*7.1.6 Polymerase chain reaction*

Polymerase chain reaction (PCR) was performed using Ampliqon (Bio-Basic Inc.) following manufacturer's instructions for FPCS of F gene and M gene fragment. The primers designed for qPCR were used.

The reaction was set up as follow


1.For amplification of M gene fragment, PCR was carried out as per cycle sequence provided below.

2.For the purpose of amplifying FPCS gene specific cDNA, PCR was performed using the cycle sequence shown below

3.The FPCS of the F gene and M gene fragment PCR products were electrophoresed at 150 V for 15 minutes in a 2.0% agarose gel and compared with a 100 bp DNA marker. The gel was then photographed and scanned using a gel doc system (Bio-Rad).

*7.1.7 Other materials/reagents for qPCR*


#### *7.1.8 qPCR*

Using SYBR® Green JumpStartTM Taq Ready MixTM (Sigma, USA, Cat # S4438) in accordance with the manufacturer's instructions, qPCR was performed. SYBR Green jumpstart Taq Ready Mix combines the convenience of an easy-to-use ready-Mix solution with the performance boost of jumpstart Taq antibody for hot start PCR with SYBR Green I. It contains a fluorescent dye and the reagents for performing highthroughput qPCR and is provided in 2 concentration.


1.The composition of reaction mix (10 μl) used for real time PCR is as follow


After each extension step, a single fluorescence `n end-point was measured. To establish the specificity of each amplification, the melting curves for PCR products were examined between 70°C and 95°C.
