a new real time PCR assay for simultaneous detection of human herpesvirus-6A and 6B

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This post by kelly came out on co-cure on Dec 7 09

Again, I like her/his comments.

NOTE: I just tried the link provided and it didn't work, and I'm too tired to look for a working one right now.

This may be of interest to the CFS and/or ME communities as HHV-6A has been implicated in some subsets of CFS. However, reliability of testing techniques and protocols have been the subject of some discussion in
scientific circles. Given the significant technological improvement - in
detection of viruses in general particularly in specificity - many studies
that did not previously link viruses to specific diseases should probably be
reproduced. The use of well defined patients groups coupled with advances in
technology may well prove to be effective in determining whether earlier
studies were indeed accurate.


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Zhonghua Er Ke Za Zhi. 2009 Jul;47(7):527-31.

Establishment and clinical application of a new real time PCR assay for
simultaneous detection of human herpesvirus-6A and human herpesvirus-6B[/B]

[Article in Chinese]3

Cai MT, Wu YD, Wu XJ, Shang SQ.

Department of Central Laboratory, Children's Hospital Affiliated to the
Medical College, Zhejiang University, Hangzhou 310003, China.

OBJECTIVE: Human herpesvirus 6 (HHV-6) isolates are classified into two
variants, HHV-6A and HHV-6B, based on distinct genetic, antigenic and
biological characteristics. HHV-6 has been associated with encephalitis in
children recently. This study aimed to establish a real time PCR assay for
simultaneous detection of the two subtypes of HHV-6, and apply this new
assay to children with suspected encephalitis, then analyze the relationship
between the infection with HHV-6 and encephalitis in children.

METHOD: The universal primers and variant-specific TaqMan probes were
designed based on the highly conserved sequences of the DNA polymerase gene
(U38) of HHV-6. The 5' end of the probes for HHV-6A and HHV-6B was labeled
with the fluorescein reporter tetrachloro-6-carboxyfluorescein and
6-carboxyfluorescein (6-FAM), separately, while the 3' end were quenched
with 6-carboxy-tetramethylrhodamine. The real time PCR assay for
simultaneous detection of HHV-6A and HHV-6B was established. Then, the
plasmids of HHV-6A and -6B which were diluted by a 10-fold series from 10(9)
to 10(0) copies/microl, together with controls were used for testing both
sensitivity and specificity of the real time PCR assay. The cerebrospinal
fluid (CSF) specimens from 445 cases of suspected encephalitis were tested
with this real time PCR and positive samples were then sequenced.

RESULT: Both HHV-6A (strain ZJ-159) and HHV-6B (strain GS) were positive on
the real time PCR assay. There were no cross-reaction with herpes simplex
virus type 1, type 2 (HSV-1, HSV-2), varicella-zoster virus (VZV),
cytomegalovirus (CMV), Epstein-Barr virus (EBV), hepatitis B virus,
Staphylococcus aureus, Mycoplasma pneumoniae and human DNA. A linear
regression curve was obtained when plotting Ct values against the log10 of
the viral DNA input for both subtypes of HHV-6. The sensitivity threshold
was 10 copies/microl for the real time PCR. HHV-6 positive rate by the real
time PCR assay was 4.72% (21/445), including 4 cases with HHV-6A infection,
16 cases of HHV-6B infection and 1 case with mixed HHV-6A and HHV-6B
infection. The new PCR assay usually took 2 to 3 hours to provide results.

CONCLUSION: This new real time PCR assay can simultaneously detect both
subtypes of HHV-6, and have high specificity and sensitivity. It will
provide an early and sensitive diagnosis of HHV-6 encephalitis in children.

PMID: 19951517 [PubMed - in process]