A Multiplex PCR Based on Mitochondrial COI Sequences for Identification of Members of the Anopheles barbirostris Complex (Diptera: Culicidae) in Thailand and Other Countries in the Region

A multiplex-PCR assay based on mitochondrial cytochrome c oxidase subunit I (COI) sequences was developed for identification of five members of the Barbirostris Complex which occur in Thailand: Anopheles barbirostris s.s., An. dissidens, An. saeungae, An. wejchoochotei and An. barbirostris species A3. Anopheles campestris was not included in the assay due to the lack of unequivocal sequences. Allele-specific primers were designed for specific nucleotide segments of COI sequences of each species. Mismatch method and addition of long GC tail were applied for some primers. The assay provided products of 706 bp for An. barbirostris s.s., 238 bp for An. dissidens, 611 bp for An. saeungae, 502 bp for An. wejchoochotei and 365 bp for An. barbirostris A3. The assay was tested using 111 wild-caught female mosquitoes from Bhutan, Cambodia, Indonesia (Sulawesi) and Thailand. The results of the multiplex PCR were in complete agreement with COI sequencing; however, one of three specimens from Bhutan and all 11 specimens from Indonesia were not amplifiable by the assay due to their distinct COI sequences. This, together with the distinct rDNA sequences of these specimens, suggests the presence of at least two additional new species in the Barbirostris Complex.

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Opisthorchis viverrini and Haplorchis taichui: Development of a multiplex PCR assay for their detection and differentiation using specific primers derived from HAT-RAPD

Experimental Parasitology ◽

10.1016/j.exppara.2012.07.007 ◽

2012 ◽

Vol 132 (2) ◽

pp. 237-242 ◽

Cited By ~ 10

Author(s):

Chalobol Wongsawad ◽

Pheravut Wongsawad

Keyword(s):

Multiplex Pcr ◽

Opisthorchis Viverrini ◽

Pcr Assay ◽

Specific Primers ◽

Multiplex Pcr Assay ◽

Haplorchis Taichui

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Development of a multiplex PCR assay for the detection of key genes associated with Pasteurella multocida subspecies

Journal of Veterinary Diagnostic Investigation ◽

10.1177/10406387211063438 ◽

2021 ◽

pp. 104063872110634

Author(s):

Barbara Ujvári ◽

Hubert Gantelet ◽

Tibor Magyar

Keyword(s):

Multiplex Pcr ◽

Pasteurella Multocida ◽

Rrna Gene ◽

Pcr Assay ◽

Biochemical Tests ◽

Specific Primers ◽

Multiplex Pcr Assay ◽

Subspecies Level ◽

Species Specific ◽

Reference Strains

The ability to distinguish among the subspecies of Pasteurella multocida isolates is important epidemiologically; however, classification at the subspecies level based on the results of conventional biochemical tests (fermentation of sorbitol and dulcitol) is reportedly not accurate in all cases. Therefore, we developed a rapid, multiplex PCR assay to differentiate among the 3 subspecies of P. multocida. The PCR assay includes the P. multocida species–specific primers KMT1SP6 and KMT1T7 as an internal amplification control, with a newly designed gatD (galactitol-1-phosphate-5-dehydrogenase)-specific primer pair (unique for subsp. gallicida), and primers targeting a 16S rRNA gene region specific for subsp. septica. The subspecies specificity of the PCR was demonstrated by applying the test to a collection of 70 P. multocida isolates, including the Heddleston serovar reference strains; all isolates and strains were assigned correctly. The PCR assay is a sensitive, specific, and highly effective method for the identification of P. multocida subspecies, and an alternative to biochemical test–based differentiation. A possible relationship was noticed between P. multocida subspecies and lipopolysaccharide (LPS) genotype; all but one of the subsp. gallicida strains were isolated only from avian hosts and represented L1 LPS genotype. Subsp. multocida and subsp. septica isolates were classified into 5 and 4 different LPS genotypes, respectively, of which L3 was the only LPS genotype shared between these 2 subspecies.

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Development of a Single-Reaction Multiplex PCR Toxin Typing Assay for Staphylococcus aureusStrains

Applied and Environmental Microbiology ◽

10.1128/aem.66.4.1347-1353.2000 ◽

2000 ◽

Vol 66 (4) ◽

pp. 1347-1353 ◽

Cited By ~ 81

Author(s):

Naresh K. Sharma ◽

Catherine E. D. Rees ◽

Christine E. R. Dodd

Keyword(s):

Multiplex Pcr ◽

Toxin Gene ◽

Gene Products ◽

Pcr Assay ◽

Specific Primers ◽

Purification Method ◽

Multiplex Pcr Assay ◽

Enterotoxin Genes ◽

Immunological Tests ◽

Single Reaction

ABSTRACT We describe here the development of a single-reaction multiplex PCR assay for the enterotoxin genes from Staphylococcus aureusthat utilizes a universal toxin gene primer in combination with toxin-specific primers to amplify characteristic toxin gene products. In combination with a new DNA purification method, the assay can detect enterotoxin genes A to E from a pure culture within 3 to 4 h. The test was used to characterize a diverse set of environmental S. aureus isolates, and a 99% correlation with toxin typing using standard immunological tests was found. The design of the assay allows it to be extended to include both newly characterized and as-yet-unknown toxin genes.

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A multiplex PCR assay for the identification of five species of the Anopheles barbirostris complex in Thailand

Parasites & Vectors ◽

10.1186/s13071-019-3494-8 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 4

Author(s):

Laura Brosseau ◽

Chanya Udom ◽

Chutipong Sukkanon ◽

Theeraphap Chareonviriyaphap ◽

Michael J. Bangs ◽

...

Keyword(s):

Multiplex Pcr ◽

Pcr Assay ◽

Multiplex Pcr Assay ◽

Anopheles Barbirostris

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A Hemi-nested, Allele Specific, Whole Blood PCR Assay for the Detection of the Factor V Leiden Mutation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656129 ◽

1997 ◽

Vol 77 (06) ◽

pp. 1154-1155 ◽

Cited By ~ 4

Author(s):

Gary D Sinclair ◽

Sandra Low ◽

Man-Chiu Poon

Keyword(s):

Whole Blood ◽

Protein C ◽

Activated Protein C ◽

Factor V Leiden ◽

Restriction Enzyme Digestion ◽

Factor V ◽

Pcr Assay ◽

Specific Primers ◽

Factor V Leiden Mutation ◽

Allele Specific

SummaryWe describe a novel hemi-nested, allele specific whole blood PCR assay for detection of the factor V Leiden mutation associated with the plasma defect, activated protein C resistance. This assay utilizes 5 μl of whole blood without prior DNA extraction. The hemi-nested design, employing an outer primer pair in combination with nested, allele specific primers obviates the need for restriction enzyme digestion. PCR reactions are analysed directly on agarose or polyacrylamide minigels. The assay confirmed the genotypes of 50 individuals previously categorized by PCR and Mnll digestion, and has been subsequently utilized in the genotyping of 445 individuals referred for thrombosis studies.

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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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