title: New universal primers for genotyping and resistance detection of low HBV DNA levels.
abstract: HBV (hepatitis B virus) genotyping is important in determining the clinical manifestation of disease and treatment response, particularly, in patients with low viral loads. Also, sensitive detection of HBV antiviral drug resistance mutations is essential for monitoring therapy response.Asensitive direct sequencing method for genotyping and the drug resistance mutation detection of low levels of HBV DNA in patients' plasma is developed by PCR amplification of the DNA with novel universal primers.The novel, common, and universal primers were identified by alignment of RT region of all the HBV DNA sequences in databases. These primers could efficiently amplify the RT region of HBV virus at low DNA levels by directly sequencing the resulting PCR products, and mapping with the reference sequence made it possible to clearly obtain the HBV subtypes and identify the resistance mutations in the samples with HBV DNA level as low as 20 IU/mL. We examined the reliability of the method in clinical samples, and found it could detect the HBV subtypes and drug resistance mutations in 80 clinical HBV samples with low HBV DNA levels ranging from 20 to 200 IU/mL.This method is a sensitive and reliable direct sequencing method for HBV genotyping and antiviral drug resistance mutation detection, and is helpful for efficiently monitoring the response to therapy in HBV patients.
authors.affiliation: Department of Clinical Laboratory Clinical Molecular Diagnostic Center, Renmin Hospital of Wuhan University Department of Pathology Affiliated Tianyou Hospital of Wuhan University of Science and Technology, Wuhan Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China Pennsylvania State University College of Medicine and Hershey Medical center, Hershey, PA New York University College of Arts and Science, New York, NY..
authors.forename: Yongqing
authors.initials: Y
authors.lastname: Tong
authors.forename: Bei
authors.initials: B
authors.lastname: Liu
authors.forename: Hui
authors.initials: H
authors.lastname: Liu
authors.forename: Hongyun
authors.initials: H
authors.lastname: Zheng
authors.forename: Jian
authors.initials: J
authors.lastname: Gu
authors.forename: Hang
authors.initials: H
authors.lastname: Liu
authors.forename: Min
authors.initials: M
authors.lastname: Lin
authors.forename: Yali
authors.initials: Y
authors.lastname: Ding
authors.forename: Chunhua
authors.initials: C
authors.lastname: Song
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
mesh_terms: D000328:Adult; D000368:Aged; D000998:Antiviral Agents / Q000627:therapeutic use; D017931:DNA Primers / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D024882:Drug Resistance, Viral / Q000235:genetics; D060005:Genotyping Techniques; D006509:Hepatitis B / Q000097:blood / Q000188:drug therapy / Q000821:virology*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D008875:Middle Aged; D010802:Phylogeny; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D016896:Treatment Outcome; D019562:Viral Load / Q000235:genetics; D055815:Young Adult
chemical_list: D000998:Antiviral Agents; D017931:DNA Primers; D004279:DNA, Viral