10.3.1. Introduction

Multiplex Ligation-dependent Probe Amplification (MLPA) technology is an amplification technique that allows simultaneous detection of up to 40 targets with the use of a single PCR primer pair. The procedure uses a series of paired oligonucleotides (half-probes), each pair specific for one target. The two half-probes; the Left Probe Oligo (LPO) and the Right Probe Oligo (RPO) lie adjacent to each other on the target genome so that they can be joined together by a ligation reaction, to produce an amplification probe (Fig. 6). In addition to a target-specific sequence, each of the half-probes contains one of two sequences recognized by a universal PCR primer, for probe amplification. Since these PCR primer sequences are common to all half-probe pairs, a single pair of PCR primers can amplify all target probes in a multiplex reaction. The half-probe pairs also contain a ‘stuffer’ fragment of variable length, allowing each amplified probe to be identified by its size, using (capillary) electrophoresis (Fig. 6). This technique was recently adopted to detect the most common honey bee viruses including CBPV, DWV (KV & VDV-1), ABPV (IAPV & KBV), BQCV, SBPV, SBV (De Smet et al., 2012). Because these are all RNA viruses, the MLPA is preceded by a reverse transcription of RNA into cDNA. Since the probes are strand-specific, this technique is highly suitable for the selective detection of either the positive-strand genomic viral RNA or the negative-strand virus replicative intermediate RNA, which is a marker for virus replication.

Since several targets are amplified at the same time, there will be competition between different targets for the amplification resources (primers, nucleotides, enzyme). This ‘competitive’ PCR allows for a measure of relative quantification between the targets, in the sense that the relative proportion of the targets after amplification should, if all targets amplify equally efficiently, reflect their initial proportions in the sample. By including one or more internal reference genes or exogenously added absolute quantification standards among the targets, the procedure can be made (semi- quantitative.

Fig. 6. Outline of the MLPA procedure for amplifying strand-specific ligated probes. LPO and RPO refer to the Left Probe Oligo and Right Probe Oligo respectively. RdRp refers to the viral RNA-dependent RNA polymerase.  Image © L De Smet.

12116PN revised Fig 6