A novel primer set for improved direct gene sequencing of human bocavirus genotype-1 from clinical samples

•HBoV is a parvovirus that causes respiratory and enteric diseases in humans.•A new primer set used in conventional PCR and direct gene sequencing was developed.•The new primer set detected all real-time positive samples.•Genotying of HBoV strains was conducted for the first time in Egypt.•High rate...

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Bibliographic Details
Published in:Journal of virological methods 2016-02, Vol.228, p.108-113
Main Authors: Abdel-Moneim, Ahmed S., Kamel, Mahmoud M., Hamed, Dina H., Hassan, Safaa S., Soliman, May S., Al-Quraishy, Saleh A., El Kholy, Amani A.
Format: Article
Language:English
Subjects:
Child, Preschool
Cross Infection - virology
DNA Primers
Egypt
Female
Genotype
Genotyping
Human bocavirus
Human bocavirus - genetics
Human bocavirus - isolation & purification
Humans
Infant
Male
Nasopharynx - virology
Nosocomial infection
Parvoviridae Infections - virology
Parvovirus
Phylogeny
Real-Time Polymerase Chain Reaction
Respiratory tract infection in children
Respiratory Tract Infections - virology
Sequence Analysis, DNA - methods
Viral Load
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Summary:•HBoV is a parvovirus that causes respiratory and enteric diseases in humans.•A new primer set used in conventional PCR and direct gene sequencing was developed.•The new primer set detected all real-time positive samples.•Genotying of HBoV strains was conducted for the first time in Egypt.•High rate of nosocomial infection with HBoV-1 was detected in children in Egypt. Human bocavirus genotype (HBoV-1) is a parvovirus associated with respiratory tract infections in children with different degrees of severity. The current study intended to improve the direct gene sequencing of the HBoV-1 using a newly developed primer set. Screening the presence of human bocavirus infection among in-patients children suffering from lower respiratory tract infections was another aim of the current study. Nasopharyngeal swab samples from in-patients children suffering from lower respiratory tract infections were examined. The real-time polymerase chain reaction was used for the initial screening as a highly sensitive method to detect the HBoV. Genotyping of real-time positive samples was attempted by direct sequencing of PCR amplicons using NP, VP1/2 and the newly developed VP/NC primers. HBoV-1 was present in 56.8% of the examined children. The newly developed primer set successfully amplified all real-time PCR positive samples, however, the other primer pairs did not reliably detect real-time PCR positive samples. The gene sequences of the detected HBoV-1 showed conserved sequences to each other with a low rate of discrepancies. The high rate of infection and the similarity between the detected strains strongly suggest nosocomial infections.
ISSN:0166-0934
1879-0984
DOI:10.1016/j.jviromet.2015.11.023