Loop-Mediated Isothermal Amplification (LAMP) for Rapid Detection and Quantification of Dehalococcoides Biomarker Genes in Commercial Reductive Dechlorinating Cultures KB-1 and SDC-9

ABSTRACTReal-time quantitative PCR (qPCR) protocols specific to the reductive dehalogenase (RDase) genesvcrA,bvcA, andtceAare commonly used to quantifyDehalococcoidesspp. in groundwater from chlorinated solvent-contaminated sites. In this study, loop-mediated isothermal amplification (LAMP) was developed as an alternative approach for the quantification of these genes. LAMP does not require a real-time thermal cycler (i.e., amplification is isothermal), allowing the method to be performed using less-expensive and potentially field-deployable detection devices. Six LAMP primers were designed for each of three RDase genes (vcrA,bvcA, andtceA) using Primer Explorer V4. The LAMP assays were compared to conventional qPCR approaches using plasmid standards, two commercially available bioaugmentation cultures, KB-1 and SDC-9 (both containDehalococcoidesspecies). DNA was extracted over a growth cycle from KB-1 and SDC-9 cultures amended with trichloroethene and vinyl chloride, respectively. All three genes were quantified for KB-1, whereas onlyvcrAwas quantified for SDC-9. A comparison of LAMP and qPCR using standard plasmids indicated that quantification results were similar over a large range of gene concentrations. In addition, the quantitative increase in gene concentrations over one growth cycle of KB-1 and SDC-9 using LAMP was comparable to that of qPCR. The developed LAMP assays forvcrAandtceAgenes were validated by comparing quantification on the Gene-Z handheld platform and a real-time thermal cycler using DNA isolated from eight groundwater samples obtained from an SDC-9-bioaugmented site (Tulsa, OK). These assays will be particularly useful at sites subject to bioaugmentation with these two commonly usedDehalococcoidesspecies-containing cultures.

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

Journal of Clinical Microbiology ◽

10.1128/jcm.01173-18 ◽

2019 ◽

Vol 57 (4) ◽

Cited By ~ 1

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.

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

Journal of Clinical Microbiology ◽

10.1128/jcm.01751-15 ◽

2016 ◽

Vol 54 (4) ◽

pp. 950-955 ◽

Cited By ~ 11

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.

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Comparison of Real-Time PCR, Reverse Transcriptase Real-Time PCR, Loop-Mediated Isothermal Amplification, and the FDA Conventional Microbiological Method for the Detection of Salmonella spp. in Produce

Applied and Environmental Microbiology ◽

10.1128/aem.00520-11 ◽

2011 ◽

Vol 77 (18) ◽

pp. 6495-6501 ◽

Cited By ~ 62

Author(s):

Guodong Zhang ◽

Eric W. Brown ◽

Narjol González-Escalona

Keyword(s):

Reverse Transcriptase ◽

Real Time ◽

Internal Control ◽

Real Time Pcr ◽

Culture Method ◽

Isothermal Amplification ◽

Molecular Methods ◽

Salmonella Spp ◽

Content Type ◽

Loop Mediated Isothermal Amplification

ABSTRACTContamination of foods, especially produce, withSalmonellaspp. is a major concern for public health. Several methods are available for the detection ofSalmonellain produce, but their relative efficiency for detectingSalmonellain commonly consumed vegetables, often associated with outbreaks of food poisoning, needs to be confirmed. In this study, the effectiveness of three molecular methods for detection ofSalmonellain six produce matrices was evaluated and compared to the FDA microbiological detection method. Samples of cilantro (coriander leaves), lettuce, parsley, spinach, tomato, and jalapeno pepper were inoculated withSalmonellaserovars at two different levels (105and <101CFU/25 g of produce). The inoculated produce was assayed by the FDASalmonellaculture method (Bacteriological Analytical Manual) and by three molecular methods: quantitative real-time PCR (qPCR), quantitative reverse transcriptase real-time PCR (RT-qPCR), and loop-mediated isothermal amplification (LAMP). Comparable results were obtained by these four methods, which all detected as little as 2 CFU ofSalmonellacells/25 g of produce. All control samples (not inoculated) were negative by the four methods. RT-qPCR detects only liveSalmonellacells, obviating the danger of false-positive results from nonviable cells. False negatives (inhibition of either qPCR or RT-qPCR) were avoided by the use of either a DNA or an RNA amplification internal control (IAC). Compared to the conventional culture method, the qPCR, RT-qPCR, and LAMP assays allowed faster and equally accurate detection ofSalmonellaspp. in six high-risk produce commodities.

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Loop-Mediated Isothermal Amplification Allows Rapid, Simple and Accurate Molecular Diagnosis of Human Cutaneous and Visceral Leishmaniasis Caused by Leishmania infantum When Compared to PCR

Microorganisms ◽

10.3390/microorganisms9030610 ◽

2021 ◽

Vol 9 (3) ◽

pp. 610

Author(s):

Ana Victoria Ibarra-Meneses ◽

Carmen Chicharro ◽

Carmen Sánchez ◽

Emilia García ◽

Sheila Ortega ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Real Time ◽

Parasite Load ◽

Isothermal Amplification ◽

Excellent Correlation ◽

Loop Mediated Isothermal Amplification ◽

Real Time Quantitative Pcr ◽

Specific Amplification ◽

Polymerase Chain ◽

Eiken Chemical

Loop-mediated isothermal amplification allows the rapid, sensitive and specific amplification of DNA without complex and expensive equipment. We compared the diagnostic performance of Loopamp™ Leishmania Detection Kit (Eiken Chemical Co., Ltd., Tokyo, Japan) with conventional and real-time polymerase chain reaction (PCR) for human cutaneous and visceral leishmaniasis caused by L. infantum. A total of 230 DNA samples from cutaneous (CL) and visceral (VL) leishmaniasis cases and controls from Spain, characterized by Leishmania nested PCR (LnPCR) were tested by: (i) the Loopamp™ Leishmania Detection Kit (Loopamp), run on Genie III real-time fluorimeter (OptiGene, UK); and (ii) real-time quantitative PCR (qPCR). The Loopamp test returned 98.8% (95% confidence interval—CI: 96.0–100.00) sensitivity and specificity of 97.7% (95% CI: 92.2–100) on VL samples, and 100% (95% CI: 99.1–100) sensitivity and 100.0% (95% CI: 98.8–100.0) specificity on CL samples. The Loopamp time-to-positivity (Tp) obtained by real-time fluorimetry showed excellent concordance (C = 97.91%) and strong correlation (r = 0.799) with qPCR’s cycle threshold (Ct). The performance of Loopamp is comparable to that of LnPCR and qPCR in the diagnosis of cutaneous and visceral leishmaniasis due to L. infantum. The excellent correlation between the Tp and Ct should be further investigated to determine the accuracy of Loopamp to quantify parasite load in tissues.

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