Simultaneous detection and differentiation of human polyomaviruses JC and BK by a rapid and sensitive PCR‐ELAHA assay and a survey of the JCV subtypes within an Australian population

Abstract

Human polyomaviruses JCV and BKV can cause several clinical manifestations in immunocompromised hosts, including progressive multifocal leukoencephalopathy (PML) and haemorrhagic cystitis. Molecular detection by polymerase chain reaction (PCR) is recognised as a sensitive and specific method for detecting human polyomaviruses in clinical samples. In this study, we developed a PCR assay using a single primer pair to amplify a segment of the VP1 gene of JCV and BKV. An enzyme linked amplicon hybridisation assay (ELAHA) using species-specific biotinylated oligonucleotide probes was used to differentiate between JCV and BKV. This assay (VP1-PCR-ELAHA) was evaluated and compared to a PCR assay targeting the human polyomavirus T antigen gene (pol-PCR). DNA sequencing was used to confirm the polyomavirus species identified by the VP1-PCR-ELAHA and to determine the subtype of each JCV isolate. A total of 297 urine specimens were tested and human polyomavirus was detected in 105 specimens (35.4%) by both PCR assays. The differentiation of JCV and BKV by the VP1-PCR-ELAHA showed good agreement with the results of DNA sequencing. Further, DNA sequencing of the JCV positive specimens showed the most prevalent JCV subtype in our cohort was 2a (27%) followed by 1b (20%), 1a (15%), 2c (14%), 4 (14%) and 2b (10%). The results of this study show that the VP1-PCR-ELAHA is a sensitive, specific and rapid method for detecting and differentiating human polyomaviruses JC and BK and is highly suitable for routine use in the clinical laboratory.

Quantification of Human Polyomaviruses JC Virus and BK Virus by TaqMan Quantitative PCR and Comparison to Other Water Quality Indicators in Water and Fecal Samples

“…Due to the health implications of BKV and JCV, several methods have been developed to rapidly detect either BKV or JCV in clinical samples (6,31,35,56). However, from an MST standpoint, it is advantageous to target both BKV and JCV.…”

Detection of Human-Derived Fecal Pollution in Environmental Waters by Use of a PCR-Based Human Polyomavirus Assay

“…The PCR mixtures were prepared using 45 l of Platinum Blue PCR SuperMix (Invitrogen, Inc., Carlsbad, CA), 200 nM of each primer, and 4 l of DNA template. The final reaction volume was adjusted to 50 l using reagent grade water (64). The PCR conditions were as follows: initial denaturation at 94°C for 2 min, followed by 45 cycles of 94°C for 20 s, 55°C for 20 s, and 72°C for 20 s, and then a final elongation at 72°C for 2 min (Eppendorf Mastercycler Thermocycler; Eppendorf International, Hamburg, Germany).…”

Human JCV Infections as a Bio-Anthropological Marker of the Formation of Brazilian Amazonian Populations

“…Among the Afro-descendants and the residents in Belem, older than 30 years of age, 65% and 66%, respectively, were infected, similarly to what has been described when testing new diagnostics kits, in Australia and among persons with non-Hodgkin lymphoma [2], [25], [28]. Although there are few studies among young individuals, the number of samples is low and the available volume of urine does not favour the amplification of DNA [21], [26], [29].…”

“…Several available studies do not inform the ages of the infected persons, except for some studying population groups from Africa [21], North America [11], [22], [23], Europe [2], [13], Australia [24], [25] and North Korea [26]. It is believed that primary infection occurs early in childhood [27], consequently, it is necessary to keep monitoring the infection among different age groups of different populations, healthy or not, once JCV is a persistent infection which is largely disseminated among human groups [1], [3], [5], [6].…”