Yonsei Med J.  2016 Jan;57(1):88-96. 10.3349/ymj.2016.57.1.88.

Comparative Evaluation of Several Gene Targets for Designing a Multiplex-PCR for an Early Diagnosis of Extrapulmonary Tuberculosis

• Raj A
• Singh N
• Gupta KB
• Chaudhary
• Yadav A
• Chaudhary
• Agarwal K
• Varma-Basil M
• Prasad R
• Khuller GK
• Mehta PK

• KMID: 2466356
• DOI: http://doi.org/10.3349/ymj.2016.57.1.88

Abstract

PURPOSE
Diagnosis of extrapulmonary tuberculosis (EPTB) poses serious challenges. A careful selection of appropriate gene targets is essential for designing a multiplex-polymerase chain reaction (M-PCR) assay.
MATERIALS AND METHODS
We compared several gene targets of Mycobacterium tuberculosis, including IS6110, devR, and genes encoding MPB-64 (mpb64), 38kDa (pstS1), 65kDa (hsp65), 30kDa (fbpB), ESAT-6 (esat6), and CFP-10 (cfp10) proteins, using PCR assays on 105 EPTB specimens. From these data, we chose the two best gene targets to design an M-PCR.
RESULTS
Among all gene targets tested, mpb64 showed the highest sensitivity (84% in confirmed cases and 77.5% in clinically suspected cases), followed by IS6110, hsp65, 38kDa, 30kDa, esat6, cfp10, and devR. We used mpb64+IS6110 for designing an M-PCR assay. Our M-PCR assay demonstrated a high sensitivity of 96% in confirmed EPTB cases and 88.75% in clinically suspected EPTB cases with a high specificity of 100%, taking clinical diagnosis as the gold standard.
CONCLUSION
These M-PCR results along with the clinical findings may facilitate an early diagnosis of EPTB patients and clinical management of disease.

Keyword

Mycobacterium tuberculosis; extrapulmonary tuberculosis; PCR; multiplex-PCR; diagnosis

MeSH Terms

Bacteriological Techniques/methods
DNA Transposable Elements/genetics
DNA, Bacterial/analysis/genetics
Early Diagnosis
Female
Gene Amplification
Humans
Male
Multiplex Polymerase Chain Reaction/*methods
Mycobacterium tuberculosis/genetics/*isolation & purification
Polymerase Chain Reaction/*methods/standards
Sensitivity and Specificity
Tuberculosis/*diagnosis
DNA Transposable Elements
DNA, Bacterial

Figure

• 

  Fig. 1 PCR gel picture of several gene targets tested on the same clinical EPTB specimens. L1, L18, L19, and L36 represent 100 bp molecular marker; L2, L6, L10, L14, L20, L24, L28, and L32 were positive controls with the purified M. tuberculosis H37RvDNA; L3, L7, L11, L15, L21, L25, L29, and L33 were negative controls without template DNA; L4, L5, L8, L9, L12, L13, L16, L17, L22, L23, L26, L27, L30, L31, L34, and L35 represent clinical EPTB specimens. EPTB, extrapulmonary tuberculosis.

• 

  Fig. 2 M-PCR: amplification of 163 bp region of mpb64 gene and 258 bp region of IS6110 of M. tuberculosis H37RvDNA in the same tube with different ratios of primers. L1 represents 100 bp molecular marker; L2, L3, L4, and L5 represent mpb64 and IS6110 primer concentrations (µM) in ratios of 0.2:0.2, 0.2:0.4, 0.4:0.4, and 0.4:0.2 with M. tuberculosis H37Rv DNA; L6, negative control (no template DNA). M-PCR, multiplex-polymerase chain reaction.

• 

  Fig. 3 M-PCR: amplification of 163 bp region of mpb64 gene and 258 bp region of IS6110 in the same tube; L1 and L11 represent 100 bp molecular marker; L2, positive control (M. tuberculosis H37Rv DNA); L3, negative control (no template DNA); L4, negative control (only PCR grade water); L5-8, representative positive clinical EPTB samples; L9-10, representative negative EPTB samples. M-PCR, multiplex-polymerase chain reaction; EPTB, extrapulmonary tuberculosis.

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