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An in-house multiplex PCR test for the detection of Mycobacterium tuberculosis, its validation & comparison with a single target TB-PCR kit.

Kulkarni S, Singh P, Memon A, Nataraj G, Kanade S, Kelkar R, Rajan MG - Indian J. Med. Res. (2012)

Bottom Line: The multiplex test was validated using sputum samples from pulmonary TB (PTB) cases.Analytical sensitivity of multiplex PCR was found to be 100 fg for 38 kDa gene and 1 fg for IS6110.Four samples showed positive PCR results only with primers for 38 kDa gene.

View Article: PubMed Central - PubMed

Affiliation: Radiation Medicine Centre, Bhaba Atomic Research Centre, Mumbai, India. savitapk@hotmail.com

ABSTRACT

Background & objectives: The conventional techniques used in TB diagnosis like AFB (acid fast bacilli) smear microscopy lack sensitivity and the gold standard, culture test takes time. A test based on multiplex polymerase chain reaction (PCR) targeting the 38 kDa gene and IS6110 insertion sequence, specific to Mycobacterium tuberculosis was developed to further increase the sensitivity of a TB-PCR kit targeting only 38 kDa gene developed earlier in the same laboratory. The multiplex test was validated using sputum samples from pulmonary TB (PTB) cases. The sensitivity and specificity were compared with AFB smear examination and Lowenstein-Jensen (LJ) culture test.

Methods: Multiplex PCR amplifying 340 and 245 bp sequence of 38 kDa gene and IS6110, respectively was standardized and analytical sensitivity was verified. Sputum samples (n=120) obtained from PTB cases were subjected to AFB smear examination, LJ culture and a multiplex as well as single target PCR test. Additionally, 72 non-TB respiratory samples were included in the study as negative controls.

Results: Analytical sensitivity of multiplex PCR was found to be 100 fg for 38 kDa gene and 1 fg for IS6110. Multiplex PCR, using both the targets, showed highest sensitivity of 81.7 per cent, followed by 69.2 per cent for L-J culture test and 53.3 per cent for AFB smear when clinical diagnosis was considered as a gold standard. The sensitivity of detection of M. tuberculosis in AFB smear positive and negative samples by multiplex PCR was 93.7 and 67.9 per cent, respectively. Sensitivity of 77.1 per cent observed for the detection of M. tuberculosis with single target PCR increased to 89.2 per cent with multiplex PCR in culture positive samples. Four samples showed positive PCR results only with primers for 38 kDa gene.

Interpretation & conclusions: Multiplex PCR increased the sensitivity of single target PCR and will be useful in diagnosing paucibacillary smear negative samples. Further, it can also be used to detect samples with M. tuberculosis strains lacking IS6110.

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Related in: MedlinePlus

PCR amplification of 340 bp region in 38 kDa gene of M. tuberculosis using KD1 and KD2 primers exhibiting the analytical sensitivity of PCR. Lane M- DNA ladder, Lanes 1 to 6 - 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg of MTB DNA, respectively, Lane 7- Neg control.
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Figure 1: PCR amplification of 340 bp region in 38 kDa gene of M. tuberculosis using KD1 and KD2 primers exhibiting the analytical sensitivity of PCR. Lane M- DNA ladder, Lanes 1 to 6 - 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg of MTB DNA, respectively, Lane 7- Neg control.

Mentions: Analytical sensitivity of single target PCR test: The detection limit for 38 kDa PCR was found to be 100 fg (Fig. 1) whereas IS6110 PCR showed amplification with even 1 fg of template DNA (Fig. 2), which is theoretically equivalent to 30 and 0.3 organisms, receptively.


An in-house multiplex PCR test for the detection of Mycobacterium tuberculosis, its validation & comparison with a single target TB-PCR kit.

Kulkarni S, Singh P, Memon A, Nataraj G, Kanade S, Kelkar R, Rajan MG - Indian J. Med. Res. (2012)

PCR amplification of 340 bp region in 38 kDa gene of M. tuberculosis using KD1 and KD2 primers exhibiting the analytical sensitivity of PCR. Lane M- DNA ladder, Lanes 1 to 6 - 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg of MTB DNA, respectively, Lane 7- Neg control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3401715&req=5

Figure 1: PCR amplification of 340 bp region in 38 kDa gene of M. tuberculosis using KD1 and KD2 primers exhibiting the analytical sensitivity of PCR. Lane M- DNA ladder, Lanes 1 to 6 - 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg of MTB DNA, respectively, Lane 7- Neg control.
Mentions: Analytical sensitivity of single target PCR test: The detection limit for 38 kDa PCR was found to be 100 fg (Fig. 1) whereas IS6110 PCR showed amplification with even 1 fg of template DNA (Fig. 2), which is theoretically equivalent to 30 and 0.3 organisms, receptively.

Bottom Line: The multiplex test was validated using sputum samples from pulmonary TB (PTB) cases.Analytical sensitivity of multiplex PCR was found to be 100 fg for 38 kDa gene and 1 fg for IS6110.Four samples showed positive PCR results only with primers for 38 kDa gene.

View Article: PubMed Central - PubMed

Affiliation: Radiation Medicine Centre, Bhaba Atomic Research Centre, Mumbai, India. savitapk@hotmail.com

ABSTRACT

Background & objectives: The conventional techniques used in TB diagnosis like AFB (acid fast bacilli) smear microscopy lack sensitivity and the gold standard, culture test takes time. A test based on multiplex polymerase chain reaction (PCR) targeting the 38 kDa gene and IS6110 insertion sequence, specific to Mycobacterium tuberculosis was developed to further increase the sensitivity of a TB-PCR kit targeting only 38 kDa gene developed earlier in the same laboratory. The multiplex test was validated using sputum samples from pulmonary TB (PTB) cases. The sensitivity and specificity were compared with AFB smear examination and Lowenstein-Jensen (LJ) culture test.

Methods: Multiplex PCR amplifying 340 and 245 bp sequence of 38 kDa gene and IS6110, respectively was standardized and analytical sensitivity was verified. Sputum samples (n=120) obtained from PTB cases were subjected to AFB smear examination, LJ culture and a multiplex as well as single target PCR test. Additionally, 72 non-TB respiratory samples were included in the study as negative controls.

Results: Analytical sensitivity of multiplex PCR was found to be 100 fg for 38 kDa gene and 1 fg for IS6110. Multiplex PCR, using both the targets, showed highest sensitivity of 81.7 per cent, followed by 69.2 per cent for L-J culture test and 53.3 per cent for AFB smear when clinical diagnosis was considered as a gold standard. The sensitivity of detection of M. tuberculosis in AFB smear positive and negative samples by multiplex PCR was 93.7 and 67.9 per cent, respectively. Sensitivity of 77.1 per cent observed for the detection of M. tuberculosis with single target PCR increased to 89.2 per cent with multiplex PCR in culture positive samples. Four samples showed positive PCR results only with primers for 38 kDa gene.

Interpretation & conclusions: Multiplex PCR increased the sensitivity of single target PCR and will be useful in diagnosing paucibacillary smear negative samples. Further, it can also be used to detect samples with M. tuberculosis strains lacking IS6110.

Show MeSH
Related in: MedlinePlus