The development of a multiplex polymerase chain reaction (PCR) method for rapid and accurate detection and typing of herpes simplex virus type 1 (HSV-1), and type-2 (HSV-2), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) is very important for clinical diagnosis to allow the deliver of therapy as early as possible. Large scale amplifications by multiplex PCR of viral DNA can lower the cost and time for viral diagnosis. In this study, therefore sensitive quadruplex PCR was achieved by optimizing parameters such as primers, and 1.5 mM magnesium and 200 uM dNTPs concentrations. The concentrations of HSV-1, HSV-2, CMV and EBV primers were 0.5, 0.3, 0.25 and 0.25 pmoles, respectively. Optimal annealing temperature was 54 degrees C. Employing these conditions, we could detect 10 copies of reconstructed template plasmid DNA, which were cloned to vectors containing target sequences of viral DNA. PCR products of 271 bp for HSV-1, 231 bp for HSV-2, 368 bp for CMV, and 326 bp for EBV were separated on 5.0% polyacrylamide gel electrophoresis and confirmed by direct sequencing. The present study showed that the quadruplex PCR assay described herein has potential application in clinical diagnosis, when rapid, accurate detection and typing of viruses HSV-1, HSV-2, CMV or EBV are necessary.
Cytomegalovirus/classification/*isolation & purification ; Herpesvirus 1, Human/classification/*isolation & purification ; Herpesvirus 2, Human/classification/*isolation & purification ; Herpesvirus 4, Human/classification/*isolation & purification ; Human ; *Polymerase Chain Reaction/methods ; Sensitivity and Specificity ; Support, Non-U.S. Gov't

BACKGROUNDHuman cytomegalovirus (HCMV) infection is an important infectious agent that results in neonatal disease and congenital deformity. HCMV infection may affect in many organs. The different symptoms and tissue tropism of HCMV infection perhaps resulted from the genetic polymorphism of HCMV. HCMV UL144 open reading frames encode a homologue of the tumor necrosis factor receptor. It seems important to study the strain-specific variability of UL144 sequence in low-passage clinical isolates and to discuss if the variability related to the clinical HCMV infection.

METHODSHCMV-UL144 gene was amplified by PCR assay in 65 low-passage clinical isolates and urine from 7 healthy children who were HCMV-DNA positive by quantitative PCR. All the positive PCR products were analyzed by Heteroduplex mobility assay and single-stranded conformation polymorphism (HMA-SSCP) and 32 of them were sequenced.

RESULTSFifty-five isolates and 5 urine specimens were HCMV-UL144 positive by PCR. Sequencing and HMA-SSCP analysis showed that significant strain-specific variability was present in the UL144 ORFs. Comparing UL144 sequences and the corresponding symptoms showed that genotype 2 did not exist in megacolon isolates. And genotype 1 and 3 were the major types among microcephaly and jaundice isolates respectively.

CONCLUSIONHCMV-UL144 existed in almost all the low passage isolates. HMA-SSCP assay is an easy and effective method to detect the genetype of HCMV-UL144 sequence. The characteristic of sequences in different isolates showed that UL144 gene may play an important role in HCMV infection.

Cytomegalovirus ; classification ; genetics ; isolation & purification ; Cytomegalovirus Infections ; virology ; Genotype ; Humans ; Infant ; Infant, Newborn ; Membrane Glycoproteins ; genetics ; Molecular Sequence Data ; Phylogeny ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; Sequence Analysis, DNA ; Viral Proteins ; genetics

OBJECTIVETo investigate the genetic polymorphism of human cytomegalovirus (HCMV) glycoprotein H (gH) from infantile clinical isolates, to analyze the genotypic distribution of gH in different diseases of HCMV infection and try to find the correlations between the diseases and genotypes.

METHODFresh urine specimens were collected from the hospitalized children with different diseases whose blood HCMV-IgM and HCMV-IgG were positive. Virus was isolated from these specimens. Glycoprotein H of harvest clinical isolates was genotyped by nested-PCR combined with restriction fragment length polymorphism (RFLP), the purified PCR products were digested by restriction endonuclease HhaI. The digested products were genotyped by polyacrylamide gel electrophoresis and silver staining. Classification and results of sequencing were compared.

RESULTTotally 102 HCMV clinical isolates were obtained. Glycoprotein H gene of these clinical isolates (43 cases had infantile hepatitis syndrome, 38 cases had anicteric hepatitis, 13 pneumonia, 7 thrombocytopenic purpura, and 1 congenital CMV infection) were positive by nested-PCR, whose positive rate was 100%. The results showed that 62 strains were gH1 genotypes (60.8%), while 40 strains were gH2 (39.2%), mixed type or new genotype was not observed. In infantile hepatitis syndrome (26 clinical isolates were gH1 genotypes, 17 clinical isolates were gH2 genotypes), anicteric hepatitis (25 were gH1, 13 were gH2) and pneumonia (9 were gH1, 4 were gH2), the distribution of HCMV gH genotypes of infantile clinical isolates was consistent with the overall trend (χ(2) = 0.357, P > 0.05). However , the gH2 was more common than gH1 in the clinical isolates of patients with thrombocytopenic purpura (6 were gH2, 1 were gH2, χ(2) = 6.083, P < 0.05).

CONCLUSIONGenotype 1 was the dominant genotype of glycoprotein H in HCMV clinical isolates from our hospital infants. There was no significant difference between the distribution of gH genotypes in infantile hepatitis syndrome, anicteric hepatitis and pneumonia. However, gH2 was the dominant genotype in thrombocytopenic purpura. These findings suggested that there may be a certain relevance between gH genotype and different clinical manifestations.

Amino Acid Sequence ; Base Sequence ; Child, Preschool ; Cytomegalovirus ; classification ; genetics ; isolation & purification ; Cytomegalovirus Infections ; virology ; DNA Primers ; DNA, Viral ; genetics ; Female ; Genotype ; Hepatitis ; virology ; Humans ; Infant ; Infant, Newborn ; Male ; Pneumonia, Viral ; virology ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length ; Urine ; virology ; Viral Envelope Proteins ; genetics