Technique for Quantitative Detection of Specific DNA Sequences Using Alternately Binding Quenching Probe Competitive Assay Combined with Loop-Mediated Isothermal Amplification

ABSTRACT This study reports the development and optimization of a real-time loop-mediated isothermal amplification (qLAMP) method for rapid detection of Acetobacter aceti strain in red wine samples. Our results showed that the primers and probes designed for 16S rRNA were effective for A. aceti detection. The quantification limit of real-time polymerase chain reaction (qPCR) and qLAMP in pure culture was 2.05 × 101 colony forming units (CFU) mL−1. qLAMP had a sensitivity of 6.88 × 101 CFU mL−1 in artificially contaminated Changyu dry red wine (CDRW) and Changyu red wine (CRW), and 6.88 × 102 CFU mL−1 in artificially contaminated Greatwall dry red wine (GDRW), which was 10 times higher than that of qPCR. In conclusion, this newly developed qLAMP is a reliable, rapid and accurate method for the detection and quantification of A. aceti species in red wine samples. Furthermore, our work provides a standard reference method for the quantitative detection of A. aceti and other acetic acid bacteria during the fermentation and storage of red wine samples.

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

Applied and Environmental Microbiology ◽

10.1128/aem.02992-10 ◽

2011 ◽

Vol 77 (8) ◽

pp. 2589-2595 ◽

Cited By ~ 27

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.00274-14 ◽

2014 ◽

Vol 80 (15) ◽

pp. 4519-4530 ◽

Cited By ~ 24

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.

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