Sample collection and DNA extraction
A total of 179 backyard poultry flocks located in different states of India
viz., Tamil Nadu, Kerala, Telangana, Haryana and Assam were included for screening of MG and MS. Pooled oropharyngeal and choanal swabs from 5 randomly selected birds from each backyard flocks of minimum size 50 were considered as single sample and were included in the study to represent each flock. The birds were either native or local breed /variety or improved backyard variety in the age group of 16-40 weeks. All the swabs were collected aseptically in sterile PBS in container and transported to laboratory under chilled condition. DNA extraction from the samples were performed either within 24 h of collection of samples or stored at -20°C until extraction.
The swabs with the PBS were centrifuged at 14,000 rpm for 10 min and 250 ml of supernatant was taken for subsequent extraction. DNA extraction was performed by using Hi PurA® Multi-sample DNA purification kit (Himedia Laboratories Pvt Ltd) following the manufacture’s instruction. The DNA from the samples was stored at -20°C until further use in PCR.
Primers design
Primers for both MG and MS were designed using NCBI Primer design tool
http://www.ncbi.nlm.nih.gov/tools/primer-blast and are presented in Table 1. The
mgc2 gene of MG and
vlhA (variable lipoprotein hemagglutinin A) gene for MS were selected as target genes for primer design and following criterion were considered while designing and selecting the primer sets. 40-60% GC content, 20-25 bp length, no hairpin loops, Tm° value in the range of 50-55°C and difference between the amplicon size of at least 200 bp between the primer sets.
Optimization of duplex PCR
The DNA extracted from live vaccines of MG and MS (Vaxsafe MG/ MS; Zoetis India Pvt Ltd) were used as template for optimization of duplex PCR assay. Different concentration of primer sets was tested using checkerboard method for getting optimal amplification of both genes from template DNA. The optimized PCR mixture is as follows: 25ml of total PCR reaction in single tube containing 2.5 ml of 10X buffer with MgCl
2, 0.25 ml of Taq polymerase (5U/ml), 0.5 ml of 10 mMol dNTPs, 0.5 ml of MG primer sets, 0.25 ml of MS primer sets, 2 ml of template DNA (50ng/ ml) and nuclease free water. The optimum annealing temperature for PCR was optimized by testing with gradient PCR method. The reaction condition optimized were as follows: initial denaturation at 94°C for 5 min followed by 35 cycles of 94 °C for 1 min, 60°C for 1 min and 72°C for 1 min, with final extension at 72°C for 10 min. The amplified specific gene PCR products were checked in 1.5% agarose gel for 150 and 787 bp respectively.
Sensitivity and specificity
To determine the sensitivity of duplex PCR primer sets, serial 10-told dilution of positive DNA template with initial concentration of 1 mg/ml was done and each dilution of both MG and MS template DNA was used for duplex PCR up to 10
-12 dilution. The highest dilution of DNA template that gives a visible target gene specific band in PCR was considered as the detection limit or sensitivity. The cross-reactivity of primer sets was tested using DNA isolated from MG and MS live vaccines. The primer set of MG was tested with DNA template from MS positive control and vice versa. The specificity of primer sets was clearly indicated by the absence of amplification in another DNA template. The gene specific products amplified with respective DNA templates were sequenced by Sanger method and obtained sequences were confirmed by BLASTN analysis for the homology.
Validation with field samples
The DNA extracted from 179 backyard poultry flock samples were subjected to duplex PCR with both MG and MS primer sets in single tube by following the optimized protocol as described earlier. The amplified PCR products were checked in 1.5% agarose gel. Presence of either 150 or 787 bp or both amplicons were considered as positive for MG / MS or both, respectively. Positive control with template DNA from vaccine and negative control were included.
Comparison with single PCR for MG and MS
The same 179 field samples were subjected to detection of MG and MS by single PCR in separate PCR reaction. Primer sets targeting the 16S rRNA gene for MG (
OIE, 2008) and
vlhA gene for MS
(Jeffery et al., 2007) described earlier were used. The amplification of 185 bp and 350 bp products indicated the samples were positive for MG and MS respectively. The results from single PCR and duplex PCR were compared for agreement between these two assays.