A new set of pure microsatellite loci in the common octopus Octopus vulgaris Cuvier, 1797 for multiplex PCR assay and their cross-amplification in O. maya Voss & Solís Ramírez, 1966

A multiplex PCR (mPCR) assay using previously known genetic markers ofShigella, Escherichia coliand Shiga-toxicEsch. coliwas standardized.uidAgene was targeted for the common detection ofEsch. coliandShigella, whereasipaHandstx1genes were used as markers for the detection ofShigellaand shiga-toxin producing strains, respectively. The standardized assays detected the target organism specifically and selectively. The mPCR developed by combining all the three reactions generated specific products. The inclusivity and exclusivity tests depicted the precise specificity of the mPCR assay. Results were interpreted on the basis of the pattern of amplicons generated: amplifications of theipaHanduidAgene fragments indicated the presence ofShigellaspp., amplification ofuidAalone revealed the presence ofEsch. coliand additional presence of verotoxin gene amplicon indicated verotoxinogenic nature of the strain. Specific patterns of bands were obtained when different strains ofEsch. coliandShigellaspp. were subjected to this assay. The reactions, individually as well as in the mPCR, could detect approximately 1 cell per 20-μl PCR assay. The protocols were validated by analyzing the coded samples of full fat milk spiked with different pathogens. In naturally contaminated raw milk samples (n=100),Esch. coliwere detected in all samples and verotoxinogenicEsch. coliin 15 samples.Shigella, however, was not detected in any of the samples. When DNA purified from the samples found positive for Shiga-toxicEsch. coliwas directly used as template for the mPCR, the results showed agreement with the enrichment based detection. The mPCR assay, standardized in this study, may be used for rapid microbiological evaluation of milk samples. Further, the study emphasizes the need for better hygienic conditions in dairies.

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Development of a multiplex PCR assay for fine‐scale population genetic analysis of the Komodo monitor Varanus komodoensis based on 18 polymorphic microsatellite loci

Molecular Ecology Resources ◽

10.1111/j.1755-0998.2011.02986.x ◽

2011 ◽

Vol 11 (3) ◽

pp. 550-556 ◽

Cited By ~ 11

Author(s):

CLAUDIO CIOFI ◽

ATHANASIA C. TZIKA ◽

CHIARA NATALI ◽

PHILLIP C. WATTS ◽

SRI SULANDARI ◽

...

Keyword(s):

Genetic Analysis ◽

Multiplex Pcr ◽

Microsatellite Loci ◽

Population Genetic ◽

Fine Scale ◽

Pcr Assay ◽

Population Genetic Analysis ◽

Multiplex Pcr Assay ◽

Scale Population ◽

Polymorphic Microsatellite

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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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