scholarly journals Detecting Potentially Virulent Vibrio vulnificus Strains in Raw Oysters by Quantitative Loop-Mediated Isothermal Amplification

2011 ◽  
Vol 77 (8) ◽  
pp. 2589-2595 ◽  
Author(s):  
Feifei Han ◽  
Fei Wang ◽  
Beilei Ge
Keyword(s):  
Pure Culture ◽  
Vibrio Vulnificus ◽  
Lamp Assay ◽  
The United States ◽  
Isothermal Amplification ◽  
Quantitative Detection ◽  
Strain Atcc ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification ◽  
Negative Results

ABSTRACTVibrio vulnificusis a leading cause of seafood-related deaths in the United States. Sequence variations in the virulence-correlated gene (vcg) have been used to distinguish between clinical and environmentalV. vulnificusstrains, with a strong association between clinical ones and the C sequence variant (vcgC). In this study,vcgCwas selected as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, sensitive, specific, and quantitative detection of potentially virulentV. vulnificusstrains in raw oysters. No false-positive or false-negative results were generated among the 125 bacterial strains used to evaluate assay specificity. The detection limit was 5.4 CFU per reaction for a virulentV. vulnificusstrain (ATCC 33815) in pure culture, 100-fold more sensitive than that of PCR. In spiked raw oysters, the assay was capable of detecting 2.5 × 103CFU/g ofV. vulnificusATCC 33815, while showing negative results for a nonvirulentV. vulnificusstrain (515-4c2) spiked at 107CFU/g. After 6 h of enrichment, the LAMP assay could detect 1 CFU/g of the virulentV. vulnificusstrain ATCC 33815. Standard curves generated in pure culture and spiked oysters suggested a good linear relationship between cell numbers of the virulentV. vulnificusstrain and turbidity signals. In conclusion, the LAMP assay developed in this study could quantitatively detect potentially virulentV. vulnificusin raw oysters with high speed, specificity, and sensitivity, which may facilitate better control ofV. vulnificusrisks associated with raw oyster consumption.

scholarly journals Rapid and Specific Detection of Escherichia coli Serogroups O26, O45, O103, O111, O121, O145, and O157 in Ground Beef, Beef Trim, and Produce by Loop-Mediated Isothermal Amplification

2012 ◽  
Vol 78 (8) ◽  
pp. 2727-2736 ◽  
Author(s):  
Fei Wang ◽  
Lin Jiang ◽  
Qianru Yang ◽  
Witoon Prinyawiwatkul ◽  
Beilei Ge
Keyword(s):  
Escherichia Coli ◽  
Pure Culture ◽  
False Negative ◽  
Ground Beef ◽  
The United States ◽  
Food Samples ◽  
Isothermal Amplification ◽  
Specific Detection ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification

ABSTRACTEscherichia coliO157 and six additional serogroups of Shiga toxin-producingE. coli(STEC) (O26, O45, O103, O111, O121, and O145) account for the majority of STEC infections in the United States. In this study, O serogroup-specific genes (wzxorwzy) were used to design loop-mediated isothermal amplification (LAMP) assays for the rapid and specific detection of these leading STEC serogroups. The assays were evaluated in pure culture and spiked food samples (ground beef, beef trim, lettuce, and spinach) and compared with real-time quantitative PCR (qPCR). No false-positive or false-negative results were observed among 120 bacterial strains used to evaluate assay specificity. The limits of detection of various STEC strains belonging to these target serogroups were approximately 1 to 20 CFU/reaction mixture in pure culture and 103to 104CFU/g in spiked food samples, which were comparable to those of qPCR. Standard curves generated suggested good linear relationships between STEC cell numbers and LAMP turbidity signals. In various beef and produce samples spiked with two low levels (1 to 2 and 10 to 20 CFU/25 g) of respective STEC strains, the LAMP assays consistently achieved accurate detection after 6 to 8 h of enrichment. In conclusion, these newly developed LAMP assays may facilitate rapid and reliable detection of the seven major STEC serogroups in ground beef, beef trim, and produce during routine sample testing.

scholarly journals Rapid Detection of Viable Salmonellae in Produce by Coupling Propidium Monoazide with Loop-Mediated Isothermal Amplification

2011 ◽  
Vol 77 (12) ◽  
pp. 4008-4016 ◽  
Author(s):  
Siyi Chen ◽  
Fei Wang ◽  
John C. Beaulieu ◽  
Rebecca E. Stein ◽  
Beilei Ge
Keyword(s):  
Pure Culture ◽  
Rapid Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Detection Methods ◽  
Sample Treatment ◽  
Propidium Monoazide ◽  
Simple Method ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification

ABSTRACTRecent outbreaks linked toSalmonella-contaminated produce heightened the need to develop simple, rapid, and accurate detection methods, particularly those capable of determining cell viability. In this study, we examined a novel strategy for the rapid detection and quantification of viable salmonellae in produce by coupling a simple propidium monoazide sample treatment with loop-mediated isothermal amplification (PMA-LAMP). We first designed and optimized a LAMP assay targetingSalmonella. Second, the performance of PMA-LAMP for detecting and quantifying viable salmonellae was determined. Finally, the assay was evaluated in experimentally contaminated produce items (cantaloupe, spinach, and tomato). Under the optimized condition, PMA-LAMP consistently gave negative results for heat-killedSalmonellacells with concentrations up to 108CFU/ml (or CFU/g in produce). The detection limits of PMA-LAMP were 3.4 to 34 viableSalmonellacells in pure culture and 6.1 × 103to 6.1 × 104CFU/g in spiked produce samples. In comparison, PMA-PCR was up to 100-fold less sensitive. The correlation between LAMP time threshold (TT) values and viableSalmonellacell numbers was high (R2= 0.949 to 0.993), with a quantification range (102to 105CFU/reaction in pure culture and 104to 107CFU/g in produce) comparable to that of PMA in combination with quantitative real-time PCR (PMA-qPCR). The complete PMA-LAMP assay took about 3 h to complete when testing produce samples. In conclusion, this rapid, accurate, and simple method to detect and quantify viableSalmonellacells in produce may present a useful tool for the produce industry to better control potential microbial hazards in produce.

scholarly journals Comparison of Loop-Mediated Isothermal Amplification and Real-Time PCR Assays for Detection ofStrongyloidesLarvae in Different Specimen Matrices

2019 ◽  
Vol 57 (4) ◽  
Author(s):  
Matthew R. Watts ◽  
Rady Kim ◽  
Vishal Ahuja ◽  
Gemma J. Robertson ◽  
Yasmin Sultana ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Chronic Infection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Percentage Agreement ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification ◽  
Stool Specimens

ABSTRACTStrongyloides stercoraliscan cause disease that ranges from asymptomatic chronic infection to fatal hyperinfection. Diagnosis from stool can be challenging because the most sensitive conventional tests require live larvae to be effective and there can be low larval output in chronic infection. Nucleic acid amplification tests (NAAT) have been developed to complement existing diagnostic methods. We compared a recently developed loop-mediated isothermal amplification (LAMP) assay with a real-time PCR that has previously been validated with larval microscopy. The limits of detection—quantified using serial dilutions of DNA extracts from singleStrongyloides rattithird-stage (L3) larvae spiked into approximately 250 µl of 5 differentS. stercoralis-negative stool specimens—were 10−3(1/5 replicates) and 10−2(1/5 replicates) dilutions for PCR and LAMP, respectively. PCR was positive for 4/5 replicates at 10−2. LAMP was compared to PCR using extracts from 396 stool specimens collected in Bangladesh and Australia, of which 53 were positive and 343 were negative by PCR. The positive percentage agreement of LAMP was 77.3% (95% score confidence interval [CI], 64.5 to 86.6). The negative percentage agreement was 100% (95% CI, 98.9 to 100). In a preliminary investigation, PCR and LAMP assays were positive using DNA extracted from serum (PCR, 3/16 extracts; LAMP, 2/16 extracts) and bronchoalveolar lavage fluid (PCR and LAMP, 2/2 extracts), demonstrating proof of concept. Compared to PCR, the lower number of positive results using the LAMP assay may have been due to reaction inhibitors and DNA degradation, and strategies to improve the LAMP assay are discussed.

scholarly journals Loop-Mediated Isothermal Amplification for the Diagnostic Detection of Meloidogyne chitwoodi and M. fallax

Plant Disease ◽  
2019 ◽  
Vol 103 (1) ◽  
pp. 12-18 ◽  
Author(s):  
Lei Zhang ◽  
Cynthia Gleason
Keyword(s):  
Pacific Northwest ◽  
Intergenic Spacer ◽  
Lamp Assay ◽  
The United States ◽  
Isothermal Amplification ◽  
Root Knot Nematode ◽  
Meloidogyne Chitwoodi ◽  
Loop Mediated Isothermal Amplification ◽  
The Pacific ◽  
Potato Nematodes

Meloidogyne chitwoodi is a root-knot nematode that parasitizes a broad range of plants. In the Pacific Northwest (PNW) of the United States, M. chitwoodi is a major potato pest. The nematodes infect roots and tubers; blemishes caused by the nematodes on the tubers significantly affect potato marketability. M. chitwoodi is a quarantine pathogen by many regulatory agencies, limiting potato trade opportunities when it is present. A loop-mediated isothermal amplification (LAMP) assay was developed to amplify the intergenic spacer (IGS2)-18S region of the ribosomal rDNA of M. chitwoodi. Using the LAMP assay, we could detect the presence of M. chitwoodi from infected Washington State soil samples. The LAMP primers showed specificity for DNA from M. chitwoodi and the closely related species M. fallax. There was no cross reaction of the LAMP primers with DNA from tropical nematodes M. incognita, M. arenaria, and M. javanica, or the Northern root-knot nematode M. hapla. The LAMP assays can be completed within 45 min, and they were 100 times more sensitive in nematode detection than conventional PCR. The LAMP assay will facilitate detection of potato nematodes M. chitwoodi and M. fallax. Knowledge of potato nematodes, particularly M. chitwoodi in PNW soils, will aid management decisions.

scholarly journals Loop-Mediated Isothermal Amplification for the Rapid Detection of the F200Y Mutant Genotype of Carbendazim-Resistant Isolates of Sclerotinia sclerotiorum

Plant Disease ◽  
2016 ◽  
Vol 100 (5) ◽  
pp. 976-983 ◽  
Author(s):  
Yabing Duan ◽  
Ying Yang ◽  
Yong Wang ◽  
Xiayan Pan ◽  
Jian Wu ◽  
...  
Keyword(s):  
Sclerotinia Sclerotiorum ◽  
Rapid Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Special Equipment ◽  
Mutant Genotype ◽  
Loop Mediated Isothermal Amplification ◽  
Negative Results ◽  
Resistant Mutants ◽  
Moderate Resistance

The point mutation at codon 200 (TTC→TAC, F200Y) confers moderate resistance to carbendazim in Sclerotinia sclerotiorum. This mutant genotype (F200Y) has been detected mainly by determining the minimum inhibitory concentration (MIC), which requires 3 to 5 days. Here, we developed a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of the F200Y mutant genotype of carbendazim-resistant isolates of S. sclerotiorum. Specific LAMP primers were designed and concentrations of LAMP components were optimized. The optimal reaction conditions were 62 to 63°C for 45 min. The new LAMP assay requires no special equipment and is highly sensitive and specific (the i.e., it generated positive results with F200Y mutant genotype but generated negative results with other carbendazim-resistant mutants and with a variety of carbendazim-resistant mutants of Botrytis cinerea and Fusarium graminearum). Inclusion of the loop backward (LB) primer reduced the reaction time to 15 min. Results were identical with LAMP and MIC determinations. The advantages of the LB-accelerated LAMP assay for detection of the F200Y mutant genotype were demonstrated by assaying sclerotia produced on rape stems that were artificially inoculated in the field. The results indicated that the new LAMP assay represents an improved way to detect the F200Y mutant genotype of carbendazim-resistant isolates of S. sclerotiorum.

scholarly journals Sensitive Detection of Xanthomonas oryzae Pathovars oryzae and oryzicola by Loop-Mediated Isothermal Amplification

2014 ◽  
Vol 80 (15) ◽  
pp. 4519-4530 ◽  
Author(s):  
Jillian M. Lang ◽  
Paul Langlois ◽  
Marian Hanna R. Nguyen ◽  
Lindsay R. Triplett ◽  
Laura Purdie ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Lamp Assay ◽  
Causal Agent ◽  
Isothermal Amplification ◽  
Plant Diseases ◽  
Xanthomonas Oryzae ◽  
Bacterial Cells ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification ◽  
Single Temperature

ABSTRACTMolecular diagnostics for crop diseases can enhance food security by enabling the rapid identification of threatening pathogens and providing critical information for the deployment of disease management strategies. Loop-mediated isothermal amplification (LAMP) is a PCR-based tool that allows the rapid, highly specific amplification of target DNA sequences at a single temperature and is thus ideal for field-level diagnosis of plant diseases. We developed primers highly specific for two globally important rice pathogens,Xanthomonas oryzaepv. oryzae, the causal agent of bacterial blight (BB) disease, andX. oryzaepv. oryzicola, the causal agent of bacterial leaf streak disease (BLS), for use in reliable, sensitive LAMP assays. In addition to pathovar distinction, two assays that differentiateX. oryzaepv. oryzae by African or Asian lineage were developed. Using these LAMP primer sets, the presence of each pathogen was detected from DNA and bacterial cells, as well as leaf and seed samples. Thresholds of detection for all assays were consistently 104to 105CFU ml−1, while genomic DNA thresholds were between 1 pg and 10 fg. Use of the unique sequences combined with the LAMP assay provides a sensitive, accurate, rapid, simple, and inexpensive protocol to detect both BB and BLS pathogens.

scholarly journals Detection of Colletotrichum acutatum Sensu Lato on Strawberry by Loop-Mediated Isothermal Amplification

Plant Disease ◽  
2016 ◽  
Vol 100 (9) ◽  
pp. 1804-1812 ◽  
Author(s):  
X. Zhang ◽  
T. C. Harrington ◽  
J. C. Batzer ◽  
R. Kubota ◽  
N. A. Peres ◽  
...  
Keyword(s):  
Pure Culture ◽  
Detection Threshold ◽  
Lamp Assay ◽  
Its Region ◽  
Isothermal Amplification ◽  
Fruit Rot ◽  
Colletotrichum Acutatum ◽  
Initial Inoculum ◽  
Loop Mediated Isothermal Amplification ◽  
Primer Sets

Colletotrichum acutatum, one of the most economically damaging pathogens of strawberry, is the primary causal agent of anthracnose fruit rot (AFR). A key challenge in managing AFR is detecting the pathogen on asymptomatic plants. To meet this need, a loop-mediated isothermal amplification (LAMP) assay was developed that incorporated two sets of primers: LITSG1, targeted on the intergenic transcribed spacer (ITS) region of ribosomal DNA, and Ltub2, on the β-tubulin 2 gene. In pure culture assays, Ltub2 was specific for detection of C. acutatum, whereas LITSG1 detected C. acutatum and two additional anthracnose pathogens, C. gloeosporioides and C. fragariae. LITSG1 had 10-fold lower detection threshold (20 pg of mycelial DNA) than Ltub2 (200 pg mycelial DNA) in detection of C. acutatum from pure culture. For detection on asymptomatic leaves, two protocols for dislodging C. acutatum for DNA extraction were compared: i) the sonicate-agitate (SA) method and ii) the freeze-incubate-sonicate-agitate (FISA) method, which initially freezes tissues, followed by 2 days of incubation at 26°C in darkness, and then, sonication and agitation. Both methods were used for greenhouse-grown plant leaves that had been spray inoculated with serial dilutions ranging from 1.5 × 106 to 1.5 conidia ml−1. The FISA method produced more repeatable results than the SA method. For the FISA method, detection limits (expressed as initial inoculum concentrations) using LITSG1 and Ltub2 were 1.5 × 101 and 1.5 × 102 conidia ml−1, respectively. For composite samples comprised of inoculated (1.5 × 106 conidia ml−1) and noninoculated leaves of greenhouse-grown strawberry, the two sets of LAMP primers were compared using the SA method. Primer set LITSG1 consistently detected the pathogen from a single inoculated leaf in bulk samples of 50 or fewer pathogen-free leaves, whereas Ltub2 consistently detected one inoculated leaf in 20 or fewer pathogen-free leaves. Using primer set LITSG1, FISA was more sensitive than SA for detecting C. acutatum on leaves of field-grown plants from Florida. In an Iowa field trial using the FISA method, both primer sets detected C. acutatum in samples of asymptomatic leaves 6 days before fruit symptoms appeared. The results indicate that the LAMP assay has potential to provide a simplified method for detection of C. acutatum on asymptomatic strawberry plants.

scholarly journals Earlyin plantadetection ofXanthomonas axonopodispv.punicaein pomegranate using enhanced loop-mediated isothermal amplification assay

2018 ◽  
Author(s):  
M.K. PrasannaKumar ◽  
P. Buela Parivallal ◽  
C. Manjunath ◽  
H.B. Mahesh ◽  
Karthik S Narayan ◽  
...  
Keyword(s):  
Post Infection ◽  
Bacterial Blight ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Economic Losses ◽  
Specific Primers ◽  
Loop Mediated Isothermal Amplification ◽  
Negative Results ◽  
Hydroxynaphthol Blue ◽  
Amplification Assay

AbstractBacterial blight in pomegranate caused byXanthomonas axonopodispv.punicae(Xap) is an increasing threat for pomegranate cultivation in India. To prevent the economic losses, it is pivotal to detect the infection in latent stages rather than in later stages. We have developed an enhanced method termed as loop-mediated isothermal amplification (LAMP) technique to evaluate for the latent detection of Xap in pomegranate using six set of specific primers. Three DNA intercalating dyes were used, such as Ethidium bromide, hydroxynaphthol blue (HNB) and SYBR Green resulted in visualising the positivity for LAMP assay. The reaction time and temperature were to be 65°C from 30 min onwards, for the dyes and its sensitivity was observed up to 10−7ng in the LAMP assay. For field applicability, LAMP assay detected Xap on 7thday post infection while the PCR amplified Xap after 11thday post infection. Finally, the specificity of LAMP assay was validated to be positive with ten Xap isolates for its accuracy and 29 non-Xap bacterial isolates showed negative results. Moreover, this method could be used as a better alternative to PCR based methods, for early detection of the pathogens.

scholarly journals Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for Rapid Detection of Aspergillus fumigatus

2016 ◽  
Vol 54 (4) ◽  
pp. 950-955 ◽  
Author(s):  
Qing Tang ◽  
Shuguang Tian ◽  
Nong Yu ◽  
Xi Zhang ◽  
Xiaodong Jia ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Rapid Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification ◽  
Real Time Quantitative Pcr ◽  
Amplification Method ◽  
Positive Results

Aspergillus fumigatusis a conditional pathogen and the major cause of life-threatening invasive aspergillosis (IA) in immunocompromised patients. The early and rapid detection ofA. fumigatusinfection is still a major challenge. In this study, the new member of the fungal annexin family, annexin C4, was chosen as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, specific, and sensitive detection ofA. fumigatus. The evaluation of the specificity of the LAMP assay that was developed showed that no false-positive results were observed for the 22 non-A. fumigatusstrains, including 5 species of theAspergillusgenus. Its detection limit was approximately 10 copies per reaction in reference plasmids, with higher sensitivity than that of real-time quantitative PCR (qPCR) at 102copies for the same target. Clinical samples from a total of 69 patients with probable IA (n =14) and possible IA (n= 55) were subjected to the LAMP assay, and positive results were found for the 14 patients with probable IA (100%) and 34 patients with possible IA (61.82%). When detection using the LAMP assay was compared with that using qPCR in the 69 clinical samples, the LAMP assay demonstrated a sensitivity of 89.19% and the concordance rate for the two methods was 72.46%. Accordingly, we report that a valuable LAMP assay for the rapid, specific, and simple detection ofA. fumigatusin clinical testing has been developed.

scholarly journals A Loop-Mediated Isothermal Amplification Assay Targeting Neisseria gonorrhoeae penA-60.001

2019 ◽  
Vol 64 (1) ◽  
Author(s):  
Ken Shimuta ◽  
Shu-ichi Nakayama ◽  
Hideyuki Takahashi ◽  
Makoto Ohnishi
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Assay System ◽  
Line Treatment ◽  
First Line ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Assay ◽  
First Line Treatment

ABSTRACT Ceftriaxone (CRO) is widely used as the first-line treatment for gonococcal infections. However, CRO-resistant Neisseria gonorrhoeae strains carrying mosaic penA-60.001 have emerged recently and disseminated worldwide. To meet the urgent need to detect these strains, we report here a loop-mediated isothermal amplification (LAMP) assay system that targets N. gonorrhoeae penA-60.001. This assay system can differentiate N. gonorrhoeae strains carrying mosaic penA-60.001 from strains carrying other penA alleles.

Sign in / Sign up

Export Citation Format

Share Document