J Vet Sci.
2014 Jun;15(2):317-325. 10.4142/jvs.2014.15.2.317.
Development of a multiplex loop-mediated isothermal amplification assay to detect shiga toxin-producing Escherichia coli in cattle
- Affiliations
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- 1BK21 PLUS Program for Creative Veterinary Science Research, Research Institute for Veterinary Science and College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea. chose@snu.ac.kr
- 2College of Veterinary Medicine and Institute of Veterinary Medical Science, Kangwon National University, Chuncheon 200-701, Korea.
- KMID: 1784654
- DOI: http://doi.org/10.4142/jvs.2014.15.2.317
Abstract
- A multiplex loop-mediated isothermal amplification (mLAMP) assay was developed for simultaneous detection of the stx1 and stx2 genes and applied for detection of shiga toxin-producing Escherichia coli (STEC) in cattle farm samples. Two target genes were distinguished based on T m values of 85.03 +/- 0.54degrees C for stx1 and 87.47 +/- 0.35degrees C for stx2. The mLAMP assay was specific (100% inclusivity and exclusivity), sensitive (with a detection limit as low as 10 fg/microL), and quantifiable (R 2 = 0.9313). The efficacy and sensitivity were measured to evaluate applicability of the mLAMP assay to cattle farm samples. A total of 12 (12/253; 4.7%) and 17 (17/253; 6.7%) STEC O157, and 11 (11/236; 4.7%) non-O157 STEC strains were isolated from cattle farm samples by conventional selective culture, immunomagnetic separation, and PCR-based culture methods, respectively. The coinciding multiplex PCR and mLAMP results for the types of shiga toxin revealed the value of the mLAMP assay in terms of accuracy and rapidity for characterizing shiga toxin genes. Furthermore, the high detection rate of specific genes from enrichment broth samples indicates the potential utility of this assay as a screening method for detecting STEC in cattle farm samples.
Keyword
MeSH Terms
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Animals
Cattle
Cattle Diseases/epidemiology/microbiology
Escherichia coli Infections/epidemiology/microbiology/*veterinary
Feces/microbiology
Multiplex Polymerase Chain Reaction/veterinary
Nucleic Acid Amplification Techniques/*veterinary
Shiga Toxin 1/*genetics/isolation & purification
Shiga Toxin 2/*genetics/isolation & purification
Shiga-Toxigenic Escherichia coli/*genetics/isolation & purification
Shiga Toxin 1
Shiga Toxin 2
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