Loop-mediated Isothermal Amplification Primer Sets Research Articles

Loop-mediated isothermal amplification: Development, validation and application of simple and rapid assays for quantitative detection of species of Arcobacteraceae family- and species-specific Aliarcobacter faecis and Aliarcobacter lanthieri.

AimThe family Arcobacteraceae formerly genus Arcobacter has recently been reclassified into six genera. Among nine species of the genus Aliarcobacter, Aliarcobacter faecis and Aliarcobacter lanthieri have been identified as emerging pathogens potentially cause health risks to humans and animals. This study was designed to develop/optimize, validate and apply Arcobacteraceae family‐ and two species‐specific (A. faecis and A. lanthieri) loop‐mediated isothermal amplification (LAMP) assays to rapidly detect and quantify total number of cells in various environmental niches.Methods and ResultsThree sets of LAMP primers were designed from conserved and variable regions of 16S rRNA (family‐specific) and gyrB (species‐specific) genes. Optimized Arcobacteraceae family‐specific LAMP assay correctly amplified and detected 24 species, whereas species‐specific LAMP assays detected A. faecis and A. lanthieri reference strains as well as 91 pure and mixed culture isolates recovered from aquatic and faecal sources. The specificity of LAMP amplification of A. faecis and A. lanthieri was further confirmed by restriction fragment length polymorphism analysis. Assay sensitivities were tested using variable DNA concentrations extracted from simulated target species cells in an autoclaved agricultural water sample by achieving a minimum detection limit of 10 cells mL−1 (10 fg). Direct DNA‐based quantitative detection, from agricultural surface water, identified A. faecis (17%) and A. lanthieri (1%) at a low frequency compared to family‐level (93%) with the concentration ranging from 2·1 × 101 to 2·2 × 105 cells 100 mL−1.ConclusionsOverall, these three DNA‐based rapid and cost‐effective novel LAMP assays are sensitive and can be completed in less than 40 min. They have potential for on‐site quantitative detection of species of family Arcobacteraceae, A. faecis and A. lanthieri in food, environmental and clinical matrices.Significance and Impact of the StudyThe newly developed LAMP assays are specific, sensitive, accurate with higher reproducibility that have potential to facilitate in a less equipped lab setting and can help in early quantitative detection and rate of prevalence in environmental niches. The assays can be adopted in the diagnostic labs and epidemiological studies.

In Situ Nucleic Acid Amplification and Ultrasensitive Colorimetric Readout in a Paper-Based Analytical Device Using Silver Nanoplates.

A rapid, highly sensitive, and quantitative colorimetric paper-based analytical device (PAD) based on silver nanoplates (AgNPls) and loop-mediated isothermal amplification (LAMP) is presented. It is shown that cauliflower-like concatemer LAMP products can mediate crystal etching of AgNPls, with a threefold signal enhancement versus linear dsDNA. Methicillin-resistant Staphylococcus aureus (MRSA), an antimicrobial resistant bacterium that poses a formidable risk with persistently high mortality, is used as a model pathogen. Due to the excellent color contrast provided by AgNPls, the PAD allows qualitative analysis by the naked eye and quantitative analysis using a smartphone camera, with detection limits down to a single copy in just 30 min, and a linear response from 1 to 104 copies (R2 = 0.994). The entire assay runs in situ on the paper surface, which drastically simplifies operation of the device. This is the first demonstration of single copy detection using a colorimetric readout, and the developed PAD shows great promise for translation into an ultrasensitive gene-based point-of-care test for any infectious disease target, via modification of the LAMP primer set.

A novel peptide nucleic acid- and loop-mediated isothermal amplification assay for the detection of mutations in the 23S rRNA gene of Treponema pallidum

Introduction. Macrolides could be a potential alternative treatment for Treponema pallidum infections in patients; however, macrolide-resistant T. pallidum is spreading rapidly worldwide.Hypothesis/Gap Statement. There are presently no alternatives to serological tests for syphilis that can be used to evaluate therapeutic effects due to the fact that T. pallidum cannot be cultured in vitro.Aim. In this study, we constructed a method for rapidly identifying T. pallidum and confirming macrolide resistance by using loop-mediated isothermal amplification (LAMP) with peptide nucleic acids (PNAs).Methodology. A set of LAMP primers was designed to span nucleotide positions 2058 and 2059 in 23S rRNA. A PNA clamping probe was also designed to be complementary to the wild-type sequence (A2058/A2059) and positioned to interfere with both the annealing of the 3' end of the backward inner primer and the concomitant extension. Prior to the LAMP assay, swab samples from suspected syphilitic lesions were boiled for DNA extraction.Results. The assay had an equivalent detection limit of 1.0×101 copies/reaction and showed specificity against 38 pathogens. In the presence of a 4 µM PNA probe, LAMP amplified up to 1.0×101 copies/reaction using plasmids harbouring the complementary mutant sequences (A2058G or A2059G), whereas amplification was completely blocked for the wild-type sequence up to a concentration of 1.0×103 copies/reaction. For the 66 PCR-positive clinical specimens, the overall detection rate via LAMP was 93.9 % (62/66). Amplification was successful for all 53 mutant samples and was incomplete for all nine WT samples by the PNA-mediated LAMP assays.Conclusion. We developed a PNA-mediated LAMP method that enabled us to rapidly identify T. pallidum and determine its macrolide susceptibility via a culture-independent protocol.

Development of a pair of real-time loop mediated isothermal amplification assays for detection of Yersinia pestis, the causative agent of plague

Yersinia pestis, the causative agent of plague mainly infects rodents, while humans are the accidental host. The conventional diagnostic methods available for Y. pestis exhibit cross-reactivity with other enteropathogenic bacteria which makes its detection difficult. Rapid and reliable point-of-care detection of Y. pestis is essential for timely initiation of medical treatment. In the present study, a pair of loop mediated isothermal amplification (LAMP) assays has been developed for rapid detection of Y. pestis. Two sets of LAMP primers, each containing 6 primers were specifically designed targeting caf1 and 3a genes located on pFra plasmid and chromosome of Y. pestis, respectively. Isothermal amplification was accomplished at 65 °C for 40 min for caf1 target, and at 63 °C for 50 min for 3a choromosomal target. The analytical sensitivity of the assay for the caf1 and 3a targets was found to be 500 fg and 100 fg genomic DNA of Y. pestis, respectively. The caf1 and 3a LAMP assays detected as few as 100 copies of caf1 and 10 copies of 3a gene targets harboured in the respective recombinant plasmids. The amplified products were detected visually under visible and UV light using SYBR Green 1 dye. The assay pair was found to be highly specific as it did not cross-react with closely related and other bacterial species.

Development of a loop-mediated isothermal amplification assay for rapid detection of African swine fever

Since the first African swine fever (ASF) outbreak was reported in China in 2018, the disease has spread rapidly to several countries in Asia. The early detection of this disease is essential for the ASF control strategy to be effective. Loop-mediated isothermal amplification (LAMP) is a nucleic acid detection assay that is rapid, simple, cost-effective and field-friendly. In this study, we have developed a colorimetric assay of LAMP to detect ASF virus (ASFV). A set of LAMP primers was designed to target the conserved region of the VP72 gene. The conditions of LAMP were optimized. The amplification products were easily detected by the naked eye using hydroxynaphthol blue (HNB). The positive LAMP reaction generated a violet to sky blue color change. The sensitivity and specificity of LAMP assay were demonstrated in comparison with the OIE-recommended real-time PCR. A total of 211 samples including 121 confiscated pork products and 90 spiked clinical specimens were tested. The optimal amplification of ASFV DNA by LAMP was incubation at 60 °C for 90 min. The analytical sensitivity of ASFV LAMP assay was at least 368 plasmid DNA copies/µL without cross-reactivity with other swine pathogens. The diagnostic sensitivity and specificity of LAMP were 88% and 100%, respectively. There was almost perfect agreement between LAMP and real-time PCR assays (Kappa value=0.84). This novel LAMP assay is deemed to be a rapid, simple, sensitive, specific diagnostic tool and suitable for early detection of ASF to minimize the likelihood of ASF spread nationwide.

Direct triplex loop-mediated isothermal amplification assay for the point-of-care molecular detection of Salmonella genus, subspecies I, and serovar Typhimurium

In this study, a novel triplex loop-mediated isothermal amplification (LAMP) assay was developed and applied to a model of representative foodborne pathogen, Salmonella, to investigate the performance of the assay with high rapidity, specificity and sensitivity, and its ability to detect a pathogen in contaminated food. Three sets of LAMP primers were designed for target genes in Salmonella genus, subspecies I, and S. Typhimurium. Multiple targets were differentiated by the simultaneous analysis of annealing curves in a single LAMP reaction. In a triplex LAMP assay, each primer amplifies the target genes only, without any cross-reactivity with non-target genes. The limit of detection of this assay was 2.5 pg of S. Typhimurium DNA. The assay was applied to chicken meat artificially inoculated with S. Typhimurium. To decrease the inhibitory effects of the food matrix on the LAMP reaction, proteinase K was used in sample preparation, and the sample was heated and used directly as a DNA template. A combination of the direct extraction method optimized for food with triplex LAMP was able to detect low levels of S. Typhimurium (6.4 × 101 CFU/g) without a pre-enrichment step. This novel triplex multiplex LAMP assay successfully identified Salmonella genus, subspecies I, and S. Typhimurium within 60 min from sampling to results. This is therefore a rapid, reliable, sensitive, specific, and easy molecular diagnostic tool for on-site pathogen detection and food safety testing.

Development of a Portable LAMP Assay for Detection of Neofabraea perennans in Commercial Apple Fruit.

Bull's eye rot (BER) is a major economic postharvest disease of apple and pear that can be caused by four Neofabraea species: N. perennans, N. alba, N. malicorticis, and N. kienholzii. In Central Washington, BER is predominantly caused by N. perennans. The fungus infects fruit preharvest, and because of the dry growing season in the region, infections remain latent with symptoms expressed only after 3 to 4 months of storage, when BER incidences as high as 20% can been seen, especially in rainy seasons and on susceptible cultivars. To ensure early and efficient infection detections before BER symptoms become visible at point-of-care locations, a portable diagnostic tool based on loop-mediated isothermal amplification (LAMP) was developed using the β-tubulin gene. The LAMP assay was optimized and tested for specificity and sensitivity using DNA extracted from pure cultures of N. perennans and seven other fungal species. The results showed that the selected LAMP primer set was specific to N. perennans and highly sensitive as it detected DNA concentrations as low as 0.001 ng/µl after only 10 min. The assay was validated for N. perennans detection on artificially inoculated apples using a portable thermocycler, Genie II, without the need for DNA extraction. The LAMP assay detected N. perennans on apples inoculated with spore suspensions 3 weeks prior to harvest at concentrations of 103 spores/ml or higher. The assay was further validated using commercial Piñata apples from organic and conventional orchards, demonstrating the ability of this technique to amplify N. perennans from asymptomatic fruit in a commercial setting 3 months before commercial maturity. The LAMP assay developed for N. perennans detection can be easily expanded to detect the other BER causal species. LAMP has potential to be used in orchards and at point-of-care facilities to better inform on BER management at different fruit growth stages, and it has potential to be utilized to better understand the epidemiology of Neofabraea spp.

Development and Evaluation of a Loop-mediated Isothermal Amplification Assay for the Rapid Detection and Identification of Pectobacterium carotovorum on Celery in the Field

Pectobacterium carotovorum is the causal agent of bacterial soft rot in a wide range of vegetable host species. Once P. carotovorum infects the plant, the spread of the disease is difficult to control. In this study, a rapid and sensitive method based on loop-mediated isothermal amplification (LAMP) was developed for detecting P. carotovorum in celery with soft rot using a primer set designed from the pmrA conserved sequence of P. carotovorum. The specificity of the LAMP primer set for P. carotovorum was extensively validated on both P. carotovorum strains and non-target strains. The sensitivity was 1 pg of P. carotovorum genomic DNA, which demonstrated 10 times more sensitive than the conventional PCR assay. LAMP was also used to detect P. carotovorum in bacterial suspension. The lowest detection concentration was 104 CFU · mL−1. In addition, a LAMP assay, in conjunction with a crude DNA extraction method, was successfully performed on P. carotovorum-infected samples derived from both artificially and naturally infected plants. In summary, the LAMP assay established in this study constitutes a simple, sensitive, and rapid method for the detection of P. carotovorum, and has potential application in the control of celery soft rot disease through early detection.

Rapid and specific detection of mango (Mangifera indica) in processed food using an isothermal nucleic acid amplification assay

Mango (Mangifera indica) is one of the most popular tropical fruits around the world. It is also widely and commonly used in many cuisines and products in the food industry. However, the anaphylactic reaction caused by mango has long been reported as a major problem for consumption in recent years. To prevent allergens in mango, the best way is to avoid mango in the diet. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of mango in food. Four specifically designed LAMP primers targeting the internal transcribed sequence 1 (ITS1) of nuclear ribosomal DNA sequence regions were used to address the LAMP reaction for amplifying mango DNA. The results demonstrated that the detection of mango DNA was specifically validated by the LAMP primer. The sensitivity of LAMP for detecting mango DNA is equivalent to that of the traditional PCR method. The LAMP primer sets showed high specificity for detecting the DNA of mango and had no cross-reactions to other species. Moreover, when mango was mixed with other fruits at different ratios, no cross-reactivity for the detection of mango DNA was manifested during LAMP. Finally, genomic DNAs extracted from different heat-processed mangos were used as templates; the detection of mango DNA by LAMP was not significantly affected and was reproducible. As to this established LAMP herein, mango ingredients can be detected, and commercial foods containing mango can also be identified. This assay will be useful and have potential for the rapid detection of mango DNA in practical food markets.

Development of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Pseudomonas syringae pv. tomato in planta

Tomato bacterial speck, caused by Pseudomonas syringae pv. tomato (Pst), is one of the most devastating bacterial diseases in tomato worldwide. To establish a rapid amplification system for the detection of Pst, a loop-mediated isothermal amplification (LAMP) method, which includes two external primers (F3/B3), two internal primers (FIP/BIP) and one backward loop primer (B-Loop), was designed based on the hrpZ gene. The specificity of the LAMP primer set was widely validated on Pst and non-target strains. The conditions for LAMP detection of Pst were optimized to complete in 60 min at 63 °C. The amplification was confirmed through gel electrophoresis or visual inspection using calcein. In the sensitivity test, the detection limit of the LAMP assay was 1.61 × 10 fg μL−1 for genomic DNA and 1.05 × 103 CFU mL−1 for bacterial suspension without DNA extraction. The novel method was also applied for the detection of Pst in artificially and naturally infected tomato leaf and stem tissue samples, and even the early onset of disease could be detected by the assay. This method can rapidly detect Pst-infected tissues without strain enrichment and complex DNA extraction, and hence, it is suitable for quarantine and field detection.

Visible DNA microarray and loop-mediated isothermal amplification (LAMP) for the identification of Cryptococcus species recovered from culture medium and cerebrospinal fluid of patients with meningitis.

Cryptococcal meningitis affects normal hosts and immunocompromised patients exhibiting high mortality rates. The objective of this study was to design two molecular assays, visible microarray platforms and loop-mediated isothermal amplification (LAMP), to identify Cryptococcus spp. and the species neoformans and gattii from the cerebral spinal fluid (CSF). To identify Cryptococcus and the two species, we designed two microarrays DNA platforms based on the internal transcribed spacer (ITS) region and CAP59 gene and LAMP assays specific for Cryptococcus species. The assays were tested using CSF from patients with cryptococcal meningitis. CSF from patients with cryptococcal meningitis was cultured in Sabouraud culture medium, and the Cryptococcus spp. grown in the culture medium were also tested for LAMP and microarray platforms. The results were compared to DNA sequencing of the same genetic regions. A total of 133 CSF samples were studied. Eleven CSFs were positive for Cryptococcus (9 C. neoformans and 2 C. gattii), 15 were positive for bacteria, and 107 were negative. The CAP59 platform correctly identified 73% of the CSF samples, while the ITS platform identified 45.5%. CAP59 platform correctly identified 100% of the Cryptococcus isolates, and ITS platform identified 70%. The two sets of LAMP primers correctly identified 100% of the Cryptococcus isolates. However, for CSF samples, the amplification occurred only in 55.5% of C. neoformans. The methodologies were reliable in the identification of Cryptococcus species, mainly for isolates from culture medium, and they might be applied as adjunctive tests to identify Cryptococcus species.

GLAPD: Whole Genome Based LAMP Primer Design for a Set of Target Genomes.

Loop-mediated isothermal amplification (LAMP) technology has been applied in a wide range of fields such as detection of foodborne bacteria and clinical pathogens due to its simplicity and efficiency. However, existing LAMP primer designing systems require a conserved gene or a short genome region as input, and they can’t design group-specific primers. With the growing number of whole genomes available, it is possible to design better primers to target a set of genomes with high specificity based on whole genomes. We present here a whole Genome based LAMP primer designer (GLAPD), a new system to design LAMP primer for a set of target genomes using whole genomes. Candidate single primer regions are identified genome wide and then combined into LAMP primer sets. For a given set of target genomes, only primer sets amplifying them and only these genomes will be output. In order to accelerate the primer designing, a GPU version is provided as well. The effectiveness of primers designed by GLAPD has been assessed for a wide range of foodborne bacteria. GLAPD can be accessed at http://cgm.sjtu.edu.cn/GLAPD/ or https://github.com/jiqingxiaoxi/GLAPD.git. A simple online version is also supplied to help users to learn and test GLAPD: http://cgm.sjtu.edu.cn/GLAPD/online/.

Application of loop-mediated isothermal amplification combined with lateral flow dipstick to rapid and sensitive detection of Alexandrium catenella.

Alexandrium catenella is one of the globally distributed toxic marine microalgae to cause paralytic shellfish poisoning that poses a great threat to marine fisheries, economy, and public health. Development of efficient and sensitive methods for accurate identification of A. catenella to minimize its damage is therefore necessary. In this study, a novel method referred to as loop-mediated isothermal amplification (LAMP) combined with lateral flow dipstick (LFD) (LAMP-LFD) was established for rapid and sensitive detection of A. catenella. The internal transcribed spacer (ITS) gene of A. catenella was cloned for sequencing and used as target for LAMP-LFD. Three sets of LAMP primers (AcLF1, AcLF2, and AcLF3) targeting the ITS were successfully designed, among which AcLF2 displaying the best performance was used in the subsequent tests. A specific LFD probe targeting the amplification region of AcLF2 was further designed. The LAMP-LFD detection system was established and the amplification conditions were optimized. Cross-reactivity tests with common marine microalgae showed that the LAMP-LFD was exclusively specific for A. catenella. The detection limits of LAMP-LFD for A. catenella genomic DNA and the plasmid containing the ITS were 4.63 × 10-4 ng μL-1 and 1.26 × 104 copies μL-1, displaying a sensitivity that is 10 times higher than that of SYBR Green I assay and 100 times higher than that of conventional PCR, respectively. Finally, the practicability of LAMP-LFD was confirmed by test with spiked samples. LAMP-LFD revealed a detection limit of approximately 0.1 cell mL-1, which was 100 times more sensitive than conventional PCR. The optimized LAMP-LFD protocol can be completed within 75 min. Therefore, the established LAMP-LFD is a specific, sensitive, and rapid method that is possibly applicable to the field monitoring of A. catenella.

Evaluation of combined effect of micronutrients and rhizobial inoculation on mungbean productivity

In present study, Loop Mediated Isothermal Amplification (LAMP) assay was conducted for diagnosis Nosema bombycis, the pebrine disease pathogen in domestic silkworm, Bombyx mori. Nine isolates of N. bombycis were collected from infected silkworms in rearing areas in Khon Kaen province, Thailand. N. bombycis genomic DNAs were extracted by boiling method and used as templates in LAMP and PCR reactions. A LAMP primer set was designed specific to N. bombycis small subunit ribosomal RNA (SSU rRNA) gene. The results revealed that the optimal condition was constantly performed at 63oC for 1 hour. The product was directly visualized by naked eye and confirmed with agarose gel eletrophoresis. LAMP assay is more sensitive than traditional PCR, since LAMP was able to detect the least 10 spores/ml while PCR needs 100 spores/ml. In addition, the present novel LAMP primer set was specific only to N. bombycis proven by the negative results when other B. mori pathogen DNAs were tested. In conclusion, the LAMP assay demonstrated a great potential alternative method in diagnosis N. bombycis with high sensitivity, rapidity and accuracy which can apply for pebrine disease surveillance.

Simple and Sensitive Quantification of Toxigenic Vibrio cholerae DNA by Real-Time Loop-Mediated Isothermal Amplification Based on ctxB Gene

Background: Loop-mediated isothermal amplification (LAMP) is a cost-effective, rapid, and specific method for the detection of bacterial pathogens within a sample. Methods: We designed and evaluated a new quantitative LAMP primer set specific to the ctxB gene for the rapid measurement of the load of toxigenic V. cholerae DNA in the reaction. Unlike the previously reported LAMP assays for V. cholerae detection, our LAMP primer set was able to detect and quantify toxigenic V. cholerae DNA with a detection limit of 2.8 pg of the ctxB gene equivalent to 8.3 copies of genomic DNA per reaction. Results: Our LAMP assay was found to be highly specific with no amplification detected in non-toxigenic V. cholerae bacterial strains. Thus, the ctxB-based LAMP assay developed in this study can serve as a simple, sensitive, specific, and quantitative molecular tool for the monitoring and epidemiological study of cholera. Conclusions: Overall, the low cost and simplicity of the LAMP assay can make it a preferable molecular method in the detection of pathogenic organisms.

Development of a loop-mediated isothermal amplification assay for rapid Helicobacter pylori detection

Infection with cagA-positive Helicobacter pylori is associated with gastric cancer. Molecular techniques are vital for accurate H. pylori diagnosis. We developed a loop-mediated isothermal amplification (LAMP) for detecting the H. pylori cagA gene and evaluated its use for clinical diagnosis. A LAMP primer set was designed to recognize the homologous regions of cagA gene sequences of 6 H. pylori strains. LAMP sensitivity was evaluated with serial dilutions of H. pylori ATCC 43504 and fecal specimens; specificity was evaluated with H. pylori ATCC 49396 and CIP 104086. The LAMP sensitivity for H. pylori specimens was 10−1 cfu/tube (reaction time, 37 min), which was 10-fold more sensitive than polymerase chain reaction. LAMP was also highly sensitive and rapid for fecal specimens. It detected cagA gene from ATCC 49396 and CIP 104086. The findings suggest LAMP can be used for diagnosing and screening of H. pylori infections to decrease gastric cancer incidence.

Monitoring Methyl Benzimidazole Carbamate-Resistant Isolates of the Cucurbit Powdery Mildew Pathogen, Podosphaera xanthii, Using Loop-Mediated Isothermal Amplification.

Powdery mildew, caused by the fungus Podosphaera xanthii, is one of the most economically important diseases affecting cucurbit crops in Spain. Currently, chemical control offers the most efficient management of the disease; however, P. xanthii isolates resistant to multiple classes of site-specific fungicides have been reported in the Spanish cucurbit powdery mildew population. In previous studies, resistance to the fungicides known as methyl benzimidazole carbamates (MBCs) was found to be caused by the amino acid substitution E198A on β-tubulin. To detect MBC-resistant isolates in a faster, more efficient, and more specific way than the traditional methods used to date, a loop-mediated isothermal amplification (LAMP) system was developed. In this study, three sets of LAMP primers were designed. One set was designed for the detection of the wild-type allele and two sets were designed for the E198A amino acid change. Positive results were only obtained with both mutant sets; however, LAMP reaction conditions were only optimized with primer set 2, which was selected for optimal detection of the E198A amino acid change in P. xanthii-resistant isolates, along with the optimal temperature and duration parameters of 65°C for 75 min, respectively. The hydroxynaphthol blue (HNB) metal indicator was used for quick visualization of results through the color change from violet to sky blue when the amplification was positive. HNB was added before the amplification to avoid opening the lids, thus decreasing the probability of contamination. To confirm that the amplified product corresponded to the β-tubulin gene, the LAMP product was digested with the enzyme LweI and sequenced. Our results show that the LAMP technique is a specific and reproducible method that could be used for monitoring MBC resistance of P. xanthii directly in the field.

Development of Rapid and Specific Detection for the Human Aichivirus A Using the Loop-Mediated Isothermal Amplification from Water Samples.

Human Aichivirus A (AiV-A) is classified as a Kobuvirus, group IV positive sense single strand RNA viruses. The first outbreak of AiV-A was reported from Aichi Prefecture, Japan in 1989. AiV-A exists not only among clinical patients, such as diarrhea, but also in a variety of water environments, as its occurrence is reported across a wide geographical range, from developing to advanced countries. For diagnose of AiV-A from water samples, mostly polymerase chain reaction (PCR) system have been developed. However, loop-mediated isothermal amplification (LAMP) assay has not been applied. In this study, developed a LAMP method to achieve a rapid, specific and highly sensitive detection of AiV-A. The method developed in this study is aimed specifically at AiV-A. Through a specific and non-specific selection and sensitivity test process for the five prepared LAMP primer sets, one primer set and optimum reaction temperature were selected. A newly developed method was more rapid (approximately 2–8 h), specific and equivalent detection of AiV-A than with the conventional PCRs. In addition, confirm system of positive LAMP reaction was developed by using the restriction enzyme Aci I and Hae III. For evaluation and verification of developing LAMP assay, a was applied to twenty cDNA from groundwater samples. This study proved rapid and specific diagnosis of AiV-A from water samples, and it is also demanded to be applicable to other environmental, clinical and food samples.Electronic supplementary materialThe online version of this article (10.1007/s12088-019-00803-3) contains supplementary material, which is available to authorized users.

Rapid detection of benzimidazole resistance in Botrytis cinerea by loop-mediated isothermal amplification

Benzimidazole fungicides (MBCs) have been widely used in agriculture since 1970s, and resistance to this class of fungicides in Botrytis cinerea is reported worldwide. Resistance to MBCs in B. cinerea is related to mutations in the target β-tubulin gene (TUB2). Compared with the mutation at codon 200, the substitutions from glutamic acid to alanine (E198A), valine (E198V), or lysine (E198K) at codon 198 are currently the predominant mutations in MBC resistant populations of B. cinerea. In this study, a loop-mediated isothermal amplification (LAMP) method was established for rapid detection of benzimidazole resistance in B. cinerea. On the basis of the three mutations at TUB2 codon 198, three sets of LAMP primers were designed, and each of these primer sets was able to specifically amplify the DNA containing its corresponding mutation, while no amplification was detected with other mutated or the wild type DNA. After optimization, the sensitivity and specificity tests illustrated that this LAMP assay had good sensitivity and specificity to detect specific resistant genotypes in B. cinerea. Result also showed that the LAMP assay had good reproducibility. Above all, boiled mycelia could be used as templates, which simplified the process and increased the efficiency of the assay. Considering its rapidity, simplicity, and high efficiency, the LAMP assay developed in this study is a promising tool for the diagnosis of benzimidazole resistance in B. cinerea, and will contribute to the monitoring of resistance development to MBCs in the future.

Species-specific loop-mediated isothermal amplification (LAMP) assay for identification of tissue of cattle origin by targeting mitochondrial gene sequences.

The present study was carried out with the objective of development of species-specific loop-mediated isothermal amplification (LAMP) assay for identification of tissue of cattle origin. The cattle-specific LAMP primer set was designed by targeting mitochondrial D-loop gene. The conditions for LAMP reaction for amplification of template DNA from cattle using designed cattle-specific primer set were optimized for the components of mixture and temperature of reaction. Amplified products were analysed using SYBR Green I dye and by agarose gel electrophoresis. The developed species-specific LAMP assay was evaluated for its specificity, sensitivity and validated in laboratory on samples from known, coded, binary meat admixture with other than cattle at relative percentage of 20%, 10%, 5% and 1%, Phire tissue direct PCR master mix treated tissues of cattle and on species-specific polymerase chain reaction assay positive samples. The developed LAMP assay using self-designed primer set was highly specific, amplifying the DNA template exclusively from cattle tissue under the optimized LAMP reaction conditions. The sensitivity assay using serially diluted DNA templates revealed lowest level of detection as 0.01ng of absolute DNA from target species. Laboratory validation substantiated the accuracy of assay in known/unknown (coded) samples and up to the 1% level of admixture in binary meat sample. DNA present in supernatant of Phire Animal tissue kit treated samples were also amplified successfully eliminating the extra step of extraction of genomic DNA. The developed assays exhibited comparable results with previously established species-specific PCR assay taken as gold standards. Thus, it was concluded that developed species-specific loop-mediated isothermal amplification assay was effective in identification of tissue of cattle origin.