Efficient differentiation of Mycobacterium abscessus complex isolates to the species level by a novel PCR-based variable-number tandem-repeat assay

Abstract

A novel duplex PCR method based on variable-number tandem-repeat targets to discriminate among Mycobacterium abscessus complex isolates was developed and evaluated in 85 clinical isolates. The assay accuracy was confirmed by a multiple-target sequence analysis. The duplex PCR assay is a one-step, reliable, and accurate assay for discriminating M. abscessus species.

Fig. 1.

Amplified products obtained by PCR using the VNTR11 and VNTR23 primer sets. (A) VNTR11 consists of one to four copy numbers (179 to 278 bp) with a 33-bp tandem repeat. (B) VNTR23 consists of one to three copy numbers (196 to 238 bp) with a 21-bp tandem repeat. In 85 clinical isolates, M. abscessus showed two to four copies of VNTR11 and one or two copies of VNTR23. In comparison, M. bolletii showed one copy of VNTR11 and two copies of VNTR23; M. massiliense showed one copy of VNTR11. M. massiliense showed no tandem repeats (non). Lanes M, molecular size markers (in base pairs); lanes 1, 2, 3, and 4, M. abscessus SM56, 25, 14, and 50, respectively; lanes 5 and 6, M. massiliense SM04 and 38, respectively; lanes 7 and 8, M. bolletii SM19 and 61, respectively.

Fig. 2.

Representative images showing duplex PCR results obtained by agarose gel (A) and capillary electrophoresis (B). Lane M, molecular size markers (in base pairs); lanes 1 to 5, M. abscessus SM56, 25, 14, 32, and 50, respectively; lane 6, M. massiliense SM04; lane 7, M. bolletii SM19. The amplified products of Mab1 and Mab2 correspond to the top and bottom bands, respectively.