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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Cai
Mei-Ting
MT
Department of Central Laboratory, Children's Hospital Affiliated to the Medical College, Zhejiang University, Hangzhou 310003, China.
Wu
Yi-Dong
YD
Wu
Xiu-Jing
XJ
Shang
Shi-Qiang
SQ
D000293:Adolescent; D002648:Child; D002675:Child, Preschool; D016172:DNA Fingerprinting; D017931:DNA Primers; D004279:DNA, Viral; D018792:Encephalitis, Viral / Q000134:cerebrospinal fluid / Q000175:diagnosis / Q000821:virology*; D005260:Female; D005470:Fluorometry; D005838:Genotype; D015654:Herpesvirus 6, Human / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004279:DNA, Viral