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

• Dong HJ
• Cho AR
• Hahn TW
• Cho S

• 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

cattle farm; E. coli O157; LAMP; shiga toxin; stx

MeSH Terms

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

Figure

• 

  Fig. 1 Discrimination of the stx1 and stx2 by distinct Tm values generated by annealing curve analysis of the mLAMP assay. The annealing curve shows temperature on the X-axis and fluorescence on the Y-axis. The peaks indicate Tm values generated by annealing curve analysis. Well 1 (red), stx1 and stx2 (STEC 43894); well 2 (green), stx1 (STEC 43890); Well 3 (blue), stx2 (STEC 43889); Well 4 (purple), NC (negative control).

• 

  Fig. 2 Detection limit and standard curve of the mLAMP assay targeting stx1 and stx2 genes of STEC. Ten-fold serial dilutions of STEC (ATCC 43894) template DNA from 100 ng/µL to 1 fg/µL tested in triplicate. (A) The amplification curve shows time in minutes on the X-axis and the amount of fluorescence detected (K) on the Y-axis. The amplification curve generated from 100 ng/µL to 10 fg/µL. (B) Discrimination of shiga toxin genes was based on the annealing curve showing 2 distinct Tm values (84.63 ± 0.30℃ for stx1 and 87.24 ± 0.25℃ for stx2). (C) The standard curve shows the amount of template DNA in log scale on the X-axis and Tp values (min:sec) on the Y-axis. DNA concentrations 1 to 8 correspond to template DNA from 10 fg/µL to 100 ng/µL in log scale.

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