Establishment And Application Of The Restriction Endonulease Patterns For Detecting The Six Major Human Herpesviruses

Human herpesviruses, particularly herpes simplex virus type l(HSV-l), and type 2(HSV-2), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster(VZV) and human herpesvirus 6(HHV6), are major agents of central nervous system and urinary system infections in children. The clinical signs and symptoms produced by the six major viruses are not viral taxon specific , furthermore, accurate etiological diagnosis of herpesviral infections is often complicated and clinical and laboratory findings are frequently non-specific. In this study, we designed two pairs of primers according to the well-conserved region of DNA polymerase gene of human herpesviruses . The detection of the six major herpesviruses is based on PCR technique with two primer pairs, followed by restriction enzyme analysis using BamHI and BstUI endonucleases. Finally, we report the results of a preliminary application that was performed to evaluate the feasibility of this technique for blood and CSF specimens.Part one Establishment of the restriction endonulease patterns for detectingHuman herpesvirusesMethods: Using the well-conserved regions of the DNA polymerse gene in human herpesviruses, We synthesized two pairs of primer, including one pair of primer designed to amplify herpes simplex virus type 1 and 2 (strain G and strain F, propagated in a vero cell line), Epstein-Barr virus(propagated in the 95-8 cell line) and cytomegalovirus(strain AD 169, cultivated in Human fibroblast cells), other pair of primer to varicella-zoster virus(strain EF, cultivated in McZ cells) and human herpesvirus 6 (strain GS, obtained from HSB2 cells )by PCR. Identification of the virus species was achieved through restriction enzyme digestion with BamHI and BstUI.Results: The products of six human herpesviruses after PCR amplification were from 510bp to 592bp and allowed characterization of herpesvirus type with restriction endonulease analysis. The sensitivity could reach 0.1 fg DNA and had no cross-reaction to human DNA, E. coil, staphylococcus aureus, hepatitis B virus(HBV), C. neoformans . According to the sequencing of the six human herpesviruses , we found that amplicons generated with the PI and P2 primer pair have high G+C contents, particularly amplicons from HSVI(66.2%), HSVII(67.5%), EBV(61.5%) to CMV(60.2%), in contrast, the amplicons generated with the P3 and P4 primer pair have much low G+C contents (from 43.2% for HHV6 to 40.5% for VZV). The CMV amplicon of 592 bp was cleaved by BstUI into nine fragments(170 bp,130 bp,80 bp,62 bp,50 bp,40 bp,30 bp,16 bp and 14 bp)and remained undigested by BamHI; the EBV amplicon of 520 bp cleaved by BamHI and BstUI into two fragments( 250 bp and 270 bp , 254 bp and 266 bp, respectively); the HSVI amplicon of 518 bp was cleaved by BstUI into eight fragments( 230 bp, 130 bp, 50 bp, 40 bp, 23 bp, 20 bp, 15 bp and 10 bp) and remained undigested by BamHI; the HSVII amplicon of 518 bp was cleaved by BamHI into two fragments(230 bp and 288 bp) and by BstUI into ten fragments (168 bp, 90 bp, 80 bp, 60bp, 40 bp, 30 bp, 20 bp, 15 bp, 10 bp and 5 bp); the VZV amplicon of 510 bp was cleaved by BstUI into two fragments (280 bp and 230 bp) and remained undigested by BamHI; the HHV6 amplicon of 513 bp was cleaved by BstUI and BamHI into two fragments (163 bp and 350 bp, 243 bp and 270 bp, respectively). After digestion and electrophoresis, each viral DNA yielded a characteristic fragment pattern that could differentiate from each other.Conclusions: The amplicons for CMV, EBV, HSVI and HSVII DNA have a high G+C content which adversely affects amplification, we found that the addition of 5% DMSO enhanced amplification of the chosen sequence efficiently. The restriction fragments unequivocally identify all viruses since all viral DNAs are cut by two enzymes and the selected endonucleases proved to perform well without the need to purify amplicons from PCR mixture. So.this PCR-RFLP was specific, sensitive, rapid and accurate in providing a new technique for the identification of herpesvirus DNA in herpesviru...