Development of a Multiplex Real-Time PCR Assay for Mycobacterium bovis BCG and Validation in a Clinical Laboratory

Vaccination against tuberculosis with bacillus Calmette-Guérin (BCG) can lead to adverse events, including a rare but life-threatening complication of disseminated BCG. This complication often occurs in young children with immunodeficiencies and is associated with an ∼60% mortality rate.

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Direct identification of mycobacteria from culture media using a multiplex real-time PCR assay: report on its application in a clinical laboratory in a region of high tuberculosis endemicity

Diagnostic Microbiology and Infectious Disease ◽

10.1016/j.diagmicrobio.2014.01.009 ◽

2014 ◽

Vol 79 (1) ◽

pp. 49-53 ◽

Cited By ~ 3

Author(s):

Choong-Hwan Cha ◽

Hae-Kyong An ◽

Jeong-Uk Kim

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Clinical Laboratory ◽

Culture Media ◽

Pcr Assay ◽

Direct Identification

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Development and Validation of a Clinical Laboratory Improvement Amendments-Compliant Multiplex Real-Time PCR Assay for Detection of mcr Genes

Microbial Drug Resistance ◽

10.1089/mdr.2018.0417 ◽

2019 ◽

Vol 25 (7) ◽

pp. 991-996 ◽

Cited By ~ 3

Author(s):

Jonathan B. Daniels ◽

Davina Campbell ◽

Sandra Boyd ◽

Uzma Ansari ◽

Joseph Lutgring ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Clinical Laboratory ◽

Pcr Assay ◽

Development And Validation ◽

Clinical Laboratory Improvement Amendments

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Development of a multiplex real-time PCR assay for BCG and validation in a clinical laboratory

10.1101/2021.06.04.21258161 ◽

2021 ◽

Author(s):

Shannon Catherine Duffy ◽

Manigandan Venkatesan ◽

Shubhada Chothe ◽

Indira Poojary ◽

Valsan Philip Verghese ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Clinical Laboratory ◽

Limit Of Detection ◽

Infected Tissue ◽

Nucleotide Polymorphisms ◽

Pcr Assay ◽

Live Attenuated Vaccine ◽

Pure Cultures ◽

Improve Patient Management

Mycobacterium bovis bacille Calmette-Guérin (BCG) is a live attenuated vaccine which can result in local or disseminated infection, most commonly in immunocompromised individuals. Differentiation of BCG from other members of the Mycobacterium tuberculosis complex (MTBC) is required to diagnose BCG disease, which requires specific management. Current methods for BCG diagnosis are based on mycobacterial culture and conventional PCR; the former is time-consuming and the latter often unavailable. Further, there are reports that certain BCG strains may be associated with a higher rate of adverse events. This study describes the development of a two-step multiplex real-time PCR assay which uses single nucleotide polymorphisms to detect BCG and identify early or late BCG strains. The assay has a limit of detection of 1 pg BCG boiled lysate DNA and was shown to detect BCG in both pure cultures and experimentally infected tissue. Performance was assessed on 19 suspected BCG clinical isolates at Christian Medical College in Vellore, India taken from January 2018 to August 2020. Of these 19 isolates, 10 were identified as BCG (6 early and 4 late strains) and 9 were identified as other MTBC members. Taken together, the results demonstrate the ability of this assay to identify and characterize BCG disease from cultures and infected tissue. The capacity to identify BCG may improve patient management and the ability to discriminate between BCG strains may enable BCG vaccine pharmacovigilance.

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Quadruplex Real-Time PCR Assay for Detection and Identification of Vibrio cholerae O1 and O139 Strains and Determination of Their Toxigenic Potential

Applied and Environmental Microbiology ◽

10.1128/aem.00517-09 ◽

2009 ◽

Vol 75 (22) ◽

pp. 6981-6985 ◽

Cited By ~ 26

Author(s):

Jianwei Huang ◽

Yumei Zhu ◽

Huixin Wen ◽

Jiafeng Zhang ◽

Shijie Huang ◽

...

Keyword(s):

Vibrio Cholerae ◽

Real Time ◽

Real Time Pcr ◽

Culture Method ◽

Toxin Gene ◽

Analytical Sensitivity ◽

Pcr Assay ◽

Detection And Identification ◽

Life Threatening ◽

Toxigenic Strains

ABSTRACT Vibrio cholerae is a natural inhabitant of the aquatic environment. However, its toxigenic strains can cause potentially life-threatening diarrhea. A quadruplex real-time PCR assay targeting four genes, the cholera toxin gene (ctxA), the hemolysin gene (hlyA), O1-specific rfb, and O139-specific rfb, was developed for detection and differentiation of O1, O139, and non-O1, non-O139 strains and for prediction of their toxigenic potential. The specificity of the assay was 100% when tested against 70 strains of V. cholerae and 31 strains of non-V. cholerae organisms. The analytical sensitivity for detection of toxigenic V. cholerae O1 and O139 was 2 CFU per reaction with cells from pure culture. When the assay was tested with inoculated water from bullfrog feeding ponds, 10 CFU/ml could reliably be detected after culture for 3 h. The assay was more sensitive than the immunochromatographic assay and culture method when tested against 89 bullfrog samples and 68 water samples from bullfrog feeding ponds. The applicability of this assay was confirmed in a case study involving 15 bullfrog samples, from which two mixtures of nontoxigenic O1 and toxigenic non-O1/non-O139 strains were detected and differentiated. These data indicate that the quadruplex real-time PCR assay can both rapidly and accurately detect/identify V. cholerae and reliably predict the toxigenic potential of strains detected.

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Duplex real-time PCR assay targeting insertion elements IS1081 and IS6110 for detection of Mycobacterium bovis in lymph nodes of cattle

Biotechnology in Animal Husbandry ◽

10.2298/bah1401045s ◽

2014 ◽

Vol 30 (1) ◽

pp. 45-59 ◽

Cited By ~ 1

Author(s):

A. Selim ◽

M. El-Haig ◽

W. Gaede

Keyword(s):

Lymph Node ◽

Lymph Nodes ◽

Real Time ◽

Mycobacterium Bovis ◽

Real Time Pcr ◽

Rapid Screening ◽

Microscopical Examination ◽

Pcr Assay ◽

Specificity And Sensitivity ◽

Insertion Elements

The development of a reliable and rapid screening test for detection of Mycobacterium bovis (M. bovis) helps in control of bovine tuberculosis. The aim of this study was evaluate a sensitive and specific assay for detecting M. bovis DNA in lymph nodes with lesions suggestive to tuberculosis taken from slaughtered cattle. A duplex real-time PCR assay was developed for the identification of M. bovis targeting insertion elements (IS) IS1081 and IS6110 in one internally controlled reaction. M. bovis DNA extraction protocols from tissue samples were evaluated. The specificity and sensitivity of the assay were evaluated for detecting serial dilutions of reference Mycobacteria strains as well as from spiked lymph node homogenate. Results revealed that microscopical examination of 600 lymph nodes with tuberculous-like lesion for detection of Acid-fast bacilli (AFB) showed a detection rate of 96.6%, compared to 98% M. bovis with duplex real-time PCR. The reproducible detection limit of the IS1081-PCR was 10 M. bovis cells/ml and the IS6110-PCR was 100 M. bovis cells/ml. Besides, both primer set of PCR protocol could detect 20 M. bovis cells/ml in spiked lymph node tissue. The assay was evaluated on 19 bacterial strains and was determined to be 100% specific for M. bovis. We suggest that the IS1081-PCR is a good candidate assay for routine screening of cattle lymph nodes and other tissue for M. bovis infection.

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