DNA Detection Of B19 And HBoV And Preliminary Study Of Specific Probe

Parvoviruses is classified into small DNA viruses.Some can be autonomous replication(such as B19,HBoV and CPV,PPV),other need depend upon a larger helper-virus for replication(such as AAV).Parvoviruses infect mankind and many other vertebrate animals and can cause a diverse kind of disease.Autonomous parvoviruses share several common features of the genomes.The overall DNA homologies by both amino acid and nucleotide of some autonomous parvoviruses are higher.Parvoviruses is host range specificity, but this specificity is changing now while some new parvoviruses variants and new parvoviruses have been found in recnt years.It is possible that animal parvoviruses may infect mankind in the future,just as coronavirus to SARS.B19 and HBoV can infect children and transmit mainly by respiratory tract,which can cause local epidemic in certain communities.B19 is the important pathogens the fifth disease,acute aplasia anemia,acute thromocytonic purpura and other diseases.B19 infection in those patient with low immunity function or immunity deficiency can be persistent and can cause chronic anemia,therefore may threaten their lives.HBoV was closely related to the bovine parvovirus and minute virus of canines(MCV),which had been classified in the genus bocavirus within subfamily of Parvoviridae.HBoV can cause acute respiratory tract infection(ARTI)of pediatric patients.The established test methods for parvoviruses detection are based on the genome analysis of single virus or prototype,not on a systemic and rapid detection.The technique of DNA microarray provide a possible alternative for clinically detecting the infection of parvovirus,which is relatively simple,sensitive,high-throughput,and much more efficient.It is necessary to establish the diagnosis microarray of parvovirus (including B19,HBoV,CPV and PPV)to simultaneously detect the infection of some kind of parvovirus and their variants when parvovirus become epidemic in large scale.Objective:To establish the method of amplifying B19 and HBoV specific probe by PCR and prepared DNA targets of B19 and HBoV labeled with biotin during PCR cycling which is the foundation of preparation and detection of DNA microarray;to detect B19's and HBoV's gene in the clinical samples to analyze clinical characteristics of B19 and HBoV and provide positive samples for clinical detection of DNA microarray.Method:1.By analyzing B19's,HBoV's,CPV's and PPV's full length gene sequence and VP2 gene sequence available in the Genbank with BLAST and clustalW software and reading the associated literature,their conserved sequences region was detemined.Probes for microarray detection of B19,HBoV, CPV and PPV was designed in these region..2.Prepare B19,HBoV,CPV and PPV probes by polymerase chain reaction (PCR)for DNA microarray.PCR product was senquenced and analyzed. 3.Prepared DNA targets of B19 and HBoV labeled with biotin during PCR cycling(the PCR substrates were labeled with biotin-dUTP).4.DNA fragements of B19 and HBoV were detected in serum,sputum and throat swab from 252 children less than 14 years old admitted for ARTI between January 2008 and December 2008 at the Department of paediatrics of Xijing Hospital in order to provide positive samples for specific detection of DNA microarray.5.Clinical characteristics and epidemiology of HBoV and B19 infection were analyzed.Results:l.B19's,HBoV's,CPV's and PPV's specific probes and target sequence were detected by agarose gel electrophoresis.There were bight and unity expected strap.2.HBoV infection was detected in 15(6.0%)of 252 study subjects.In 8(53.3%)of the HBoV positive children,co-infections with other respiratory viruses were present.One of 9 PCR positive products were randomly selected for sequence analysis.The 291bp product shared high nucleotide sequence homology with the two prototype strains-Stockholm1 1(st1,99.3%)and Stockholm 2(st2,99.7%)and two Beijing strains(No.DQ988934.2 and No.DQ988933.1,99.7%)when compared with their sequences.HBoV infection was not detected in the serum sample.3.B19 DNA was detected in the throat swab and sputum sample from four patient with ARTI and three had rash in the course of disease.Conclutions:1.For simple and small genome parvoviruses,the use of PCR technology to prepare parvoviruses-specific DNA microarray probe and target sequence is a fast,simple,low-cost and practical method.2.HBoV is detected in sputum and throat swab of 15 children hospitalized for ARTI,and was associated with acute lower respiratory tract infection.Co-infections were frequent and clinically similar to single infections.HBoV DNA was not detected in the serum sample.3.The positive rate of B19 was lower(1.6%)in the respiratory tract secretions,but the method of detecting B19 infection with non-blood sample was worth to apply,expecially for the detection of etiological agents of rash illlness in children.