IS6110-based PCR methods for detection of Mycobacterium tuberculosis

Abstract

IS6110-Based PCR Methods for Detection of Mycobacterium tuberculosis Kent et al. (3) detected Mycobacterium tuberculosis by a nested PCR test that amplified a 181-bp target located in insertion sequence IS6110, but they also encountered many false-positive results when the test was applied to isolates of mycobacteria that do not belong to the M. tuberculosis complex. Because the investigators attributed the false positives to homology between IS6110 and genomic DNA of the other mycobacterial species, they warned that false-positive PCR results could be a problem with some IS6110-based methods for detection of M. tuberculosis complex. Our recent experience in using IS6110-based amplifications for detection of M. tuberculosis complex is informative on this issue. We selected two such amplifications that others had developed, with respective 123-bp (2) and 317-bp (1, 4) targets, and after slightly modifying the procedures incorporated them into a dual-PCR clinical protocol that we applied to mycobacterial colonies grown on Löwenstein-Jensen or Middlebrook 7H11 medium. The modifications that we made included use of dUTP (rather than dTTP) in both reaction mixes and use of a cycling program that was the same for both amplfications (30 cycles; cycle 1: 94ЊC for 5 min, 68ЊC for 45 s, 72ЊC for 90 s; cycles 2 through 30: 94ЊC for 45 s, 68ЊC for 45 s, 72ЊC for 90 s). The two amplifications were performed in separate tubes in a single thermal-cycler run, and the two amplified products were then electrophoresed in separate lanes of the same ethidium bromide-stained 2% agarose gel. In initial validation studies and in subsequent routine clinical examinations, we used this dual-PCR procedure in blindly testing 116 mycobacterial isolates, all of which were also identified to the species (or complex) level by the Mycobacteriology Laboratory of the New Jersey State Department of Health on the basis of their colony characteristics, chemical reactions, patterns shown on highpressure liquid chromatography, and DNA-rRNA hybridizations. The results produced by the dual-PCR procedure and the final identifications by the state laboratory were in total agreement for all 116 isolates: they were positive for M. tuberculosis complex in 46 cases and negative in the remaining 70. Identifications made by the state laboratory on the 70 isolates negative for M. tuberculosis complex were as follows: M. avium-M. intracellulare complex, 50; M. xenopi, 7; M. gordonae, 7; M. fortuitum, 5; and M. kansasii, 1. (In the entire series of 116 cultures, there was a single specimen for which the initial PCR results were discrepant, i.e., the 123-bp target was amplified but the 317-bp target was not. An advantage of the dual-PCR approach was demonstrated in th is case, in that duplicate repeats with the two sets of applicable primer pairs produced amplification of both targets, thus identifying the culture as positive for M. tuberculosis complex. This diagnosis was later confirmed by the state laboratory.) Kent et al.'s call for careful validation of any IS61...

Specificity of IS 6110 -Based DNA Fingerprinting and Diagnostic Techniques for Mycobacterium tuberculosis Complex

“…In a subsequent study by Hellyer et al (9) no DNA amplification of any of the 27 NTM isolates analyzed, 26 of which had been used in the study by Kent et al (13), was observed. Similar results were obtained by Mulcahy et al (15).…”

False positive diagnosis of meningococcal infection by the IS1106 PCR ELISA

“…IS elements are highly mobile and capable of spreading among species and genera [5,7]. IS-based PCR methods in some circumstances have been used serendipitously for the detection of organisms other than for which they were originally described, with varying success.…”

“…The meningococcal IS1106 PCR has proved to be highly sensitive identifying 91 cases following evaluation during the last quarter of 1995 [4]. Unfortunately the suitability of IS elements for the detection of speci¢c pathogens is compromised by their inherent genetic mobility resulting in potential spread to other species and genera [5]. Subsequently during the evaluation period of the IS1106 PCR enzyme-linked immunosorbent assay (ELISA), a number of cases occurred where organisms other than N. meningitidis were cultured from the blood of patients whose clinical samples repeatedly tested positive in the IS1106 PCR ELISA.…”

False positive diagnosis of meningococcal infection by the IS1106PCR ELISA

“…IS elements are highly mobile and capable of spreading among species and genera [5, 7]. IS‐based PCR methods in some circumstances have been used serendipitously for the detection of organisms other than for which they were originally described, with varying success.…”

“…The meningococcal IS 1106 PCR has proved to be highly sensitive identifying 91 cases following evaluation during the last quarter of 1995 [4]. Unfortunately the suitability of IS elements for the detection of specific pathogens is compromised by their inherent genetic mobility resulting in potential spread to other species and genera [5]. Subsequently during the evaluation period of the IS 1106 PCR enzyme‐linked immunosorbent assay (ELISA), a number of cases occurred where organisms other than N. meningitidis were cultured from the blood of patients whose clinical samples repeatedly tested positive in the IS 1106 PCR ELISA.…”