Loop-mediated Isothermal Amplification Primers Research Articles

Rapid detection of a novel B1-\u03b2-lactamase gene, blaAFM-1 using a loop-mediated isothermal amplification (LAMP) assay

BackgroundBlaAFM-1 (GenBank Accession No. 143105.1) is a new B1 subclass metallo-β-lactamase gene discovered by our group, and isolated from an Alcaligenes faecalis plasmid that renders carbapenem antibiotics ineffective. In this study, we generated a fast and reliable assay for blaAFM-1 detection.MethodsWe designed optimum loop-mediated isothermal amplification (LAMP) primers and constructed a recombinant plasmid AFM-1 to specifically detect blaAFM-1. Optimal LAMP primers were used to assess sensitivity of the recombinant plasmid AFM-1 and blaAFM-1-supplemented samples (simulated sputum and simulated feces). Fifty two samples, without blaAFM-1, were used to assess LAMP real-time assay specificity; these samples were verified by conventional PCR and sequencing for the absence of blaAFM-1. Three hundred clinical Gram-negative carbapenem-resistant strains were tested by LAMP assay for strains carrying blaAFM-1, which were confirmed by conventional PCR and Sanger sequencing. We calculated the sensitivity and its 95% confidence interval (95% CI), specificity and its 95% CI, and predictive values of the LAMP assay and conventional PCR/sequencing by investigating positive and negative clinical strains.ResultsThe lowest limit of detection for the recombinant plasmid AFM-1 and blaAFM-1-supplemented samples (in both simulated sputum and simulated feces) was 101 copies/reaction. All amplification curves of the 52 blaAFM-1-free bacteria strains were negative, suggesting the LAMP assay had excellent specificity for detecting blaAFM-1. Among the 300 clinical strains, eight were positive for blaAFM-1 using LAMP. These LAMP results were consistent with conventional PCR and Sanger sequencing data. As with conventional PCR/sequencing, the LAMP method exhibits 100% sensitivity (95% CI 59.8–100%) and 100% specificity (95% CI 98.4–100%) for blaAFM-1 detection. The LAMP assay is also time-efficient (1 h) for blaAFM-1 detection.ConclusionsWe established a new LAMP assay with high sensitivity and specificity to detect the novel B1-β-lactamase gene, blaAFM-1.

Probing Loop-Mediated Isothermal Amplification (LAMP) targeting two gene-fragments of rose rosette virus.

This study explores the development of Loop-mediated isothermal amplification (LAMP) for detection of rose rosette virus (RRV), a technique with the potential to be translated to rose nurseries. RRV is a negative-sense, single-stranded RNA virus which is a member of the genus Emaravirus (Family Fimoviridae) and the causal agent of the rose rosette disease (RRD). Although RRV symptoms are characteristics, early visual diagnosis of RRD can be misleading and confusing since it may appear like herbicide damage. Moreover, it may take incubation time for symptoms to appear after virus infection. Two sets of RRV gene sequences RNA3 and RNA4 were analyzed and two sets of four LAMP primers were designed. The direct antigen-capture method for direct trapping of RRV in plastic was used for RNA extraction followed by cDNA synthesis. RT-LAMP reactions were for 1 hour at 64°C (RRV-P3) and 66.5°C (RRV-P4) using either a thermocycler or a portable dry bath. RT-qLAMP was also optimized using DNA polymerase GspSSD LD using the same RRV sets of primers. RRV was detected in symptomatic and non-symptomatic RRD tissue from Oklahoma. The limit of detection (LoD) was 1pg/μL and 1 fg/μL using Bst 2.0 LAMP and GspSSD LD quantitative LAMP, respectively. In visual colorimetric pre- and post-reactions, the LoD was 10 pg/μL and 0.1 pg/μL using hydroxy naphthol blue (HNB, 120 μM) and SYBR green I (1:10 dilution), respectively. No cross-reactivity was detected in the RT-LAMP reaction testing cDNAs of eight commonly co-infecting rose viruses and one virus taxonomically related to RRV. Four different dyes were tested, and visible colorimetric reactions were obtained with RT-LAMP Bst 2.0 combined with SYBR I or HNB. RT-qLAMP with GspSSD2.0 offers LoD equal to RT-PCR and it is faster since it works with RNA directly.

A Label-Based Polymer Nanoparticles Biosensor Combined with Loop-Mediated Isothermal Amplification for Rapid, Sensitive, and Highly Specific Identification of Brucella abortus.

Brucella abortus (B. abortus), an important zoonotic pathogen in Brucella spp., is the major causative agent of abortion in cattle (namely, bovine brucellosis). Currently, although the isolation and identification of the Brucella abortus were commonly accepted as the gold standard method, it cannot meet the requirements for early diagnostic strategies. Conventional PCR techniques and immunological tests can realize rapid detection of B. abortus, but the demands for PCR thermal cyclers and/or specific antibodies hinder their application in basic laboratories. Thus, rapid, sensitive, and specific diagnostic strategies are essential to prevent and control the spread of the bovine brucellosis. In this work, a novel detection method for the rapid identification of B. abortus, which uses loop-mediated isothermal amplification (LAMP) combined with a label-based polymer nanoparticles lateral flow immunoassay biosensor (LFIA), was established. One set of specific B. abortus-LAMP primers targeting the BruAb2_0168 gene was designed by the online LAMP primer design tool. The B. abortus-LAMP-LFIA assay was optimized and evaluated using various pathogens and whole blood samples. The optimal amplification temperature and time for B. abortus-LAMP-LFIA were determined to be 65°C and 50 min, respectively. The B. abortus-LAMP-LFIA method limit of detection (LoD) was 100 fg per reaction for pure genomic DNA of B. abortus. Meanwhile, the detection specificity was 100%, and there was no cross-reactivity for other Brucella members and non-Brucella strains. Furthermore, the entire procedure, including the DNA preparation for whole blood samples (30 min), isothermal incubation (50 min), and LFIA detection (2–5 min), can be completed in approximately 85 min. Thus, the B. abortus-LAMP-LFIA assay developed was a simple, rapid, sensitive, and reliable detection technique, which can be used as a screening and/or diagnostic tool for B. abortus in the field and basic laboratories.

Genome-informed loop-mediated isothermal amplification assay for specific detection of Pectobacterium parmentieri in infected potato tissues and soil

Pectobacterium parmentieri (formerly Pectobacterium wasabiae), which causes soft rot disease in potatoes, is a newly established species of pectinolytic bacteria within the family Pectobacteriaceae. Despite serious damage caused to the potato industry worldwide, no field-deployable diagnostic tests are available to detect the pathogen in plant samples. In this study, we aimed to develop a reliable, rapid, field-deployable loop-mediated isothermal amplification (LAMP) assay for the specific detection of P. parmentieri. Specific LAMP primers targeting the petF1 gene region, found in P. parmentieri but no other Pectobacterium spp., were designed and validated in silico and in vitro using extensive inclusivity (15 strains of P. parmentieri) and exclusivity (94 strains including all other species in the genus Pectobacterium and host DNA) panels. No false positives or negatives were detected when the assay was tested directly with bacterial colonies, and with infected plant and soil samples. Sensitivity (analytical) assays using serially diluted bacterial cell lysate and purified genomic DNA established the detection limit at 10 CFU/mL and 100 fg (18–20 genome copies), respectively, even in the presence of host crude DNA. Consistent results obtained by multiple users/operators and field tests suggest the assay’s applicability to routine diagnostics, seed certification programs, biosecurity, and epidemiological studies.

LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) ASSAY FOR DETECTION OF ASIAN FISH TAPEWORM, SCHYZOCOTYLE ACHEILOGNATHI (YAMAGUTI, 1934) [SYN. BOTHRIOCEPHALUS ACHEILOGNATHI

The Asian fish tapeworm (Schyzocotyle acheilognathi syn. Bothriocephalus acheilognathi) (AFT) is an invasive parasite that can infect many species of fish, although most hosts are primarily members of Cyprinidae. Pathogenicity has most often been reported in aquaculture settings in fry and fingerling stages of carp (Cyprinus spp.). More recently, it has been shown to cause growth retardation in the endangered bonytail chub (Gila elegans) and found to be widespread in populations of endangered humpback chub (Gila cypha) in the Colorado River, Grand Canyon, Arizona. AFT spreads most often through the transport of infected fish, particularly baitfish. Despite its harmful potential, there is no efficient or accurate ante mortem test to detect AFT in water or fish samples before transport. Herein, we report on the development of a sensitive and specific loop-mediated isothermal amplification (LAMP) assay to detect the parasite in under 30 min from laboratory prepared samples. Six LAMP primers were designed to amplify a variable region of the 18S ribosomal RNA gene in AFT with the detection and quantification of DNA on a real-time fluorometer. The limit of detection was 1 × 101 copies/μl of DNA extracted from as few as 2 AFT eggs. Future application of our assay would be a low-cost test to rapidly and accurately detect AFT DNA from environmental samples on-site so that preventive actions can be taken to halt the spread of the AFT through the movement of infected fish.

Rapid Detection of Airborne Inocula of Grapevine Mildews Using PCR and LAMP Assay

Grapes powdery mildew and downy mildew caused by Erysiphe necator and Plasmopara viticola respectively are the most devastating diseases worldwide resulting in significant loss of yield and quality. Epidemics of grapevine mildews are caused by airborne inocula such as conidia and sporangia. Rapid detection of airborne inocula will help to face up timely management strategies under field conditions. The aim of the current study was to design a suction spore trap to trap the airborne mildew inocula and their early detection by molecular methods of PCR and LAMP assay. A total of twelve airborne inocula samples were collected the weekly intervals from 3 to 14 standard weeks of 2021 during the cropping season. The presence of airborne inocula of E. necator was detected on standard weeks 3,6,10 and 13 through PCR assay which yielded an amplicon of 470 bp. Similarly, airborne inocula of P.viticola were detected on standard week 6 only through PCR which yielded an amplicon of 520 bp. A rapid, highly specific , sensitive Loop mediated isothermal amplification (LAMP) assay was performed to detect the E. necator and P. viticola using six sets of LAMP primers constructed by targeting rDNA region of ITS and the 5S rRNA and CesA4 a gene, respectively. LAMP assay efficiently detected the presence of airborne inocula of E.necator in most of the samples collected from standard week 3 – 14 except 7, 8, and 9. However, the presence of airborne inocula of P.viticola from standard week 3 – 14 was confirmed by LAMP assay. The LAMP assay is absolutely the best in identifying airborne inocula of grapevine mildews compared to PCR and phenotypic microscopic observation.

Development of LAMP Assays Using a Novel Target Gene for Specific Detection of Pythium terrestris, Pythium spinosum, and 'Candidatus Pythium huanghuaiense'.

Pythium terrestris, Pythium spinosum, and 'Candidatus Pythium huanghuaiense' are closely related species and important pathogens of soybean that cause root rot. However, the sequences of commonly used molecular markers, such as rDNA internal transcribed spacer 2 and cytochrome oxidase 1 gene, are similar among these species, making it difficult to design species-specific primers for loop-mediated isothermal amplification (LAMP) assays. The genome sequences of these species are also currently unavailable. Based on a comparative genomic analysis and de novo RNA-sequencing transcript assemblies, we identified and cloned the sequences of the M90 gene, a conserved but highly polymorphic single-copy gene encoding a Puf family RNA-binding protein among oomycetes. After primer design and screening, three LAMP assays were developed that specifically amplified the targeted DNA sequences in P. terrestris and P. spinosum at 62°C for 70 min and in 'Ca. Pythium huanghuaiense' at 62°C for 60 min. After adding SYBR Green I, a positive yellow-green color (under natural light) or intense green fluorescence (under ultraviolet light) was observed by the naked eye only in the presence of the target species. The minimum concentration of target DNA detected in all three LAMP assays was 100 pg·μl-1. The assays also successfully detected the target Pythium spp. with high accuracy and sensitivity from inoculated soybean seedlings and soils collected from soybean fields. This study provides a method for identification and cloning of candidate detection targets without a reference genome sequence and identified M90 as a novel specific target for molecular detection of three Pythium species causing soybean root rot.

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for quick identification of the Japanese salamander Hynobius tokyoensis.

Species identification using molecular techniques has recently become common for various taxa. Loop-mediated isothermal amplification (LAMP) is one of the easiest and least expensive molecular identification methods. Although few studies have developed LAMP assays for amphibians, we believe that LAMP is also useful for identifying endangered amphibians. Hynobius tokyoensis and H. lichenatus occur in Honshu, Japan, and have parapatric distributions. They are similar morphologically, especially at early developmental stages, including eggs and larvae. Hynobius tokyoensis has been listed as a national endangered species in Japan since 2020, and unambiguous identification of these species is therefore important for their conservation and management. In this study, we developed a LAMP primer set for the mitochondrial cytochrome b region to detect H. tokyoensis, and we evaluated the LAMP assay using total genomic DNA from four H. tokyoensis and three H. lichenatus individuals from across most of their ranges. Our LAMP primer set could distinguish these two species. This study should help to establish LAMP assays for other endangered species and morphologically similar species.

LAMP Detection and Identification of the Blackleg Pathogen Leptosphaeria biglobosa 'brassicae'.

Blackleg of oilseed rape is a damaging invasive disease caused by the species complex Leptosphaeria maculans (Lm)/L. biglobosa (Lb), which is composed of at least two and seven phylogenetic subclades, respectively. Generally, Lm is more virulent than Lb, but under certain conditions, Lb can cause a significant yield loss in oilseed rape. Lb 'brassicae' (Lbb) has been found to be the causal agent for blackleg of oilseed rape in China, whereas Lm and Lb 'canadensis' (Lbc) were frequently detected in imported seeds of oilseed rape, posing a risk of spread into China. To monitor the blackleg-pathogen populations, a diagnostic tool based on loop-mediated isothermal amplification (LAMP) was developed using a 615-bp-long DNA sequence from Lbb that was derived from a randomly amplified polymorphic DNA assay. The LAMP was optimized for temperature and time, and tested for specificity and sensitivity using the DNA extracted from Lbb, Lbc, Lm, and 10 other fungi. The results showed that the optimal temperature and time were 65°C and 40 min, respectively. The LAMP primer set was specific to Lbb and highly sensitive as it detected the Lbb DNA as low as 132 fg per reaction. The LAMP assay was validated using the DNA extracted from mycelia and conidia of a well-characterized Lbb isolate, and its utility was evaluated using the DNA extracted from leaves, stems, pods, and seeds of oilseed rape. The LAMP assay developed herein will help for monitoring populations of the blackleg pathogens in China and in developing strategies for management of the blackleg disease.

Rapid and sensitive detection of Salmonella species targeting the hilA gene using a loop-mediated isothermal amplification assay.

Salmonella species are among the major pathogens that cause foodborne illness outbreaks. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of Salmonella species. We designed LAMP primers targeting the hilA gene as a universal marker of Salmonella species. A total of seven Salmonella species strains and 11 non-Salmonella pathogen strains from eight different genera were used in this study. All Salmonella strains showed positive amplification signals with the Salmonella LAMP assay; however, there was no non-specific amplification signal for the non-Salmonella strains. The detection limit was 100 femtograms (20 copies per reaction), which was ~1,000 times more sensitive than the detection limits of the conventional polymerase chain reaction (PCR) assay (100 pg). The reaction time for a positive amplification signal was less than 20 minutes, which was less than one-third the time taken while using conventional PCR. In conclusion, our Salmonella LAMP assay accurately detected Salmonella species with a higher degree of sensitivity and greater rapidity than the conventional PCR assay, and it may be suitable for point-of-care testing in the field.

Development of a LAMP-Based Molecular Species Diagnosis Method for Four Major Agricultural Pests in the Genus Spodoptera (Lepidoptera: Noctuidae).

Simple SummaryFour major Spodoptera pests, S. exigua, S. frugiperda, S. litura, and S. littoralis, are widely distributed polyphagous pests affecting various crops. Despite different distribution areas, these four species cause serious damage to agriculture worldwide. As these species are morphologically similar at the larval stage, diagnostic methods have been developed and utilized for their identification. Here, we developed a loop-mediated isothermal amplification (LAMP) assay for rapid and effective species diagnosis, along with PCR, to identify Korean field-collected or overseas samples. The optimal conditions for the LAMP assay were 61 °C for 60 min with four LAMP primers. Additional loop primers increased the amplification efficiency in S. exigua, whereas increased non-specific amplification was found in other species. A broad range of DNA concentrations was observed in the LAMP assay, and the minimum detectable DNA concentration was 1 pg. The DNA release method for LAMP involved incubation of larval or adult tissue samples for 5 min at 95 °C, without a DNA extraction step. Considering the gradual diversification invasive pest incidence, this simple and accurate LAMP assay can be used for intensive field monitoring of invasive pests and integrated management of these species.Molecular-based species identification tools are helpful to identify tiny insect and lepidopteran pests that show morphological similarities in the larval stage and are essential for quarantine as well as agricultural research. Here, we focused on four major Spodoptera pests: S. exigua, S. frugiperda, S. litura, and S. littoralis. S. exigua and S. litura mitochondrial genome sequences were newly identified and species-specific sequence regions were identified in the cytochrome c oxidase subunit II and III regions. Species primers were designed and applied in loop-mediated isothermal amplification (LAMP) and PCR to identify Korean field-collected or overseas samples. The optimal incubation conditions for LAMP were 61 °C for 60 min with four LAMP primers. Additional loop primers increased the amplification efficiency for S. exigua, and the nonspecific amplification for other species. The LAMP assay could detect a wide range of DNA concentrations, with the range 1 ng–1 pg in dependence of four LAMP primers. The DNA-releasing technique, without DNA extraction, in the LAMP assay involved larval or adult tissue sample incubation at 95 °C for 5 min. The entire process takes approximately 70 min. This new molecular diagnostic method is simple and accurate, with application in the field and laboratory and for monitoring and ecological studies.

A Rapid colorimetric assay for field-portable authentication of Amanita sepiacea using the beta-tubulin gene sequence

Globally, mushroom poisoning is becoming one of the most serious food safety-related problems. To investigate the source of mushroom poisoning, a rapid method for identifying toxic mushrooms is urgently required. This study aimed to establish a colorimetric method based on loop-mediated isothermal amplification (LAMP) for rapid detection of Amanita sepiacea based on the analysis of the beta-tubulin gene. Specific LAMP primers for species specificity identification were designed. The primers designed by the colorimetric method did not cross-react with 41 non-target species mushrooms, and 1 ng of A. sepiacea DNA was detected. The detection method established herein can be used to identify A. sepiacea when present at concentrations as low as 1% in mixed fresh, cooked, or fried mushroom samples. The whole detection process only takes 30 min and requires heating and no complex instrumental help. Therefore, this method can be used in the field detection of poisonous mushrooms.

G-Quadruplex-Probing CRISPR-Cas12 Assay for Label-Free Analysis of Foodborne Pathogens and Their Colonization In Vivo.

Foodborne pathogen infection is a key issue of food safety. Herein, we developed a label-free assay for Salmonella enterica (S. enterica) detection based on the G-quadruplex-probing CRISPR-Cas12 system (termed G-CRISPR-Cas), allowing highly sensitive detection of S. enterica and investigation of their colonization in chickens. The introduction of the G-quadruplex probe serving as the substrate of Cas 12a realized a label-free analysis for foodborne pathogens. Due to the amplification process induced by loop-mediated isothermal amplification (LAMP), G-CRISPR-Cas assay can detect S. enterica as low as 20 CFU. Specificity for pathogenic gene detection was guaranteed by the dual recognition process via LAMP primers and Cas 12a-guided RNA binding. The G-CRISPR-Cas assay was applied to explore S. enterica colonization in the intestinal tract and organs of chickens and showed the risk of S. enterica infection outside of the intestinal tract. The G-CRISPR-Cas assay is promising for on-site diagnosis of the infection or contamination of foodborne pathogens outside the laboratories, such as abattoirs and markets.

WarmStart colorimetric loop-mediated isothermal amplification for the one-tube, contamination-free and visualization detection of Shigella flexneri

ObjectivesShigella flexneri (S. flexneri) is prevalent worldwide and the most common Shigella in many countries, causing highly contagious diarrhea, which seriously threatens public health. This study aimed to develop a colorimetric loop-mediated isothermal amplification (LAMP) for the rapid, accurate, and visualization detection of S. flexneri. MethodsAccording to the screened specific genes of S. flexneri, three groups of LAMP primers were designed and evaluated, and the colorimetric LAMP reaction volume was optimized. The specificity of the colorimetric LAMP was validated by 20 S. flexneri and 96 non-S. flexneri clinical isolates. In addition, the sensitivity of the developed assay was evaluated by the serial 10-fold dilutions of plasmid DNA. ResultsA colorimetric LAMP assay was developed based on the specific S. flexneri hypothetical protein gene (Accession: AE014073 Region: 4170556.4171068). The colorimetric LAMP method had good specificity for detecting S. flexneri and enabled detection of S. flexneri within 30 minutes, with a plasmid detection limit of 7*10° copies/μL. The results of amplification could be easily identified by color. ConclusionsThis colorimetric LAMP assay could be used for rapid and accurate diagnosis of S. flexneri infection, especially in remote hospitals and laboratories with under-equipped medical facilities, and in situations where an urgent diagnosis is needed.

Development and validation of LAMP primer sets for rapid identification of Aspergillus fumigatus carrying the cyp51A TR46 azole resistance gene

Infections due to triazole-resistant Aspergillus fumigatus are increasingly reported worldwide and are associated with treatment failure and mortality. The principal class of azole-resistant isolates is characterized by tandem repeats of 34 bp or 46 bp within the promoter region of the cyp51A gene. Loop-mediated isothermal amplification (LAMP) is a widely used nucleic acid amplification system that is fast and specific. Here we describe a LAMP assay method to detect the 46 bp tandem repeat insertion in the cyp51A gene promoter region based on novel LAMP primer sets. It also differentiated strains with TR46 tandem repeats from those with TR34 tandem repeats. These results showed this TR46-LAMP method is specific, rapid, and provides crucial insights to develop novel antifungal therapeutic strategies against severe fungal infections due to A. fumigatus with TR46 tandem repeats.

Detection of the Benthic Dinoflagellates, Ostreopsis cf. ovata and Amphidinium massartii (Dinophyceae), Using Loop-Mediated Isothermal Amplification

For the in situ and sensitive detection of benthic dinoflagellates, we have established an integrated loop-mediated isothermal amplification (LAMP) assay based on Ostreopsis cf. ovata and Amphidinium massartii. To detect the two species, a set of species-specific primers was constructed between the ITS gene and D1–D6 LSU gene, and the reaction temperature, time, and buffer composition were optimized to establish this method. In addition, the specificity of the LAMP primers was verified both in strains established in the laboratory and in field samples collected from the Jeju coastal waters, Korea. With the LAMP assay, the analysing time was within 45 to 60 min, which may be shorter than that with the conventional PCR. The detection sensitivity of the LAMP assay for O. cf. ovata or A. massartii was comparable to other molecular assays (PCR and quantitative PCR (qPCR)) and microscopy examination. The detection limit of LAMP was 0.1 cell of O. cf. ovata and 1 cell of A. massartii. The optimized LAMP assay was successfully applied to detect O. cf. ovata and A. massartii in field samples. Thus, this study provides an effective method for detecting target benthic dinoflagellate species, and could be further implemented to monitor phytoplankton in field surveys as an altenative.

A paper-based colorimetric molecular test for SARS-CoV-2 in saliva

Herein, we describe the development of a paper-based device to detect nucleic acids of pathogens of interest in complex samples using loop-mediated isothermal amplification (LAMP) by producing a colorimetric response visible to the human eye. To demonstrate the utility of this device in emerging public health emergencies, we developed and optimized our device to detect SARS-CoV-2 in human saliva without preprocessing. The resulting device was capable of detecting the virus within 60 min and had an analytical sensitivity of 97% and a specificity of 100% with a limit of detection of 200 genomic copies/μL of patient saliva using image analysis. The device consists of a configurable number of reaction zones constructed of Grade 222 chromatography paper separated by 20 mil polystyrene spacers attached to a Melinex® backing via an ARclean® double-sided adhesive. The resulting device is easily configurable to detect multiple targets and has the potential to detect a variety of pathogens simply by changing the LAMP primer sets.

Literature Review: A potential detection method for SARS-CoV-2

acid cleavage. There, Cas13 was found to have significantly higher nucleic acid detection ability at a relatively faster speed. However, Cas13's detection duration was 60 minutes at a femtomolar level, but this is not sufficient for a point-of-care diagnostic. To tackle this, costeffective Loop-mediated isothermal amplification (LAMP) was used to amplify nucleic acid with high sensitivity in the attomolar level in under 20 minutes. Despite the rapid, sensitive nucleic acid detection of LAMP, they have non-specific interaction resulting in non-template control (NTC) signal that needs to be addressed. This NTC signal is due to the dimerization of LAMP primer sets which might lead to high false-positive. To work around this, Cas13 was used as a secondary probe for specific target recognition. For Cas13 to avoid false-positive detections, the guide RNAs must not overlap with the primers. Short and highly overlapping LAMP primer raised amplicons -size ranged from 1-60 nt -to avoid...

Rapid and Sensitive Detection of the Colistin Resistance Gene mcr-3 by Loop-Mediated Isothermal Amplification and Visual Inspection

Objective: The distribution of colistin resistance in mcr-carrying bacteria poses a threat to global public health. In particular, the newly identified mcr-3 allele has spread globally, especially in China, second only to mcr-1. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay for rapid, sensitive, and visual detection of the presence of the mcr-3 gene. Materials and Methods: A total of 13 clinical bacterial strains and 11 negative strains were used in this study. We designed LAMP Primers, optimized reaction conditions, used three different methods to detect LAMP amplification products: (1) agarose gel electrophoresis, (2) LAMP-hydroxy naphthol blue (HNB) detection, (3) LAMP-SYBR Green I (LAMP-SGI) visual inspection, and evaluated its specificity and sensitivity. Results: The amplification reaction was completed in 1 hr at 62°C under isothermal conditions. The final optimized mixtures contained 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton X-100, 1.2 μL HNB, and 0.5 μL SYBR Green I as additives to the initial reaction mixture. LAMP detection, including two visual methods, LAMP-HNB and LAMP-SGI, of mcr-3 possessed the same specificity and a 10-fold higher sensitivity compared with a conventional polymerase chain reaction assay using the same samples. Conclusion: We successfully established an mcr-3 LAMP detection with portability and rapidity of the reaction by the easily distinguishable color changes in the reaction tubes. This visual LAMP assay for mcr-3 detection was simple, time saving, and economical, especially suited to field laboratories.

Development of a rapid loop-mediated isothermal amplification (LAMP) assay for visual detection of porcine parvovirus (PPV) and its application.

Porcine parvovirus (PPV) infection is one of the most important causes of reproductive failure in pigs impacting the piggery industry globally with huge economic losses. A cost-effective, simple, rapid, specific, and sensitive method is critical for monitoring PPV infection on pig farms. The main aim of the present study was to develop and evaluate a loop-mediated isothermal amplification (LAMP) assay for rapid visual detection of porcine parvovirus (PPV) in pigs. A set of six LAMP primers including two outer primers, two inner primers, and two loop primers were designed utilizing the conserved region of capsid protein VP2 gene sequences of PPV and was applied for detection of PPV from porcine samples. Time and temperature conditions for amplification of PPV genes were optimized to be 30min at 63°C. The developed assay was ten-fold more sensitive than conventional PCR with analytical sensitivity of 20pg and 200pg, respectively. This is the first report of detection of PPV by LAMP assay from India. The assay did not cross-react with porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), or classical swine fever virus (CSFV). The LAMP assay was assembled into a LAMP assay kit of 20 reactions and was validated in different laboratories in India. The newly developed LAMP assay was proved to be a specific, sensitive, rapid, and simple method for visual detection of PPV which does not require even costly equipments for performing the test. It complements and extends previous methods for PPV detection and provides an alternative approach for detection of PPV.