期刊
LETTERS IN APPLIED MICROBIOLOGY
卷 75, 期 4, 页码 857-868出版社
OXFORD UNIV PRESS
DOI: 10.1111/lam.13758
关键词
diagnosis; GeneXpert; IS6110; M-nested PCR; M-PCR; mpt64; UGTB
资金
- Indian Council of Medical Research, New Delhi [5/8/5/38/2014-ECD-I]
- Council of Scientific and Industrial Research (CSIR), New Delhi [21(1068)/19/EMR-II]
- CSIR, New Delhi
A multiplex-nested PCR (M-nested PCR) targeting mpt64 (Rv1980c) + IS6110 was designed for the detection of Mycobacterium tuberculosis (Mtb) DNA in urine, endometrial biopsies, and menstrual blood samples of UGTB patients. The M-nested PCR showed a lower detection limit and higher sensitivities compared to conventional M-PCR. Incorporation of an internal amplification control helped confirm true PCR-negative results. GeneXpert MTB/RIF assay had lower sensitivity but high specificity compared to M-nested PCR. The M-nested PCR shows promise as a potential diagnostic tool for UGTB.
A multiplex-nested PCR (M-nested PCR) targeting mpt64 (Rv1980c) + IS6110 was designed to detect Mycobacterium tuberculosis (Mtb) DNA within urine (n = 35), endometrial biopsies (n = 22) and menstrual blood (n = 3) of male/female UGTB patients, and results were compared with M-PCR using the same targets. Detection limit of the purified Mtb DNA was found to be 1 fg by M-nested PCR, which was 10(6)-fold lower than M-PCR. Moreover, sensitivities of 100% and 81 center dot 8% were obtained in confirmed (n = 5) and clinically suspected UGTB (n = 55) cases, respectively, by M-nested PCR, with a specificity of 97 center dot 1% (n = 70). Sensitivities attained by M-nested PCR were significantly higher (p < 0 center dot 05) than M-PCR in both clinically suspected and total UGTB (n = 60) cases. To confirm the true PCR-negative results, an internal amplification control, that is, human beta-globin gene (hbb) was incorporated in the M-nested PCR/M-PCR assays, wherein all the clinical specimens (positive/negative for mpt64/IS6110) were found to be positive for hbb. Some UGTB specimens (n = 35) were also subjected to GeneXpert (R) MTB/RIF assay that revealed a significantly lower (p < 0 center dot 001) sensitivity (17 center dot 1 vs 88 center dot 6%) than M-nested PCR, although high specificity (100%) was attained with GeneXpert. After validating the results in a higher number of UGTB specimens, our M-nested PCR may be translated into an attractive diagnostic kit.
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