title: Overcoming the errors of in-house PCR used in the clinical laboratory for the diagnosis of extrapulmonary tuberculosis.
abstract: 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.
authors.affiliation: Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand. ramkn@mahidol.ac.th
authors.forename: M
authors.initials: M
authors.lastname: Kunakorn
authors.forename: K
authors.initials: K
authors.lastname: Raksakai
authors.forename: R
authors.initials: R
authors.lastname: Pracharktam
authors.forename: C
authors.initials: C
authors.lastname: Sattaudom
mesh_terms: D015982:Bias; D015139:Blotting, Southern / Q000379:methods* / Q000592:standards*; D001826:Body Fluids / Q000382:microbiology; D019411:Clinical Laboratory Techniques / Q000592:standards*; D002985:Clinical Protocols; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D005189:False Positive Reactions; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D011785:Quality Assurance, Health Care; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D004269:DNA, Bacterial