**4.14 Real-time polymerase chain reaction (qPCR)**

This technique can be employed to quantitate CPV-2 in samples using either TaqMan probe technology or SYBR Green method. It is used for strain differentiation of concurrent infection using Multiplex Real-time PCR; and also, to differentiate vaccine strain from wild CPV strains. Different multiplex assays real-time PCR has been validated for the presence of CPV, FPV and PPV [67].

### **4.15 Amplification refractory mutation system PCR (ARMS-PCR)**

It is used for the detection and typing of the known point mutations/single nucleotide polymorphism based on variable size of PCR-amplified products specific to a particular allele. In this PCR basically 2 pairs of primers are used (2 inner and 2 outer specific primers matching to individual allele type) in a single PCR tube and there are

no post-PCR protocols used as restriction enzyme digestion (PCR-RFLP) and sequencing therefore they provide an economical confirmation. ARMS-PCR is a well-known technique frequently employed for phenotypic association and single nucleotide polymorphism (SNP) studies. This has been used for CPV detection and its antigenic typing [54].
