title: TaqMan hydrolysis probe based real time PCR for detection and quantitation of camelpox virus in skin scabs.
abstract: The study describes the development of TaqMan hydrolysis probe based real time PCR (rt-PCR) assay targeting the ankyrin repeat protein (C18L) gene sequences for the detection and quantitation of camelpox virus (CMLV) nucleic acid and its comparison with established conventional and SYBR green rt-PCR assays. The assay was specific with an efficiency of 99.4%. The analytical sensitivity was 4 × 10¹ and 0.35 in terms of copy number and picogram of virus genomic DNA, respectively. The assay was linear with an acceptable intra (0.9-2.83% and 0.9-2.3%) and inter-assay (0.46-2.3% and 0.9-3.3) variations, when standard plasmid DNA and genomic DNA from purified CMLV respectively were tested. The assay was rapid, specific and sensitive as that of SYBR green and 1000 times more sensitive than the conventional PCR. It is suitable for the detection of CMLV nucleic acid directly from clinical samples. Further, the assay was evaluated using cell culture adapted CMLV isolates (n=11) and clinical samples (n=23) from camels and humans suspected of camelpox. This is an improved technique over conventional and SYBR green rt-PCR methods for the detection and quantitation of CMLV from skin scabs.
authors.affiliation: Pox Virus Laboratory, Division of Virology, Indian Veterinary Research Institute, Nainital Distt., Mukteswar 263138, Uttarakhand, India. gnanamvirol@gmail.com
authors.forename: G
authors.initials: G
authors.lastname: Venkatesan
authors.forename: V
authors.initials: V
authors.lastname: Bhanuprakash
authors.forename: V
authors.initials: V
authors.lastname: Balamurugan
authors.forename: M
authors.initials: M
authors.lastname: Prabhu
authors.forename: A B
authors.initials: AB
authors.lastname: Pandey
mesh_terms: D000818:Animals; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics; D018155:Orthopoxvirus / Q000235:genetics / Q000302:isolation & purification*; D011213:Poxviridae Infections / Q000662:veterinary* / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012867:Skin / Q000821:virology*; D013997:Time Factors; D019562:Viral Load / Q000379:methods*
chemical_list: D015345:Oligonucleotide Probes