Division I.3.2 Polymerase chain reaction (PCR)

Several PCR protocols have been developed and published.

Two PCR protocols targeting the small subunit (SSU) rDNA may be used:

1. (a)

   the first one is a conventional PCR that amplify several members of the Haplosporidia including Bonamia spp. Primers designated Bo and Boas are 5′-CAT-TTA-ATT-GGT-CGG-GCC-GC-3′ and 5′-CTG-ATC-GTC-TTC-GAT-CCC-CC-3′ respectively, and amplify a 300 bp product. PCR mixtures contain buffer (500 mM KCl, 100 mM Tris/HCl [pH 9,0 at 25 °C] and 1 % Triton® X-100), 2,5 mM MgCl2, 0,2 mM dNTP mix, 1 μM forward and reverse primers, 0,02 units μl^{– 1} Taq DNA polymerase, and 0,2 ng μl^{– 1} of the DNA template in a total volume of 50 μl. Samples shall be denatured in a thermocycler for five minutes at 94 °C before being submitted to 30 cycles (94 °C for one minute, 55 °C for one minute, 72 °C for one minute) followed by a final extension of 10 minutes at 72 °C.

   Positive controls shall consist of genomic DNA from a highly infected host or plasmidic DNA including the target region.

   Negative controls shall consist of genomic DNA from non-infected hosts and PCR reactives without target DNA.

   A positive result shall be positive PCR amplification at the expected size (namely, 300 bp), with all negative controls being negative and all positive controls being positive;

2. (b)

   the second PCR protocol is a SYBR® Green Real time PCR assay. It allows the specific detection of B. ostreae (described below) and may be combined with a SYBR® Green Real time PCR assay allowing the specific detection of B. exitiosa (Ramilo et al. 2013).

   Primers BOSTRE-F (5′- TTACGTCCCTGCCCTTTGTA-3′) and BOSTRE-R (5′- TCGCGGTTGAATTTTATCGT-3′) amplify a 208 bp product. PCR mixtures contain SYBR® Green Master Mix (1X), 0,3 μM forward and reverse primers, and 200 ng of extracted DNA. Samples shall be denatured in a Real Time Detection System for 10 minutes at 95 °C before being submitted to 35 cycles (95 °C for 30 seconds, 55 °C for 45 seconds and 72 °C for one minute). Melting temperature curve analysis shall be carried out with temperature increments of 0,5 °C/s starting at 55 °C and ending at 95 °C and recording fluorescence at each change of temperature.

   Positive controls shall consist in genomic DNA from a highly infected host or plasmidic DNA including the target region.

   Negative controls shall consist in genomic DNA from non-infected hosts and PCR reactives without target DNA.

   A positive result shall be positive PCR amplification with a unique melting temperature peak (78,25 ± 0,25 °C in the conditions published by Ramilo et al. 2013), with all negative controls being negative and all positive controls being positive.