Development and application of a novel multiplex polymerase chain reaction (PCR) assay for rapid detection of various types of Staphylococci strains

Abstract Background: Canine Kobuvirus (CaKoV) and Canine Circovirus (CaCV) are viruses that infect dogs causing diarrheal symptoms that are very similar. However, there is no clinical method to detect a co-infection of these two viruses.Results: In this study, a duplex SYBR Green I-based quantitative real-time polymerase chain reaction (PCR) assay for the rapid and simultaneous detection of CaKoV and CaCV was established. CaKoV and CaCV were distinguished by their different melting temperature which was 86℃ for CaKoV and 78℃ for CaCV. The assay was highly specific, with no cross-reactivity with other common canine viruses and demonstrated high sensitivity. The detection limits of CaKoV and CaCV were 8.924 × 101 copies/μL and 3.841 × 101 copies/μL, respectively. The highest intra- and inter-assay Ct value variation coefficients (CV) of CaKoV were 0.40% and 0.96%, respectively. For CaCV, the highest intra- and inter-assay Ct value variation coefficients were 0.26% and 0.70%, respectively. In 57 clinical samples, positive detection rates of CaKoV and CaCV were 8.77% (7/57) and 15.79% (9/57), respectively. The co-infection rate was 7.02% (4/57). Conclusions: The duplex SYBR Green I-based real-time PCR assay established in this study is a fast, efficient, and sensitive method for the simultaneous detection of the two viruses and provides a powerful tool for the rapid detection of CaKoV and CaCV in clinical practice.

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Application of a Multiplex Polymerase Chain Reaction Assay for the Simultaneous Confirmation of Listeria monocytogenes and Other Listeria Species in Turkey Sample Surveillance†

Journal of Food Protection ◽

10.4315/0362-028x-65.5.780 ◽

2002 ◽

Vol 65 (5) ◽

pp. 780-785 ◽

Cited By ~ 30

Author(s):

IRENE V. WESLEY ◽

KAREN M. HARMON ◽

JAMES S. DICKSON ◽

ANN RAMOS SCHWARTZ

Keyword(s):

Polymerase Chain Reaction ◽

Listeria Monocytogenes ◽

Multiplex Pcr ◽

Multiplex Polymerase Chain Reaction ◽

Rrna Gene ◽

Chain Reaction ◽

Pcr Assay ◽

Listeria Species ◽

Multiplex Pcr Assay ◽

Polymerase Chain

A multiplex polymerase chain reaction was developed to simultaneously identify Listeria monocytogenes and species of the genus Listeria. Two sets of primers were used, with the first amplifying a 938-bp region of the 16S rRNA gene that is highly conserved in all Listeria species and the second amplifying a 174-bp region of the listeriolysin (hlyA) gene of L. monocytogenes. Thus, isolates of Listeria spp. yield a single 938-bp product, whereas L. monocytogenes isolates yield both the 938-bp product and a 174-bp product. The specificity of the assay was verified with all six Listeria species and 11 serotypes of L. monocytogenes, as well as nonrelated bacteria. The multiplex PCR assay was used to determine the incidence of Listeria spp., especially L. monocytogenes, in mechanically separated turkey samples (n = 150 samples). L. monocytogenes strains were selected by using the University of Vermont two-step enrichment protocol and plating to selective Palcam agar. The multiplex PCR assay was used for verification of presumptive Listeria colonies. Approximately 38% of mechanically separated turkey samples (57 of 150) yielded L. monocytogenes; an additional 18% of these samples (27 of 150) harbored other Listeria spp. Fifty-one percent (29 of 57) of the L. monocytogenes isolates were of serogroup 1, 44% (25 of 57) were of serogroup 4, and 2% (1 of 57) were assigned to serogroups other than 1 and 4.

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Development of multiplex polymerase chain reaction assay for rapid detection ofStaphylococcus aureusand selected antibiotic resistance genes in bovine mastitic milk samples

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638711416964 ◽

2011 ◽

Vol 23 (5) ◽

pp. 894-901 ◽

Cited By ~ 20

Author(s):

Jian Gao ◽

Miro Ferreri ◽

Xiu Q. Liu ◽

Li B. Chen ◽

Jing L. Su ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Antibiotic Resistance ◽

Resistance Genes ◽

Rapid Detection ◽

Multiplex Polymerase Chain Reaction ◽

Antibiotic Resistance Genes ◽

Chain Reaction ◽

Milk Samples ◽

Polymerase Chain ◽

Mastitic Milk

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Multiplex polymerase chain reaction as a mastitis screening test for Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis in bulk milk samples

Journal of Dairy Research ◽

10.1017/s0022029903006010 ◽

2003 ◽

Vol 70 (2) ◽

pp. 149-155 ◽

Cited By ~ 33

Author(s):

Patchara Phuektes ◽

Glenn F Browning ◽

Garry Anderson ◽

Peter D Mansell

Keyword(s):

Polymerase Chain Reaction ◽

Multiplex Polymerase Chain Reaction ◽

Streptococcus Dysgalactiae ◽

Chain Reaction ◽

Pcr Assay ◽

Milk Samples ◽

Streptococcus Uberis ◽

Bulk Milk ◽

Polymerase Chain ◽

Total Bacteria

Effective diagnostic tools for screening herds for mastitis pathogens are important in development and monitoring of mastitis control programmes. A multiplex polymerase chain reaction (PCR) assay for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis was used in preliminary studies to assess its applicability as an alternative method for monitoring mastitis caused by these organisms at the herd level. PCR was used to detect the presence of these organisms in bulk milk samples. Correlations with bulk milk somatic cell counts (BMCC), total bacteria counts and thermoduric bacteria counts were evaluated. A total of 176 bulk milk samples were collected from 42 herds on five consecutive occasions at approx. 10-d intervals. Str. uberis was the most common organism in these bulk milk samples. There was no relationship between presence of either Staph. aureus, Str. dysgalactiae or Str. uberis and BMCC, total bacteria counts or thermoduric bacteria counts. However, presence of Str. agalactiae was associated with high BMCC and total bacteria counts. The results of this study show that regular analysis of bulk milk using this multiplex PCR assay may be a useful tool for monitoring herd status with respect to Str. agalactiae, but is of less value for monitoring occurrence of Staph. aureus, Str. dysgalactiae and Str. uberis. Further investigations are needed to clarify the relationship between positive PCR results and the prevalence of infected cows in the herd.

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