scholarly journals Evaluation of an internal control in a multiplex-PCR assay for sex determination of <i>in vitro</i>-produced bovine embryos

2011 ◽  
Vol 02 (06) ◽  
pp. 456-462
Author(s):  
Fabiana de Almeida Rufino ◽  
Marcelo Marcondes Seneda ◽  
Alice Fernandes Alfieri ◽  
Katia Cristina Silva-Santos ◽  
Karina Cristina Puggesi Rubin ◽  
...  
Keyword(s):  
Sex Determination ◽  
Multiplex Pcr ◽  
Internal Control ◽  
Bovine Embryos ◽  
Pcr Assay ◽  
Multiplex Pcr Assay

Evaluation of a Multiplex PCR Assay for the Identification of Salmonella Serovars Enteritidis and Typhimurium Using Retail and Abattoir Samples

2017 ◽  
Vol 80 (2) ◽  
pp. 295-301 ◽  
Author(s):  
Dele Ogunremi ◽  
Susan Nadin-Davis ◽  
Andrée Ann Dupras ◽  
Imelda Gálvan Márquez ◽  
Katayoun Omidi ◽  
...  
Keyword(s):  
Salmonella Typhimurium ◽  
Multiplex Pcr ◽  
Internal Control ◽  
Broiler Chickens ◽  
Salmonella Enteritidis ◽  
Bacterial Species ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Retail Outlets ◽  
Dna Fragment

ABSTRACT A multiplex PCR was developed to identify the two most common serovars of Salmonella causing foodborne illness in Canada, namely, serovars Enteritidis and Typhimurium. The PCR was designed to amplify DNA fragments from four Salmonella genes, namely, invA gene (211-bp fragment), iroB gene (309-bp fragment), Typhimurium STM 4497 (523-bp fragment), and Enteritidis SE147228 (612-bp fragment). In addition, a 1,026-bp ribosomal DNA (rDNA) fragment universally present in bacterial species was included in the assay as an internal control fragment. The detection rate of the PCR was 100% among Salmonella Enteritidis (n = 92) and Salmonella Typhimurium (n = 33) isolates. All tested Salmonella isolates (n = 194) were successfully identified based on the amplification of at least one Salmonella-specific DNA fragment. None of the four Salmonella DNA amplicons were detected in any of the non-Salmonella isolates (n = 126), indicating an exclusivity rate of 100%. When applied to crude extracts of 2,001 field isolates of Salmonella obtained during the course of a national microbiological baseline study in broiler chickens and chicken products sampled from abattoir and retail outlets, 163 isolates, or 8.1%, tested positive for Salmonella Enteritidis and another 80 isolates, or 4.0%, tested as Salmonella Typhimurium. All isolates identified by serological testing as Salmonella Enteritidis in the microbiological study were also identified by using the multiplex PCR. The new test can be used to identify or confirm pure isolates of the two serovars and is also amenable for integration into existing culture procedures for accurate detection of Salmonella colonies.

scholarly journals Evaluation of Semiautomated Multiplex PCR Assay for Determination of Streptococcus pneumoniae Serotypes and Serogroups

2003 ◽  
Vol 41 (2) ◽  
pp. 601-607 ◽  
Author(s):  
E. R. Lawrence ◽  
D. B. Griffiths ◽  
S. A. Martin ◽  
R. C. George ◽  
L. M. C. Hall
Keyword(s):  
Streptococcus Pneumoniae ◽  
Multiplex Pcr ◽  
Pcr Assay ◽  
Multiplex Pcr Assay

scholarly journals Comparative Genomics to Develop a Specific Multiplex PCR Assay for Detection of Clavibacter michiganensis

2020 ◽  
Vol 110 (3) ◽  
pp. 556-566
Author(s):  
Shree P. Thapa ◽  
Michael O’Leary ◽  
Marie-Agnès Jacques ◽  
Robert L. Gilbertson ◽  
Gitta Coaker
Keyword(s):  
Comparative Genomics ◽  
Multiplex Pcr ◽  
Internal Control ◽  
Management Strategies ◽  
Detection Methods ◽  
Clavibacter Michiganensis ◽  
Pcr Assay ◽  
Tomato Seed ◽  
Plant Materials ◽  
Multiplex Pcr Assay

Clavibacter michiganensis is a Gram-positive bacterial pathogen that proliferates in the xylem vessels of tomato, causing bacterial wilt and canker symptoms. Accurate detection is a crucial step in confirming outbreaks of bacterial canker and developing management strategies. A major problem with existing detection methods are false-positive and -negative results. Here, we report the use of comparative genomics of 37 diverse Clavibacter strains, including 21 strains sequenced in this study, to identify specific sequences that are C. michiganensis detection targets. Genome-wide phylogenic analyses revealed additional diversity within the genus Clavibacter. Pathogenic C. michiganensis strains varied in plasmid composition, highlighting the need for detection methods based on chromosomal targets. We utilized sequences of C. michiganensis-specific loci to develop a multiplex PCR-based diagnostic platform using two C. michiganensis chromosomal genes (rhuM and tomA) and an internal control amplifying both bacterial and plant DNA (16s ribosomal RNA). The multiplex PCR assay specifically detected C. michiganensis strains from a panel of 110 additional bacteria, including other Clavibacter spp. and bacterial pathogens of tomato. The assay was adapted to detect the presence of C. michiganensis in seed and tomato plant materials with high sensitivity and specificity. In conclusion, the described method represents a robust, specific tool for detection of C. michiganensis in tomato seed and infected plants.

Design of a multiplex PCR assay for the simultaneous detection and confirmation of Neisseria gonorrhoeae

2010 ◽  
Vol 63 (5) ◽  
pp. 431-433 ◽  
Author(s):  
Isabelle O'Callaghan ◽  
Daniel Corcoran ◽  
Brigid Lucey
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Multiplex Pcr ◽  
Internal Control ◽  
Limit Of Detection ◽  
Simultaneous Detection ◽  
Pcr Primers ◽  
Real Potential ◽  
Pcr Assay ◽  
Neisseria Species ◽  
Multiplex Pcr Assay

ObjectivesTo improve the detection of Neisseria gonorrhoeae by designing a multiplex PCR assay using two N gonorrhoeae-specific genes as targets, thereby providing detection and confirmation of a positive result simultaneously.MethodsPCR primers were designed to detect two N gonorrhoeae genes, namely porA and pgi1. Primers for an internal control targeting the ompW gene of Vibrio cholerae were also designed and incorporated in the assay. The DNA of 45 clinical isolates including 33 N gonorrhoeae isolates, seven non-gonococcal Neisseria species, and five non-Neisseria species was tested using the multiplex PCR assay.ResultsAll 33 N gonorrhoeae isolates were successfully detected by the assay and none of the non-gonococcal isolates was detected. The assay showed a sensitivity and specificity of 100%, and a limit of detection of 1.25 ng of DNA.ConclusionThis multiplex PCR assay offers a sensitive and specific assay suitable for the detection of N gonorrhoeae, and offers real potential for diagnostic use.

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

2016 ◽  
Vol 1 (2) ◽  
pp. 38-42 ◽  
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

scholarly journals Sex Determination of Bovine Embryos Using H-Y Antibodies

1989 ◽  
Vol 30 (2) ◽  
pp. 155-164 ◽  
Author(s):  
Birthe Avery ◽  
Mette Schmidt
Keyword(s):  
Sex Determination ◽  
Bovine Embryos

scholarly journals Sex Determination of Bovine Embryos Based on Embryonic Cleavage Rates

1989 ◽  
Vol 30 (2) ◽  
pp. 147-153
Author(s):  
Birthe Avery ◽  
Mette Schmidt ◽  
Torben Greve
Keyword(s):  
Sex Determination ◽  
Bovine Embryos ◽  
Embryonic Cleavage
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