Use of a single, triplicate arbitrarily primed-PCR procedure for molecular fingerprinting of lactic acid bacteria

Abstract

Arbitrarily primed (AP)-PCR can be used to generate characteristic DNA fingerprint patterns. However, small changes in reaction conditions can cause band irreproducibility. In this study, a single methodology encompassing triplicate reactions, which were intentionally exposed to three different annealing temperatures, enabled bands that were reproducibly generated to be recognized. A single triplicate AP-PCR (TAP-PCR) procedure, using an 18-mer primer, was developed and used to fingerprint representative isolates from the major genera of lactic acid bacteria and Bifidobacterium to the strain level.

FIG. 1

TAP-PCR-generated fingerprints of various LAB by using primer P32 and 1.5 mM MgCl₂. Lanes a, b, and c, annealing temperatures of 38, 40, and 42°C, respectively; lane M, 1-kb ladder (BRL). Molecular sizes (right) are in kilobases.

FIG. 2

(a) Fingerprints generated from three different batches (1, 2, and 3) of degenerate primer P32 by using L. lactis JS102 DNA. The arrow shows a major product difference between batches 2 and 3. Lane M, 1-kb ladder (BRL). (b) Fingerprints generated with P32, P32-A, P32-T, and various ratios of P32-A and P32-T by using L. lactis JS102. Arrows indicate different classes of bands (see text). Lane M, 1-kb ladder (BRL).

FIG. 3

TAP-PCR-generated fingerprints of various Bifidobacterium breve and B. infantis strains using a 1-to-1 ratio of P32-A to P32-T. Lanes a, b, and c, annealing temperatures of 38, 40, and 42°C, respectively; lane M, 1-kb ladder (BRL). Molecular sizes (left) are in kilobases. The arrow points to a band which is discussed in the text.