The relative test performance characteristics of two commercial assays for the detection of Mycobacterium tuberculosis complex in paraffin-fixed human biopsy specimen

Since December of 2000, the Central Public Health Laboratory (CPHL) of the Ontario Public Health Laboratories, Ministry of Health and Long-Term Care has utilized an inhouse nucleic acid amplification testing followed by Sanger sequencing for the detection of Mycobacterium tuberculosis complex (MTBC) in formalin-fixed paraffinimbedded tissue specimens. Although this methodology has allowed for the characterization of approximately 250 tissue specimens, it is labor intensive and requires the use of an expensive sequencing protocol to ensure assay specificity. During this time, several commercial kits and other published methodologies have become available which allow for the molecular diagnosis of MTBC in biopsy specimens and utilize specific mechanisms to ensure assay specificity. The targets of these assays include IS6110 [1] and MPB64 [2] using either traditional endpoint PCR or real-time PCR. Traditional endpoint PCR is often thought to lack the analytical sensitivity and specificity when compared to realtime PCR technology [3,4]. Although real-time PCR may offer increased sensitivity and specificity, several articles have described failures of real-time PCR assays to detect MTBC due to slight changes in primer and probe sequences [5]. When using end-point PCR reactions, specificity can be increased through the use of sequencing technology or restriction enzyme analysis of PCR prodPublished: 8 September 2008 Diagnostic Pathology 2008, 3:37 doi:10.1186/ Received: 18 April 2008 Accepted: 8 September 2008 This article is available from: © 2008 Drews et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Diagnostic Pathology 2008, 3:37 Page 2 of 4 (page number not for citation purposes) ucts. Dual-priming oligonucleotide technology (DPO) has also been recently described in the literature as a method that increases both the sensitivity and specificity of endpoint PCR reactions [6]. As a result, this methodology provides a means of confirming sequences without the requiring real-time PCR or extensive processing of PCR products for use in sequencing reactions or restriction enzyme analysis [6]. Recently, a commercial DPObased assay (Seegene, Rockville, MD) utilizing nested PCR for MTBC specific target genes IS6110 and MPB64, became available. This manuscript describes the verification of the Seeplex™ TB Detection-2 assay (Seegene, Rockville, MD) and the artus® M. tuberculosis TM assay (Qiagen, Mississauga, ON, Canada) on formalin-fixed paraffin-embedded tissue specimens that had been previously tested using the inhouse assay, and an analysis of the turn-around-time (TAT) and workflow required for performance of the assays [1]. Purified M. tuberculosis H37Rv DNA was used as standard for analysis of limit of detection for multiple methods. Theoretical limits of detection for each methodology were determined using serial 10-fold dilutions of M. tuberculosis H37Rv DNA in PCR grade water. The theoretical number of copies of M. tuberculosis H37Rv DNA was calculated according to a generally accepted conversion formula. Formalin-fixed paraffin-embedded tissue sample blocks are submitted to the CPHL with a copy of the corresponding histopathology report. Depending of the size of the tissue block, either the entire tissue, or portions of the tissue block (determined histopathologically) were de-paraffinized and DNA extraction prepared using QiaAMP spin column (QIAGEN, Mississauga, Ontario) protocol [7]. DNA was frozen at -80°C until further use in PCR protocols. The in-house reference method was a modification of a previously published method [1] and has been used as a diagnostic tool at the authors' facility since December 2000. Briefly, 5 μl of extracted DNA were used in a nonnested PCR reaction utilizing the DNA minikit (QIAGEN, Mississauga, Ontario) procedure, 0.3 μM primer IS-1 (5'- CCT GCG AGC GTA GGC GTC GG-3') and 0.3 μM IS-2 (5'-CTC GTC CAG CGC CGC TTC GG-3') using the PCR4 reaction (one 4 minute denaturation step at 95°; 45 cycles of 30 seconds at 95°C, 30 seconds at 63°C, 30 seconds at 72°C; and a 7 minute extension at 72°C) [1]. In addition, amplicons of the appropriate molecular mass were identified by EtBr staining of a 1% agarose gel electrophoresis and due to the presence of some non-specific banding, amplicons of the appropriate size were confirmed by Sanger sequence analysis using the ABI BigDye® Terminator v3.1 Cycle Sequencing Kit on an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA). IS6110 sequences were compared to accessioned sequences using the Basic Local Alignment Search Tool (BLAST) and the NCBI database B. Sequences were considered to be identified as IS6110 if they shared the highest identity