Identification and genotyping of molluscum contagiosum virus from genital swab samples by real-time PCR and Pyrosequencing.
BACKGROUND
Laboratory diagnosis of molluscum contagiosum virus (MCV) is important as lesions can be confused with those caused by Cryptococcus neoformans, herpes simplex virus, human papillomavirus, and varicella-zoster virus.


OBJECTIVE
To develop a rapid method for identifying patients infected with MCV via swab sampling.


METHODS
Two dual-labeled probe real-time PCR assays, one homologous to the p43K gene and one to the MC080R gene, were designed. The p43K PCR was designed to be used in conjunction with Pyrosequencing for confirmation of PCR products and discrimination between MCV1 and MCV2.


RESULTS
Both PCR assays were optimized with respect to reaction components, thermocycling parameters, and primer and probe concentrations. The specificities of both PCR assays were confirmed by non-amplification of 38 known human pathogens. Sensitivity assays demonstrated detection of as few as 10 copies per reaction. Testing 703 swabs, concordance between the two real-time PCR assays was 99.9%. Under the developed conditions, Pyrosequencing of the p43K PCR product was capable of providing enough nucleotide sequence to definitively differentiate MCV1 and MCV2.


CONCLUSIONS
These real-time PCR assays can be used for the rapid, sensitive, and specific detection of MCV and, when combined with Pyrosequencing, can further discriminate between MCV1 and MCV2.
Trama
Jason P
JP
Molecular and Cellular Biology Division, Medical Diagnostic Laboratories LLC, 2439 Kuser Road, Hamilton, NJ 08690, United States. jtrama@mdlab.com
Adelson
Martin E
ME
Mordechai
Eli
E
D001483:Base Sequence; D017931:DNA Primers; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D005260:Female; D005836:Genitalia, Female / Q000821:virology*; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D008976:Molluscum Contagiosum / Q000175:diagnosis / Q000821:virology*; D008977:Molluscum contagiosum virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers; D015342:DNA Probes; D004279:DNA, Viral