Detection and Genotyping of Varicella-Zoster Virus by TaqMan Allelic Discrimination Real-Time PCR

Abstract

A proportion of individuals vaccinated with live attenuated Oka varicella-zoster virus (VZV) vaccine subsequently develop attenuated chicken pox and/or herpes zoster. To determine whether postvaccination varicella infections are caused by vaccine or wild-type virus, a simple method for distinguishing the vaccine strain from wild-type virus is required. We have developed a TaqMan real-time PCR assay to detect and differentiate wild-type virus from Oka vaccine strains of VZV. The assay utilized two fluorogenic, minor groove binding probes targeted to a single nucleotide polymorphism in open reading frame 62 that distinguishes the Oka vaccine from wild-type strains. VZV DNA could be genotyped and quantified within minutes of thermocycling completion due to real-time monitoring of PCR product formation and allelic discrimination analysis. The allelic discrimination assay was performed in parallel with two standard PCR-restriction fragment length polymorphism (RFLP) methods on 136 clinical and laboratory VZV strains from Canada, Australia, and Japan. The TaqMan assay exhibited a genotyping accuracy of 100% and, when compared to both PCR-RFLP methods, was 100 times more sensitive. In addition, the method was technically simpler and more rapid. The TaqMan assay also allows for high-throughput genotyping, making it ideal for epidemiologic study of the live attenuated varicella vaccine. Varicella-zoster virus (VZV) is the etiological agent of varicella chicken pox and herpes zoster (HZ). Both varicella and HZ are self-limiting infections in healthy individuals but may cause more severe disease in immunocompromised hosts (2,8,23,38).A live attenuated Oka vaccine (V-Oka) strain was created in 1974 by the repeated passage of a Japanese clinical isolate in tissue culture (34). The vaccine was licensed in the United States in 1995 (2, 4) and in Canada in 1998 (27) and is currently used in many other countries (3, 40). Extensive clinical trials have shown the vaccine to be highly effective in both healthy and immunocompromised individuals (11,19,30,33). However, a low incidence of superinfection with wild-type (WT) strains after immunization, termed breakthrough varicella, has been well documented (3,10,21,39). Vaccine-associated rash occurs in 4 to 7% of healthy individuals (4, 40) and in up to 40% of leukemic vaccinees (10, 11). The vaccine strain does establish neuronal latency, and hence, reactivation of vaccine virus in and transmission from healthy and immunocompromised individuals is possible (11,20,21). As the number of vaccinees is increasing worldwide (31), the clinical management of HZ is becoming more complex. Given the fact that the vaccine virus is less pathogenic (39) and also less transmissible than WT virus (37), a timely differentiation of vaccine or WT disease would be clinically useful in situations with pregnant or immunocompromised household or hospital contacts, where prompt postexposure treatment may be indicated.The original method used to differentiate WT and vaccine VZV was restriction fragment leng...

TaqMan allelic discrimination assay for A1 and A2 alleles of the bovine CSN2 gene

“…The TaqMan assay had a 100% genotyping accuracy, rapid performance and a 100-times greater sensitivity than the standard ACRS-PCR method. These findings are also in agreement with results of Campsall et al (2004). Losing post-amplification steps, possible mismatches and other technical problems are avoided.…”

Identification of Five Major and Two Minor Genotypes of Varicella-Zoster Virus Strains: a Practical Two-Amplicon Approach Used To Genotype Clinical Isolates in Australia and New Zealand

“…VZV genome loads were assessed using real-time quantitative PCR analysis with the minor groove binding (MGB) Eclipse probe system for allelic discrimination (Epoch Biosciences, Inc.). In this test additional VZV Oka vaccine-specific SNPs were evaluated (MspI/NaeI sites; VZV genome position 107252) using a modification of the method described by Campsall et al (3). Briefly, modified MGB Eclipse primer mixes contained the forward (5Ј-GCCCAAAAACACTTTATC CTAC) and reverse (5Ј-GTTGTTGGAGAAGGGTGA) primers for amplification and the following MGB Dark Quencher Eclipse probes for detection and SNP discrimination: a wild-type probe (GCCTTTGCCAGC) labeled with 6-carboxyfluorescein and a mutant probe (5Ј-GAGCCTTTGCCGG) labeled with tetrachloro-6-carboxyfluorescein by using a controlled-pore glass column.…”

Microbiology laboratory and the management of mother-child varicella-zoster virus infection

“…They are more sensitive than viral cultures, DFA and Tzanck smears particularly when primers for genes 28 and 29 are used [30,428,429,440,449,450,457] . Molecular biology tests are also useful in the case of varicella appearing 7-42 d after vaccination, in the case of herpes zoster appearing 42 d after vaccination and, in the case of the suspected transmission of the vaccine virus, can distinguish virus vaccine, wild-type virus and potential recombinants of vaccine and wild-type viruses [30,451,452,454,455,[458][459][460][461][462][463] , although some tests have proved to be less appropriate over time for these purposes [30] . There are also some multiplex assays that can differentiate HSV-1, HSV-2 and VZV in cutaneous and mucocutaneous samples, making them very useful in the case of skin lesions of unclear etiology or immunosuppressed patients [429,464,465] .…”

“…Microbiology laboratory in mother-child VZV infection Molecular biology tests: Molecular biology tests based on in vitro nucleic acid amplification (PCR) are now considered the new platinum tests [20,30,429,449,450] . Various types of PCR are used to diagnose varicella and herpes zoster [13,421,422,[450][451][452][453][454][455][456] , including nested PCR, which is particularly sensitive, but susceptible to contamination leading to false positive results [348] . However, the latest real-time PCR tests are not only rapid, easy to perform, and as sensitive as nested PCR, but have also reduced the risk of contamination [348] .…”