Multiplex RT-nested PCR differentiation of gill-associated virus (Australia) from yellow head virus (Thailand) of Penaeus monodon

Abstract

A multiplex RT-nested PCR has been developed to detect and differentiate the closely related prawn viruses, gill-associated virus (GAV) from Australia and yellow head virus (YHV) from Thailand. RT-PCR using primers to conserved sequences in the ORF1b gene amplified a 794 bp region of either GAV or YHV. Nested PCR using a conserved sense primer and either a GAV- or YHV-specific antisense primer to a divergent sequence differentially amplified a 277 bp region of the primary PCR amplicon. Multiplexing the YHV antisense primer with a GAV antisense primer to another divergent sequence allowed the viruses to be distinguished in a single nested PCR. Nested PCR enhanced detection sensitivity between 100- and 1000-fold and GAV or YHV RNA was detectable in approximately 10 fg lymphoid organ total RNA. The multiplex RT-nested PCR was also able to co-detect GAV and YHV RNA mixed over a wide range of concentrations to simulate potential dual-infection states. The robustness of the test was examined using RNA samples from Penaeus monodon prawns infected either chronically or acutely with GAV or YHV and collected at different locations in Eastern Australia and Thailand between 1994 and 1998. GAV- (406 bp) or YHV-specific (277 bp) amplicons were differentially generated in all cases, including five YHV RNA samples in which no primary RT-PCR amplicon was detected. Sequence analysis of GAV and YHV PCR amplicons identified minor variations in the regions targeted by the virus-specific antisense primers. However, none occurred at positions that critically affected the PCR.

Fig. 1

GAV and YHV nucleotide and amino acid sequences in the ORF1b gene region targeted by primers used in the multiplex RT-nested PCR. YHV sequence differences are indicated above and below the GAV sequences and the primer targets are highlighted (arrows). Numbering is taken from a GAV–YHV sequence comparison reported previously (Cowley et al., 1999).

Fig. 2

Amplification of GAV and YHV RNA by RT-PCR followed by nested PCR with various primer combinations. (a) PCR amplification (primer pair GY1–GY4) of a 794 bp product from cDNA synthesised from reference GAV (lane 1) and YHV (lane 2) RNA using primer GY5. (b) Nested PCR amplification of RT-PCR products from GAV (lanes 1–3) and YHV (lanes 4–6) using primer pairs GY2–Y3 (lanes 1 and 4), GY2–G3 (lanes 2 and 5) and GY2–G6 (lanes 3 and 6). (c) Nested PCR amplification of a 406 bp GAV-specific product (lane 1) or 277 bp YHV-specific product (lane 2) using the multiplexed primer set GY2–Y3/G6. PCR products (10 μl) were resolved in a 2% agarose–TAE gel containing 0.5 μg/ml ethidium bromide. M=1 kb DNA ladder (Invitrogen).

Fig. 3

Detection limits of the RT-PCR and nested PCR primer combinations using titrations of LO total RNA from P. monodon infected with the reference GAV or YHV isolates. cDNA synthesised using primer GY5 and serial 10-fold RNA dilutions (100 ng/μl to 1 fg/μl, lanes 1–9, respectively) diluted in 10 ng/μl P. japonicus LO RNA, which was also used as a negative control (lane 10), was amplified by PCR using primer pair GY1–GY4. Nested PCRs were performed using primer pairs GY2–G3 and GY2–G6 for GAV and GY2–Y3 for YHV. Multiplex nested PCR (primer set GY2–Y3/G6) was also performed using 1:1 mixtures of the GAV and YHV RT-PCRs. PCR products (10 μl) were resolved as in Fig. 2. M=1 kb DNA ladder.

Fig. 4

Detection of RNA from different GAV and YHV samples using the multiplex RT-nested PCR. The origin of P. monodon from which RNA was isolated is described in Table 1. Isolates GAV#1 to GAV#10 correspond to GAV lanes 1–10, respectively, and isolates YHV#1 to YHV#9 correspond to YHV lanes 1–9, respectively. GAV lane 11 and YHV lane 10 were negative controls comprising LO RNA from uninfected P. japonicus. (a) cDNA synthesised from 100 ng RNA using primer GY5 amplified by PCR using the primer pair GY1–GY4. (b) RT-PCR products amplified by nested PCR using the multiplex primer set GY2-Y3/G6. (c) For selected YHV samples (#3 (lane 1), #5 (lane 2), #6 (lane 3), #7 (lane 4) and #8 (lanes 5–7)) which generated little or no 794 bp PCR amplicon, the multiplex nested PCR was repeated using RT-PCRs performed using 2 μl instead of 1 μl input cDNA. cDNA from P. japonicus RNA was used as a negative control (lane 8). For isolate YHV#8, 5 μl (lane 6) and 10 μl (lane 7) of the primary PCR were also amplified by nested PCR. PCR products (10 μl) were resolved as in Fig. 2. M=1 kb or 1 kb plus DNA ladder (Invitrogen).

Fig. 5

Multiplex RT-nested PCR amplification of GAV and YHV RNA mixed at various ratios to simulate dual-infection states. RNA was mixed 1:1 at concentrations of either (a) 100 ng/μl (lane 5) or (b) 1 ng/μl (lane 6) and serial 10-fold dilutions of virus RNA were prepared in a diluent of reciprocal virus RNA at these two concentrations. (a) YHV RNA amounts 100 fg, 10 pg, 100 pg, 1 ng (lanes 1–4, respectively) and 100 ng (lanes 5–9), GAV RNA amounts 100 ng (lanes 1–5) and 1 ng, 100 pg, 10 pg, 100 fg, (lanes 6–9, respectively) and P. japonicus negative control RNA (lane 10). (b) YHV RNA amounts 10 fg, 100 fg, 1 pg, 10 pg, 100 pg (lanes 1–5, respectively) and 1 ng (lanes 6–11), GAV RNA amounts 1 ng (lanes 1–6) and 100 pg, 10 pg, 1 pg, 100 fg, 10 fg (lanes 7–11, respectively). In both tests, cDNA synthesised using primer GY5 was amplified by PCR (primer pair GY2–GY4) followed by nested PCR with the multiplex primer set GY2–Y3/G6. PCR products (10 μl) were resolved as in Fig. 2. M=1 kb DNA ladder.

Fig. 6

Nucleotide sequences of genome regions targeted by primers G3/Y3 and G6 in the nine GAV and eight YHV field samples compared to the reference isolates (GAV#6 and YHV#1). The GAV#6 amino acid sequence is shown, as are differences between this sequence and that of YHV#1. Nucleotide variations to the GAV#6 sequence are shown (bold), as are variations (bold) to the YHV#1 sequence.