Overcoming the errors of in-house PCR used in the clinical laboratory for the diagnosis of extrapulmonary tuberculosis

• Main Authors: Mongkol Kunakorn, Kanchana Raksakait, Roongnapa Pracharktam, Chavachol Sattaudom
• Other Authors: Mahidol University
• Format: Article
• Published: 2018
• Subjects: Medicine
• Online Access: https://repository.li.mahidol.ac.th/handle/123456789/25679

Our experiences from 1993 to 1997 in the development and use of IS6110 base PCR for the diagnosis of extrapulmonary tuberculosis in a routine clinical setting revealed that error-correcting processes can improve existing diagnostic methodology. The reamplification method initially used had a sensitivity of 90.91% and a specificity of 93.75%. The concern was focused on the false positive results of this method caused by product-carryover contamination. This method was changed to single round PCR with carryover prevention by uracil DNA glycosylase (UDG), resulting in a 100% specificity but only 63% sensitivity. Dot blot hybridization was added after the single round PCR, increasing the sensitivity to 87.50%. However, false positivity resulted from the nonspecific dot blot hybridization signal, reducing the specificity to 89.47%. The hybridization of PCR was changed to a Southern blot with a new oligonucleotide probe giving the sensitivity of 85.71% and raising the specificity to 99.52%. We conclude that the PCR protocol for routine clinical use should include UDG for carryover prevention and hybridization with specific probes to optimize diagnostic sensitivity and specificity in extrapulmonary tuberculosis testing.