Standardization of a TaqMan-Based Real-Time PCR for the Detection of Mycobacterium tuberculosis-Complex in Human Sputum

Abstract

Abstract. Real-time polymerase chain reaction (qPCR) was optimized for detecting Mycobacterium tuberculosis in sputum. Sputum was collected from patients (N = 112) with suspected pulmonary tuberculosis, tested by smear microscopy, decontaminated, and split into equal aliquots that were cultured in Lö wenstein-Jensen medium and tested by qPCR for the small mobile genetic element IS6110. The human ERV3 sequence was used as an internal control. 3 of 112 (3%) qPCR failed. For the remaining 109 samples, qPCR diagnosed tuberculosis in 79 of 84 patients with cultureproven tuberculosis, and sensitivity was greater than microscopy (94% versus 76%, respectively, P 0.05). The qPCR sensitivity was similar (P = 0.9) for smear-positive (94%, 60 of 64) and smear-negative (95%, 19 of 20) samples. The qPCR was negative for 24 of 25 of the sputa with negative microscopy and culture (diagnostic specificity 96%). The qPCR had 99.5% sensitivity and specificity for 211 quality control samples including 84 non-tuberculosis mycobacteria. The qPCR cost~5US$ per sample and provided same-day results compared with 2-6 weeks for culture.

Rapid Detection of Mycobacterium tuberculosis Strains Resistant to Isoniazid and/or Rifampicin: Standardization of Multiplex Polymerase Chain Reaction Analysis

“…Second, these assays also gave qPCR products for some of the NTM reference strains; therefore, they could only be applied after M. tuberculosis presence confirmation, for instance with our previous qPCR protocol for TB detection. 25 In summary, the qPCR methods proposed in this study showed high specificity and sensitivity for the targeted mutations, short turn-around time, and relatively low cost that shows its potential for improving TB diagnosis and treatment. Therefore, further evaluation is needed to determine its diagnostic reliability in specimens in operational settings, and potential usefulness for routine clinical practice in settings with qPCR facilities.…”

“…Thus, both multiplex assays must be run after TB confirmation, for example, by the PCR previously standardized by our laboratory. 25 Abnormal or double melting peaks should alert for a possibly unnoticed presence of heteroresistance or mixture with NTM.…”

“…First, the use of a simplified DNA extraction using only heating and ethanol precipitation, 25 potentially facilitating implementation in resource-poor settings and avoiding the high cost of commercial kits for DNA isolation. Second, to avoid sole-source reagents and equipment that can be difficult to import, afford, and sustain in some settings, we used a SYBR Green real-time PCR assay and obtained sensitivity better than the much slower LJ culture.…”

“…Genomic DNA (gDNA) was obtained from freshly grown LJ slants for the WT H37Ra strain by a simple heat inactivation method 25 and the four mutants (katGS315T, rpoBS531L, inhA-15C!T, and rpoBH526Y) by the cetyltrimethylammonium bromide method. 26 DNA concentration was measured with a NanoDrop 2000 UV-Vis -2 × 10 1 genome copies) were prepared in triplicate.…”

“…The use of qPCR provided diagnostic information within 24 hours from the receipt of the sample. However, it can only be applied after TB confirmation with the TaqMan-based real-time PCR previously standardized in our laboratory 25 given the reaction with some NTM. Therefore, excluding the expense of the qPCR thermocycler device self and taking in account an additional qPCR for TB detection, the total reagent costs of our cascade qPCR would be approximately US$ 8.00 (Supplemental Table 4).…”