3.2. Identification

Often the first step in the identification of P. larvae growing on solid media is the verification of its growth rate and colony morphology. Visible colonies may appear on the second day of incubation. However, if no colonies emerge it is advisable to extend the incubation time for a few more days. Two serial subcultures should be grown to insure culture purity. Pure P. larvae colonies have a characteristic morphology but this appears to be highly dependent on the medium that was used (see OIE, 2008). Using P. larvae reference strains is highly advisable. Some non-molecular identification protocols exist and provide a good alternative for diagnostic purposes when sophisticated equipment is lacking (see OIE, 2008). However, for research purposes we recommend a PCR-based identification of P. larvae. Several PCR methods have been described (reviewed by de Graaf et al., 2006a), but one in particular based on the 16S rRNA gene (Dobbelaere et al., 2001) has proven its robustness in the past decade. A detailed description is given here below. Primers are listed in Table 3.

Table 3. Primer sets for identification and genotyping of P. larvae by PCR.

Name

Sequence

PCR-product size

Reference

AFB-F

AFB-R

 

5'-CTTGTGTTTCTTTCGGGAGACGCCA-3'

5'-TCTTAGAGTGCCCACCTCTGCG-3'

1106 bp

Dobbelaere et al., 2001

Primer 1

Primer 2

 

5’-AAGTCGAGCGGACCTTGTGTTTC-3’

5-’TCTATCTCAAAACCGGTCAGAGG-3’

973 bp

Govan et al., 1999

 

ERIC1R

ERIC2

 

5´-ATGTAAGCTCCTGGGGATTCAC-3´

5´-AAGTAAGTGACTGGGGTGAGCG-3´

Several amplicons

Versalovic et al., 1994

BOXA1R

 

 

5´-CTACGGCAAGGCGACGCTGACG-3

Several amplicons

Versalovic et al., 1994

MBO-REP1

 

 

5´-CCGCCGTTGCCGCCGTTGCCGCCG-3

Several amplicons

Versalovic et al., 1994

3.2.2. Polymerase chain reaction