectiveness of RT LAMP in testing field-grown plants compared favorably with DAS ELISA and RT-PCR; under the tested conditions, RT-LAMP was about 1000-fold more sensitive than DAS ELISA and lateral flow assay (LFA) and about 10-fold more sensitive than RT PCR. Thus, this fluorescent RT-LAMP assay has great potential for routine detection of PVY.

Early detection of pathogens is crucial for the effective surveillance of diseases. Many efforts have been made to explore methods which can detect these pathogens within a short period of time without requiring a tedious protocol. However, these developed methods have disadvantages such as they are relatively time-consuming or require specialized laboratory facilities. In this work, we have developed an integrated microfluidic system for rapid and automatic detection of viruses by direct analysis from fresh Phalaenopsis orchid leaves. The entire protocol, including ribonucleic acid (RNA) purification, reverse transcription loop-mediated-isothermal-amplification (RT-LAMP) and optical detection by measuring changes in turbidity was performed on a single chip. This is the first time that an integrated microfluidic system for the detection of viruses infecting the Phalaenopsis orchid has been demonstrated. The sensitivity of the developed system was also explored in this study to validate its performance.

Twenty two isolates of Pasteurella multocida were obtained from different tissues of dead birds and animals (cattle, buffalo, sheep, and goat) suspected of fowl cholera and haemorrhagic septicaemia. The isolates were confirmed as P. multocida by various biochemical tests and PM PCR. An attempt was made to standardize Loop mediated isothermal amplification (LAMP) using newly designed primer sequences of KMT1 gene. Loop mediated isothermal amplification was conducted using 6 sets of primers at 65°C for 30 minutes and the result was confirmed by visual observation using SYBR green fluorescence dye as marker of positive reaction under UV transilluminator. On electrophoretic analysis of the products on 2% agarose gel, a ladder like pattern was observed, which suggested a positive amplification, whereas no amplification was observed in negative controls. Additionally, product of positive reaction yielded a green fluorescence following addition of SYBR green under UV transilluminator. It was observed that LAMP is a more sensitive test than polymerase chain reaction (PCR), as the former could detect DNA to lower limit of 22.8 pg/µl, while the latter could detect DNA to lower limit of 2.28 ng/ µl, thus LAMP could detect 100 times lesser concentration of DNA in comparison to PCR. Loop mediated isothermal amplification is a rather newer molecular technique, which can be used for rapid detection of infectious agent at field level and which does not require sophisticated instrument, i.e. thermal cycler. Furthermore, unlike the conventional PCR technique, LAMP requires lesser time to perform and result can be read visually.

This study presents a novel automatic assay for targeted ribonucleic acid (RNA) extraction and a one-step reverse transcription loop-mediated-isothermal-amplification (RT-LAMP) process for the rapid detection of viruses from tissue samples by utilizing an integrated microfluidic system. By utilizing specific probeconjugated magnetic beads, target RNA samples can be specifically recognized and hybridized onto the surface of the magnetic beads which are mixed with whole tissue lysates, followed by the synthesis of complementary deoxyribonucleic acid (cDNA) and isothermal amplification of target genes simultaneously with the incorporation of two specific primer sets. The nervous necrosis virus (NNV), the most common aquaculture pathogen, with a mortality rate in infected fish ranging from 80% to 100%, has been selected to verify the performance of the developed miniature system. Experimental results showed that the sensitivity of the integrated microfluidic LAMP system is about 100-fold higher when compared to a conventional one-step reverse-transcript polymerase chain reaction (RT-PCR) process. Significantly, the entire protocol from sample pre-treatment to target gene amplification can be completed within 60 min in an automatic manner without cross-reactions with other tested virus, bacteria and eukaryotic cells. Consequently, this integrated microfluidic LAMP system may provide a powerful platform for rapid purification and detection of virus samples.

A limit of detection of 200 CFU/mL of Salmonella typhi spiked in various sample matrices were achieved in 30 min. The sample matrices were raw/unprocessed milk, commercially available milk, juice from packed bottles, fresh juice from carts, potable water, turbid water and calf serum. The complete protocol comprised of three steps: (a) cell lysis (b) nucleic acid amplification and (c) an in situ optical detection. The cell lysis was carried out using a simple heating based protocol, while the loop-mediated isothermal amplification of DNA was carried out by an in-house designed and fabricated system. The developed system consists of an aluminum block fitted with two cartridge heaters along with a thermocouple. The system was coupled to a light source and spectrometer for a simultaneous in situ detection. Primers specific for STY2879 gene were used to amplify the nucleic acid sequence, isolated from S. typhi cells. The protocol involves 15 min of cell lysis and DNA isolation followed by 15 min for isothermal amplification and simultaneous detection. No cross-reactivity of the primers were observed at 106 CFU/mL of Escherichia coli, Vibrio cholerae, Salmonella typhimurium, Salmonella paratyphi A, Pseudomonas aeruginosa, Bacillus cereus, Lysteria monocytogenes, Clostridium botulinum, Staphylococcus aureus and Salmonella havana. In addition, the system was able to detect S. typhi of 200 CFU/mL in a concoction of 106 CFU/mL of E. coli, 106 CFU/mL of V. cholerae, and 106 CFU/mL of hepatocyte-derived cellular carcinoma HUH7 cells. The proposed rapid diagnostic system shows a promising future in the field of food and medical diagnostics.

Objectives: Loop-mediated isothermal amplification (LAMP) is a newly developed molecular method that can be performed isothermally. We developed and evaluated a LAMP assay using novel primers to diagnose tuberculosis directly from clinical samples. Materials: Primers were designed to amplify the specific novel esat-6 gene target of Mycobacterium tuberculosis (MTB). Quantitated DNA was used to determine analytical sensitivity and specificity was evaluated by testing 29 NTM and 37 other bacterial species. After standardization, its sensitivity and specificity were evaluated on samples from 118 TB suspected and 31 non-TB patients and compared it with smear, culture and mPCR methods. Results: LAMP was able to detect 5 fg DNA (one MTB) within 21 min and found to be 10 times more sensitive than mPCR and showed 100% specificity against NTM and other bacterial species. In clinical samples, LAMP showed highest MTB detection rate (52.5%) as compared to mPCR (44%) and culture (30.5%). On culture positive and mPCR positive samples, the sensitivity of LAMP was found to be 100% (95% CI 90.2e100) and 96.1% (95% CI 86.7e99.5) respectively with 93.5% (95% CI 78.5e99.2) of overall specificity. Conclusion: LAMP was found to be more sensitive than culture and mPCR for the detection of MTB. It showed specificity comparable to mPCR but was rapid and cost effective.

In recent years, many improvements have been made in foodborne pathogen detection methods to reduce the impact of food contamination. Several rapid methods have been developed with biosensor devices to improve the way of performing pathogen detection. This paper presents an automated endpoint detection system for amplicons generated by loop mediated isothermal amplification (LAMP) on a microfluidic compact disk platform. The developed detection system utilizes a monochromatic ultraviolet (UV) emitter for excitation of fluorescent labeled LAMP amplicons and a color sensor to detect the emitted florescence from target. Then it processes the sensor output and displays the detection results on liquid crystal display (LCD). The sensitivity test has been performed with detection limit up to 2.5 × 10−3 ng/µL with different DNA concentrations of Salmonella bacteria. This system allows a rapid and automatic endpoint detection which could lead to the development of a point-of-care diagnosis device for foodborne pathogens detection in a resource-limited environment.

Thrips palmi (from the order Thysanoptera) is a serious insect pest of various crops, including vegetables, fruits and ornamental plants, causing significant economic losses. Its presence constitutes a double threat; not only does T. palmi feed on the plants, it is also a vector for several plant viruses. T. palmi originated in Asia, but has spread to North and Central America, Africa, Oceania and the Caribbean in recent decades. This species has been sporadically noted in Europe and is under quarantine regulation in the European Union. For non-specialists its larval stages are indistinguishable morphologically from another widespread and serious insect pest Frankliniella occidentalis (a non-quarantine species in the European Union) as well as other frequently occurring thrips. In this study, we have developed a loop-mediated isothermal amplification protocol to amplify rDNA regions of T. palmi. The results were consistent whether isolated DNA or crushed insects were used as template , indicating that the DNA isolation step could be omitted. The described method is species specific and sensitive and provides a rapid diagnostic tool to detect T. palmi in the field.

In recent years, many improvements have been made in foodborne pathogen detection methods to reduce the impact of food contamination. Several rapid methods have been developed with biosensor devices to improve the way of performing pathogen detection. This paper presents an automated endpoint detection system for amplicons generated by loop mediated isothermal amplification (LAMP) on a microfluidic compact disk platform. The developed detection system utilizes a monochromatic ultraviolet (UV) emitter for excitation of fluorescent labeled LAMP amplicons and a color sensor to detect the emitted florescence from target. Then it processes the sensor output and displays the detection results on liquid crystal display (LCD). The sensitivity test has been performed with detection limit up to 2.5 × 10-3 ng/µL with different DNA concentrations of Salmonella bacteria. This system allows a rapid and automatic endpoint detection which could lead to the development of a point-of-care diagnosis d...

This report describes the development of a one-step reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for the detection of swine vesicular disease virus (SVDV). The assay detects the virus rapidly, within 30-60 min and the result is visualised either by gelelectrophoresis or by the naked eye through the addition of SybrGreen. A collection of 28 SVDV isolates were tested positive, while heterologous viruses such as foot-and-mouth disease virus and vesicular stomatitis virus remained negative. The performance of the RT-LAMP was compared directly with real-time PCR using RNA from clinical samples including nasal swabs, serum and faeces. For nasal swabs and serum the sensitivity of the RT-LAMP was shown to be at least equivalent to real-time PCR. Interestingly, for faecal samples the RT-LAMP assay was shown to be even more sensitive than real-time PCR, possibly because it is less sensitive to inhibitory substances. This RT-LAMP assay provides a number of benefits for the diagnosis of SVD, since the assay is sensitive and rapid, and the isothermal amplification strategy used is not reliant upon expensive equipment it is particularly suited for "front line" diagnosis of SVD in modestly equipped laboratories, in field stations or in mobile diagnostic units.

Psocids of the genera Liposcelis and Lepinotus (Psocoptera) are well known small, soft-bodied storedproduct pests that are difficult to identify using morphological characteristics, particularly the immature stages. Methods for quick, sensitive, and accurate identification of stored-product psocid species belonging to these genera are required for identification, discrimination, pest management, and inspection, and quarantine of imported grain. A specific primer set, RetCO1F/RetCO1R, was designed by targeting consensus sequences from multiple alignments of the CO1 gene of Lepinotus reticulatus for use in end point PCR, SYBR Green qPCR, and HDA. Lepinotus and Liposcelis species were tested to confirm the primer specificity. The described primer set allowed accurate identification of L. reticulatus, producing an amplicon of 119 bp in the three described assays. The primer set yielded a detection limit as low as 10 pg, 100 fg, and 1 ng using end point PCR, SYBR Green real time PCR, and HDA, respectively. All tests are accurate, rapid, sensitive, and useful for L. reticulatus identification and have multiple applications including biosecurity and forensic entomology.

E. coli O157:H7 is one of the most important foodborne pathogen that causes some
human illnesses such as bloody diarrhea, hemolytic-uremic syndrome, and kidney
failure. We developed a loop-mediated isothermal amplification (LAMP) assay with six
special primers that target a highly specific 299-bp region of the Z3276 gene for the
detection of E. coli O157:H7. Among 117 bacterial strains tested in this study, positive
results were only obtained from E. coli O157:H7 strains. The sensitivity level of the
Z3276-LAMP assay was determined to be 5 CFU/reaction tube in pure bacterial
culture. Moreover, the LAMP assay was successfully applied to artificially
contaminated ground beef with a sensitivity level of 103 CFU/mL without preenrichment and 10 CFU/mL after a 4-hour pre-enrichment. In conclusion, the present
LAMP assay would be a useful and powerful tool for the rapid, sensitive, and specific
diagnosis of E. coli O157:H7 strains in resource limited laboratories.

Objectives: Loop-mediated isothermal amplification (LAMP) is a newly developed molecular method that can be performed isothermally. We developed and evaluated a LAMP assay using novel primers to diagnose tuberculosis directly from clinical samples. Materials: Primers were designed to amplify the specific novel esat-6 gene target of Mycobacterium tuberculosis (MTB). Quantitated DNA was used to determine analytical sensitivity and specificity was evaluated by testing 29 NTM and 37 other bacterial species. After standardization, its sensitivity and specificity were evaluated on samples from 118 TB suspected and 31 non-TB patients and compared it with smear, culture and mPCR methods. Results: LAMP was able to detect 5 fg DNA (one MTB) within 21 min and found to be 10 times more sensitive than mPCR and showed 100% specificity against NTM and other bacterial species. In clinical samples, LAMP showed highest MTB detection rate (52.5%) as compared to mPCR (44%) and culture (30.5%). On culture positive and mPCR positive samples, the sensitivity of LAMP was found to be 100% (95% CI 90.2e100) and 96.1% (95% CI 86.7e99.5) respectively with 93.5% (95% CI 78.5e99.2) of overall specificity. Conclusion: LAMP was found to be more sensitive than culture and mPCR for the detection of MTB. It showed specificity comparable to mPCR but was rapid and cost effective.

'Torrado' disease caused by tomato torrado virus (ToTV) is responsible for considerable losses in tomato production. Therefore, a one-step reverse transcription loop-mediated isothermal amplification protocol for early and fast detection of ToTV isolates has been developed. The RNA extracted from ToTV-infected plants was tested using this protocol with a set of six primers specific for the Vp35 coat protein gene sequence. The amplified products were analyzed using amplification curves, electrophoresis, and direct staining of DNA. The sensitivity of the protocol was tenfold higher than that of conventional RT-PCR. This new protocol is inexpensive, rapid, simple, and very sensitive.

Loop-mediated isothermal amplification (LAMP) method amplifies DNA with high specificity, sensitivity and rapidity. In this study, we used a conserved sequence in the 200-to 300-fold repetitive 529 bp gene of Toxoplasma gondii to design primers for LAMP test. Detection limit of T. gondii LAMP assay with the primers is 1 pg/lL of T. gondii DNA, which was evaluated using 10-fold serially diluted DNA of cultured parasites. Furthermore, LAMP and conventional PCR methods were applied for amplification of the T. gondii DNA extracted from the lymph nodes taken from pigs which were suspected to be Toxoplasma infection. As a result, 76.9% (70/91) and 85.7% (78/91) of the samples were positive on PCR and LAMP analyzes, respectively. Therefore, the LAMP has a potential to be applied as an alternative molecular diagnostic tool for detection of T. gondii infection from veterinary samples. This is the first study, which applies the LAMP method to diagnose Toxoplasma from veterinary samples.

Loop-mediated isothermal amplification (LAMP) is a novel method that amplifies target nucleic acids under isothermal conditions. It is a rapid, specific, and sensitive method, which does not require costly thermal cyclers for the detection of nucleic acids. Thus, it is suitable for on-site detection assays under low-resource settings. It can also be integrated on compact lab-on-a-chip devices for the development of micro-total analysis systems. This review discusses LAMP-based methods, as well as LAMP-based centrifugal, microfluidic, and other fluid-handling devices, which have been developed for the assessment of meat quality parameters that are related to the presence or absence of nucleic acids, for example, animal species identification and microbiological quality. Advances in improving the rapidity, specificity, and sensitivity of LAMP techniques for the assessment of these meat quality parameters are also discussed in this review.

Aims: Enterocytozoon hepatopenaei is an emerging microsporidian parasite that has been linked to recent losses caused by white faeces syndrome (WFS) in cultivated giant or black tiger shrimp Penaeus (Penaeus) monodon and whiteleg shrimp Penaeus (Litopenaeus) vannamei in Asia. To more accurately assess its impact on shrimp production and to determine reservoir carriers for control measures, our objective was to establish a loop-mediated isothermal amplification (LAMP) assay combined with colorimetric nanogold (AuNP) for rapid, sensitive and inexpensive detection of this parasite. Methods and Results: A set of six specific primers was designed to successfully detect the SSU rRNA gene of E. hepatopenaei by a LAMP reaction of 45 min at 65°C combined with visual detection of the amplification product via hybridization at 65°C for 5 min with a ssDNA-labelled nanogold probe, followed by salt-induced AuNP aggregation (total assay time, approximately 50 min). This method gave similar results to LAMP followed by electrophoresis or spectrophotometric detection, and it was more sensitive (0Á02 fg total DNA) than a conventional nested PCR (0Á2 fg total DNA). The new method gave negative results with shrimp DNA templates extracted from diseased shrimp containing other pathogens, indicating that the LAMP-AuNP assay was specific for E. hepatopenaei. Conclusions: Without sacrificing sensitivity or specificity, the new LAMP-AuNP assay significantly reduced the time, ease and cost for molecular detection of E. hepatopenaei in shrimp. Significance and Impact of the study: The new method employs simple, inexpensive equipment and involves simple steps making it applicable for small field laboratories. Wider application of the method to screen broodstock before use in a hatchery, to screen postlarvae before stocking shrimp ponds, to test for natural carriers and to monitor shrimp in rearing ponds would help to assess and reduce the negative impact of this parasite in shrimp farming.

Background. Hepatitis B virus (HBV) is an important blood-borne pathogen that causes hepatic inflammation and can lead to liver cirrhosis and hepatocellular carcinoma. Conventional methods of HBV detection are time consuming and require highly trained personnel and elaborate equipment. This report describes the development of a rapid, simple, specific, and sensitive loop-mediated isothermal amplification assay (LAMP) for detection of HBV genotypes A, B, C, D, E, and F in blood samples.

BACKGROUND: Bemisia tabaci, the sweetpotato whitefly, is a globally invasive pest that causes serious agricultural damage by transmitting plant viruses. This pest forms a cryptic species complex that displays morphologically indistinguishable biotypes. Among them, the B and Q biotypes are the most important pests worldwide. Because they have different levels of insecticide resistance, these biotypes must be identified in order to achieve proper pest control. Therefore, a convenient, rapid and specific detection method for identifying the two biotypes is necessary.

A simple, rapid, and efficient method for isolating genomic DNA from germinated seeds of wheat that is free from polysaccharides and polyphenols is reported. DNA was extracted, treated with RNase, measured and tested for completeness using agarose gel electrophoresis. DNA purification from wheat grains yielded abundant, amplifiable DNA with yields typically between 100 and 200 ng DNA/mg. The effectiveness and reliability of the method was tested by assessing quantity and quality of the isolated DNA using three PCR-based markers. Inter-simple sequence repeats (ISSRs) were used to assess the genetic diversity between different wheat varieties. Specific PCR primer pair Tox5-1/Tox5-2 and a loop-mediated isothermal amplification (LAMP) procedure were used to detect genomic DNA of Fusarium graminearum in contaminated wheat seeds. In this method there is no need to use liquid nitrogen for crushing germinated seedlings. The protocol takes approximately one hour to prepare high quality DNA. In

Avian reovirus (ARV) is an important pathogen of poultry and causes significant economic losses to the poultry industry. To develop a rapid and sensitive method for the surveillance of ARV, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was established using a set of six primers specific to the S1 gene segment of ARV. The established assay was performed at 62oC for 60 min in a thermal block, and the result was visualized directly under daylight or ultraviolet light. The detection limit of the RT-LAMP assay was 10 fg total RNA, which was 100-fold higher than that of reverse transcriptase polymerase chain reactions. The specificity of the assay was supported by the lack of cross-reaction with other avian pathogens. Furthermore, viral RNAs of field isolates were successfully detected by the assay. Overall, the newly established RT-LAMP assay is simple, rapid, sensitive, specific, and can visually detect ARV without the use of any specialized equipment.

To establish a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid detection of porcine reproductive and respiratory syndrome virus (PRRSV), four primers specific to six regions of the N gene were designed. After amplification in an isothermal water bath for 1 h, samples containing PRRSV generated the expected ladder-like products while porcine parvovirus, porcine circovirus, classic swine fever virus, pseudorabies virus, and swine testis cells generated no product. The sensitivity and specificity of the RT-LAMP assay were evaluated by comparison with reverse-transcription polymerase chain reaction (RT-PCR) and real-time PCR. Because it is specific and simple, the RT-LAMP assay will be useful for the diagnosis of PRRSV infection.

Loop-mediated isothermal amplification (LAMP) is a technique capable of rapidly amplifying specific nucleic acid sequences without specialized thermal cycling equipment. In addition, several detection methods that include dye fluorescence, gel electrophoresis, turbidity and colorimetric change, can be used to measure or otherwise detect target amplification. To date, publications have described the requirement for some form of sample nucleic acid extraction (boiling, lysis, DNA purification, etc.) prior to initiating a LAMP reaction. We demonstrate here, the first LAMP positive results obtained from vegetative cells and spores of Bacillus anthracis without nucleic acid extraction. Our data show that the simple addition of cells or spores to the reaction mixture, followed by heating at 63°C is all that is required to reproducibly amplify and detect target plasmid and chromosomal DNA via colorimetric change. The use of three primer sets targeting both plasmids and the chromosome of B. anthracis allows for the rapid discrimination of non-pathogenic bacteria from pathogenic bacteria within 30 min of sampling. Our results indicate that direct testing of B. anthracis spores and cells via LAMP assay will greatly simplify and shorten the detection process by eliminating nucleic acid purification. These results may allow more rapid detection of DNA from pathogenic organisms present in field and environmental samples.

Aims: The present study was aimed to develop a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of Vibrio cholerae. Methods and Results: A set of five designed primers that recognized specifically the V. cholerae ompW gene was used. The optimized time and temperature conditions for the LAMP assay were 75 min at 65°C, respectively. The LAMP method accurately identified 16 isolates of V. cholerae but did not detect 28 non-cholerae Vibrio isolates and 37 non-Vibrio bacterial isolates. The sensitivity of LAMP for V. cholerae detection in pure cultures was 2AE2 · 10 3 CFU ml )1 or equivalent to 8 CFU per reaction. In the case of spiked shrimp samples without enrichment, the detection limit for V. cholerae was 2AE2 · 10 4 CFU g )1 or equivalent to 20 CFU per reaction, while that of PCR was 100 CFU per reaction. Conclusion: The developed LAMP assay targeting ompW gene was rapid, specific and sensitive for V. cholerae detection. Significant and Impact of the study: The developed LAMP assay appears to be precise, accurate and a valuable tool for detection of V. cholerae. This assay can replace laborious biochemical tests for the identification of V. cholerae in contaminated food sample.

Psocids of the genera Liposcelis and Lepinotus (Psocoptera) are well known small, soft-bodied storedproduct pests that are difficult to identify using morphological characteristics, particularly the immature stages. Methods for quick, sensitive, and accurate identification of stored-product psocid species belonging to these genera are required for identification, discrimination, pest management, and inspection, and quarantine of imported grain. A specific primer set, RetCO1F/RetCO1R, was designed by targeting consensus sequences from multiple alignments of the CO1 gene of Lepinotus reticulatus for use in end point PCR, SYBR Green qPCR, and HDA. Lepinotus and Liposcelis species were tested to confirm the primer specificity. The described primer set allowed accurate identification of L. reticulatus, producing an amplicon of 119 bp in the three described assays. The primer set yielded a detection limit as low as 10 pg, 100 fg, and 1 ng using end point PCR, SYBR Green real time PCR, and HDA, respectively. All tests are accurate, rapid, sensitive, and useful for L. reticulatus identification and have multiple applications including biosecurity and forensic entomology.

Point-of-care (POC) genetic diagnostics critically depends on miniaturization and integration of sample processing, nucleic acid amplification, and detection systems. Polymerase chain reaction (PCR) assays have extensively applied for the diagnosis of genetic markers of disease. Microfluidic chips for microPCR with different materials and designs have been reported. Temperature cycling systems with varying thermal masses and conductivities, thermal cycling times, flow-rates, and cross-sectional areas, have also been developed to reduce the nucleic acid amplification time. Similarly, isothermal amplification techniques (e.g., loop-mediated isothermal amplification or LAMP), which are still are emerging, have a better potential as an alternative to PCR for POC diagnostics. Isothermal amplification techniques have: (i) moderate incubation temperature leading to simplified heating and low power consumption, (ii) yield high amount of amplification products, which can be detected either visually or by simple detectors, (iii) allow direct genetic amplification from bacterial cells due to the superior tolerance to substances that typically inhibit PCR, (iv) have high specificity, and sensitivity, and (v) result in rapid detection often within 10-20 min. The aim of this review is to provide a better understanding of the advantages and limitations of microPCR and microLAMP systems for rapid and POC diagnostics.

Objective: Mycoplasmas are major contaminants of cell culture and affect in vitro biological and diagnostic tests. Mycoplasma detection is conducted using culture and molecular methods. These methods vary in terms of accuracy, reliably and sensitivity. Loop-mediated isothermal amplification (LAMP) is used to amplify target DNA in a highly specific and rapid manner. This study aimed to develop a LAMP method for rapid detection of Mycoplasma in culture samples. Materials and Methods: In this descriptive laboratory study, for LAMP detection of Mycoplasma contaminations in cell culture, we used primers specifically designed for targeting the 16S rRNA conserved gene of Mycoplasma spp. For a positive control structure, 16S rRNA amplified based on PCR, was cloned in a plasmid vector and sequenced. The assay specificity was evaluated using Mycoplasma genomic DNA and a panel containing genomes of gram-positive and gram-negative organisms. Results: In this study, the method developed for detection of Mycoplasma contamination of cell cultures was a rapid, sensitive and cost-effective LAMP approach. The results demonstrated that this method benefits from high specificity (100%) for amplification of Mycoplasma strains and high speed (multiplication within 60 minutes), while it does not require expensive laboratory equipment compared to those needed for polymerase chain reaction (PCR)-based detection. Conclusion: Our study is the first report about application of LAMP assay based on 16S rRNA gene for detection of Mycoplasma strains; this technique could be considered a useful tool for rapid detection of contamination of cell culture.

Loop-mediated isothermal amplification (LAMP) is a promising nucleic acid assay for rapid and cost-effective detection of pathogen-specific sequences within a sample. Development of an appropriate taxonomic group-specific LAMP assay highly relies on the design of proper primers to cover all major members of the taxon. Regarding this fact, we designed and evaluated a new LAMP primer set specific to prt (rfbS) gene for rapid identification of Salmonella serogroup D serotypes. Unlike the previously reported LAMP assay for serogroup D which detects solely the non-typhoidal serotypes; the new LAMP primers set detects both typhoidal and non-typhoidal serotypes of this serogroup with a detection limit of 10 CFU/rection. Furthermore, the technique was successfully applied to artificially contaminated meat samples with an inoculation level of 1–5 CFU/250 ml of Salmonella Enteriti-dis, following a 5-h pre-enrichment step in tryptic soy broth. Overall, the new LAMP assay and its optimized setup would be useful for fast diagnosis of food poisoning incidents caused by these bacteria.

In recent years, many improvements have been made in foodborne pathogen detection methods to reduce the impact of food contamination. Several rapid methods have been developed with biosensor devices to improve the way of performing pathogen detection. This paper presents an automated endpoint detection system for amplicons generated by loop mediated isothermal amplification (LAMP) on a microfluidic compact disk platform. The developed detection system utilizes a monochromatic ultraviolet (UV) emitter for excitation of fluorescent labeled LAMP amplicons and a color sensor to detect the emitted florescence from target. Then it processes the sensor output and displays the detection results on liquid crystal display (LCD). The sensitivity test has been performed with detection limit up to 2.5 × 10 −3 ng/µL with different DNA concentrations of Salmonella

Ebola virus (EBOV) causes severe hemorrhagic fever in humans and nonhuman primates with high mortality rates. Rapid identification of the virus is required to prevent spread of the infection. In this study, we developed and evaluated a one-step simple reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for the rapid detection of Zaire ebolavirus (ZEBOV), the most virulent species of EBOV, targeting the trailer region of the viral genome. The assay could detect 20 copies of the artificial ZEBOV RNA in 26 min with a real time-monitoring detection, and also detect 10 −3 FFU of the cell-culture propagated viruses. The reaction time needed to detect 10 4 FFU of ZEBOV was only 20 min. In addition, the assay was highly specific for ZEBOV. The RT-LAMP assay developed in this study is rapid, simple, highly specific, and sensitive for the detection of ZEBOV, and so may be an effective diagnostic tool. Furthermore, as this technique does not require sophisticated instrumentation, it seems very suitable for diagnosis in the field or laboratories in Ebola outbreak areas such as Central Africa.

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid method whereby DNA is amplified with high specificity, efficiency, and rapidity under isothermal conditions using a set of four specifically designed primers and a DNA polymerase with strand displacement activity. In this study, we used LAMP primer sets designed from EMA-1 and Bc 48 genes for detection of Theileria equi and Babesia caballi infections, respectively. These primer sets specifically amplified DNA of the respective parasites. Both primer sets amplified T. equi and B. caballi up to 10 À6 dilution of 10-fold serially diluted samples. Furthermore, DNA extracted from blood collected from a horse experimentally infected with T. equi was amplified by a T. equi LAMP primer set from days 2 to 35 post-infection, demonstrating the high sensitivity of these primers. Of 55 samples collected from China, 81.8% and 56.3% were positively detected by LAMP for T. equi and B. caballi infections, respectively. In contrast, 91.8% and 45.9% of the 37 samples collected from South Africa were LAMP positive for T. equi and B. caballi, respectively. These results suggest that LAMP could be a potential diagnostic tool for epidemiological studies of equine piroplasmosis. #

Isothermal nucleic acid amplifications (iNAATs) have become an important alternative to PCR for in vitro molecular diagnostics in all fields. Amongst iNAATs Loop-mediated amplification (LAMP) has gained much attention over the last decade because of the simplicity of hardware requirements. LAMP demonstrates performance equivalent to that of PCR, but its application has been limited by the challenging primer design. The design of six primers in LAMP requires a selection of eight priming sites with significant restrictions imposed on their respective positioning and orientation. In order to relieve primer design constraints we propose an alternative approach which uses Stem primers instead of Loop primers and demonstrate the application of STEM-LAMP in assaying for Clostridium difficile, Listeria monocytogenes and HIV. Stem primers used in LAMP in combination with loop-generating and displacement primers gave significant benefits in speed and sensitivity, similar to those offered by Loop primers, while offering additional options of forward and reverse orientations, multiplexing, use in conjunction with Loop primers or even omission of one or two displacement primers, where necessary. Stem primers represent a valuable alternative to Loop primers and an additional tool for IVD assay development by offering more choices for primer design at the same time increasing assay speed, sensitivity, and reproducibility.

A simple, rapid, and efficient method for isolating genomic DNA from germinated seeds of wheat that is free from polysaccharides and polyphenols is reported. DNA was extracted, treated with RNase, measured and tested for completeness using agarose gel electrophoresis. DNA purification from wheat grains yielded abundant, amplifiable DNA with yields typically between 100 and 200 ng DNA/mg. The effectiveness and reliability of the method was tested by assessing quantity and quality of the isolated DNA using three PCR-based markers. Inter-simple sequence repeats (ISSRs) were used to assess the genetic diversity between different wheat varieties. Specific PCR primer pair Tox5-1/Tox5-2 and a loop-mediated isothermal amplification (LAMP) procedure were used to detect genomic DNA of Fusarium graminearum in contaminated wheat seeds. In this method there is no need to use liquid nitrogen for crushing germinated seedlings. The protocol takes approximately one hour to prepare high quality DNA. In