Comparison of a capillary gel electrophoresis-based multiplex PCR assay and ribosomal intergenic transcribed spacer-2 amplicon sequencing for identification of clinically important Candida species

Abstract A detection method for genetically modified foods is an essential regulatory requirement for many countries. The present study is aimed at developing a qualitative method for detection of genetically modified organisms by combining PCR methodology with capillary gel electrophoresis (PCR-CGE) in a sequencing platform to detect Bacillus thuringiensis (Bt)-cotton (MON 531)and Roundup Ready (RR) soybean (GTS 40-3-2). A sensitive duplex PCR-CGE method was developed in which target DNA sequences (35S and Nos) were separated both by size and color to detect 0.01% Cry1Ac DNA (w/w) in Bt-cotton. A multiplex PCR-CGE method was developed to simultaneously detect fourtargets such as Sad1, Cry1Ac, 35S, and Nos in Bt-cotton. Four novel PCR primers were designed to customize amplicon size for multiplexing for better visualizationof multiple peaks. The LOD for Cry1Ac DNA specific PCR was 0.01% for Bt-cotton. The LOD for multiplex PCR assay was 0.05% for Bt-cotton. A singleplex PCR-CGE method was developed to detect Lec, 35S and Nos in a trace sample of RR soybean grainpowder (0.1%, w/w). This study demonstrates aPCR-CGE-based method for the qualitative detection of35S, Nos and Cry1Ac targets associated with genetically modifiedproducts.

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Multiplex PCR Assay Simplifies Serotyping and Sequence Typing of Listeria monocytogenes Associated with Human Outbreaks

Journal of Food Protection ◽

10.4315/0362-028x-68.9.1907 ◽

2005 ◽

Vol 68 (9) ◽

pp. 1907-1910 ◽

Cited By ~ 22

Author(s):

WEI ZHANG ◽

STEPHEN J. KNABEL

Keyword(s):

Listeria Monocytogenes ◽

Multiplex Pcr ◽

Gel Electrophoresis ◽

Virulence Genes ◽

Rapid Identification ◽

Pulsed Field Gel Electrophoresis ◽

Pcr Assay ◽

Pulsed Field ◽

Typing Scheme ◽

Multiplex Pcr Assay

Listeria monocytogenes serotypes 1/2a and 4b are responsible for the majority of cases of human listeriosis worldwide. In this study, a multiplex PCR assay was developed to allow rapid identification and easily interpretable differentiation of serotypes 1/2a and 4b from other serotypes of L. monocytogenes by simultaneously targeting two virulence genes (inlB and inlC) and two serotype-specific genes (ORF2372 and lmo0171). A subsequent gel extraction and sequence typing analysis of the highly polymorphic intragenic regions in inlB and inlC simplified a previously developed multi–virulence-locus sequence typing scheme and provided discriminatory power for subtyping L. monocytogenes similar to pulsed-field gel electrophoresis analysis.

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Usefulness of a Multiplex PCR for Detection of Diarrheagenic Escherichia coli in a Diagnostic Microbiology Laboratory Setting

Bangladesh Journal of Medical Microbiology ◽

10.3329/bjmm.v1i2.21506 ◽

2016 ◽

Vol 1 (2) ◽

pp. 38-42 ◽

Cited By ~ 4

Author(s):

Khairun Nessa ◽

Dilruba Ahmed ◽

Johirul Islam ◽

FM Lutful Kabir ◽

M Anowar Hossain

Keyword(s):

Escherichia Coli ◽

Multiplex Pcr ◽

Clinical Laboratory ◽

Proteinase K ◽

Microbiology Laboratory ◽

Pcr Assay ◽

E Coli ◽

Diarrheagenic Escherichia Coli ◽

Multiplex Pcr Assay ◽

Diagnostic Microbiology

A multiplex PCR assay was evaluated for diagnosis of diarrheagenic Escherichia coli in stool samples of patients with diarrhoea submitted to a diagnostic microbiology laboratory. Two procedures of DNA template preparationproteinase K buffer method and the boiling method were evaluated to examine isolates of E. coli from 150 selected diarrhoeal cases. By proteinase K buffer method, 119 strains (79.3%) of E. coli were characterized to various categories by their genes that included 55.5% enteroaggregative E. coli (EAEC), 18.5% enterotoxigenic E. coli (ETEC), 1.7% enteropathogenic E. coli (EPEC), and 0.8% Shiga toxin-producing E. coli (STEC). Although boiling method was less time consuming (<24 hrs) and less costly (<8.0 US $/ per test) but was less efficient in typing E. coli compared to proteinase K method (41.3% vs. 79.3% ; p<0.001). The sensitivity and specificity of boiling method compared to proteinase K method was 48.7% and 87.1% while the positive and negative predictive value was 93.5% and 30.7%, respectively. The majority of pathogenic E. coli were detected in children (78.0%) under five years age with 53.3% under one year, and 68.7% of the children were male. Children under 5 years age were frequently infected with EAEC (71.6%) compared to ETEC (24.3%), EPEC (2.7%) and STEC (1.4%). The multiplex PCR assay could be effectively used as a rapid diagnostic tool for characterization of diarrheagenic E. coli using a single reaction tube in the clinical laboratory setting.Bangladesh J Med Microbiol 2007; 01 (02): 38-42

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