Dataset Information

Development of a high sensitivity TaqMan-based PCR assay for the specific detection of Mycobacterium tuberculosis complex in both pulmonary and extrapulmonary specimens.

ABSTRACT: Tuberculosis (TB) causes a heavy health burden worldwide, especially in developing countries. The need for the rapid and accurate diagnosis of TB has not been satisfied, especially for extra-pulmonary specimens or specimens with acid fast stain (AFS)-negative condition. Development and validation of a novel, sensitive and specific assay for diagnosing TB is essential. We developed IS4 primer/probe based on insertion sequence 6110 (IS6110). A qPCR assay was designed for detecting a specific region in IS6110 by BLAST. The IS4 primer/probe concentration, qPCR efficiency and various of PCR additives were evaluated and optimized. Thirty-four species of commonly isolated microorganisms were used for evaluating the analytical specificity. Moreover, 130 clinical specimens were collected for evaluating the performance versus Cobas TaqMan MTB (CTM) assay kit and culture. The amplification efficiencies of IS4 were 99.61% and 102.61% without and with internal control DNA (Bacteriophage Lambda), respectively. Dimethyl sulfoxide outperformed glycerol or BSA for eliciting the most effective amplification and the lowest limit of detection. In evaluating the clinical performance, various specimen types were collected. IS4 demonstrated a high degree of agreement (kappa?=?0.71) with CTM. The clinical sensitivity and specificity of IS4 and CTM were 92.11% (35/38), 82.61% (76/92), 84.21% (32/38) and 95.65% (88/92), respectively. The clinical sensitivity and specificity of IS4 were similar for both pulmonary [92.00% (23/25) and 76.92% (30/39), respectively] and extrapulmonary [92.31% (12/13) and 86.79% (46/53), respectively] specimens. Among AFS-negative cases, the clinical sensitivity and specificity remained 90.48% (19/21) and 83.91% (73/87), respectively, with culture as the gold standard. We concluded that IS4, a new primer/probe pair for TaqMan based qPCR assay, was developed, optimized, and validated for the sensitive and specific detection of TB among various specimen types. The performance was not compromised under AFS-negative conditions.

SUBMITTER: Wang HY

PROVIDER: S-EPMC6331544 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Development of a high sensitivity TaqMan-based PCR assay for the specific detection of Mycobacterium tuberculosis complex in both pulmonary and extrapulmonary specimens.

Wang Hsin-Yao HY Lu Jang-Jih JJ Chang Ching-Yu CY Chou Wen-Pin WP Hsieh Jason Chia-Hsun JC Lin Chien-Ru CR Wu Min-Hsien MH

Scientific reports 20190114 1

Tuberculosis (TB) causes a heavy health burden worldwide, especially in developing countries. The need for the rapid and accurate diagnosis of TB has not been satisfied, especially for extra-pulmonary specimens or specimens with acid fast stain (AFS)-negative condition. Development and validation of a novel, sensitive and specific assay for diagnosing TB is essential. We developed IS4 primer/probe based on insertion sequence 6110 (IS6110). A qPCR assay was designed for detecting a specific regio ...[more]

PMID: 30643154

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Project description:BACKGROUND:Although leprosy is efficiently treated by multidrug therapy, resistance to first-line (dapsone, rifampin) and second-line (fluoroquinolones) drugs has been described worldwide. However, the characteristics of drug resistance in Southwest China remain unknown. Furthermore, the sensitivity of polymerase chain reaction (PCR)/sequencing for resistance detection is limited, especially for paucibacillary (PB) leprosy patients. The current study aimed to develop a nested PCR/sequencing and TaqMan SNP Genotyping Assay to increase the sensitivity of the method used to detect drug resistance in Mycobacterium leprae and to reveal the nature of M. leprae drug resistance in Southwest China. METHODOLOGY/PRINCIPAL FINDINGS:Seventy-six specimens, including skin biopsy (n = 64), formalin-fixed paraffin-embedded (FFPE) (n = 11) and skin-slit smear (SSS) (n = 1) samples from multibacillary (MB, n = 70) and PB (n = 6) leprosy patients from Southwest China, were included in this study. The presence of mutations in drug resistance-determining regions (DRDRs) of the rpoB, folP1, and gyrA genes, which are associated with rifampicin, dapsone, and quinolone resistance, respectively, was detected by PCR/sequencing, as recommended by the WHO, and the nested PCR and TaqMan SNP Genotyping Assay developed in this study. Mutations in the folP gene were detected in 19 (25.00%) samples, indicating dapsone-resistant M. leprae, with one (1.31%) sample showing mutations in two genes, folP and gyrA, reflecting multidrug-resistant strains to dapsone and ofloxacin. However, no rpoB mutation was detected. Compared with PCR/sequencing, nested PCR increased the sensitivity of detecting rpoB (from 51.39% to 78.94% for leprosy patients and from 0.00% to 50.00% for PB), gyrA (from 75.00% to 80.26% for leprosy patients and from 50.00% to 66.67% for PB), and folP1 (from 5.26% to 84.21% for leprosy patients and from 0.00% to 66.67% for PB). Moreover, the TaqMan SNP Genotyping Assay showed greater sensitivity for folP1 detection (from 5.26% to 78.94-86.84% for leprosy patients and from 0.00% to 33.33%-83.33% for PB patients) than the PCR/sequencing method. In addition, the latter method was able to more easily distinguish heterozygous genotypes and mutant homozygous genotypes from homozygous genotypes. CONCLUSIONS/SIGNIFICANCE:Nested PCR/sequencing and the TaqMan SNP Genotyping Assay are rapid and highly sensitive methods for detecting drug resistance in leprosy cases. The current study revealed that diamino-diphenylsulfone (DDS; also known as dapsone) resistance in M. leprae, as indicated by folP1 gene detection, is still the most concerning form of drug resistance in leprosy patients from Southwest China.

Dataset Information

Development of a high sensitivity TaqMan-based PCR assay for the specific detection of Mycobacterium tuberculosis complex in both pulmonary and extrapulmonary specimens.

Publications

Development of a high sensitivity TaqMan-based PCR assay for the specific detection of Mycobacterium tuberculosis complex in both pulmonary and extrapulmonary specimens.

Similar Datasets

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