Establishment and Clinical Application of LNA-PCR Assay Detecting Hepatitis B Virus Adefovir Dipivoxil Resistance

doi:10.13523/j.cb.20180907

China Biotechnology

2018, Vol. 38

Issue (9): 48-54 DOI: 10.13523/j.cb.20180907

Orginal Article

Establishment and Clinical Application of LNA-PCR Assay Detecting Hepatitis B Virus Adefovir Dipivoxil Resistance

Xiao-yong ZHANG¹,Qian-cheng LUO^2,^**(

)

¹ Shanghai MOLE Diagnostics Co. Ltd, Shanghai 201203,Chain
² Gongli Hospital Affiliated to the Second Military Medical University, Shanghai 200135,Chain

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Abstract

Objective: To develop a simple, quick and sensitive method based on the LNA (locked nucleic acid) PCR for detection of rtA181V and rtN236T mutations associated with adefovir dipivoxil (ADV) resistance of hepatitis B virus (HBV). Method: Built wild strains and the mutant recombinant plasmids of ADV rt181 and rt236 by gene sequencing screening positive samples, then designed the specific primers and LNA fluorescent probes (rtA181V, rtN236T) to construct recombinant plasmid for standard real-time fluorescent PCR reaction system, and determined the feasibility and accuracy of the detection method through the parallel to gene sequencing to detect serum samples. Result: LNA-PCR assay could detect 10 ² copies/ml mutant template, with high specificity. Eighty-nine HBV DNA positive samples were from patients with ADV therapy over one year. LNA-PCR detected two (2.24%) rtA181V and rtN236T dual mutations, eight (8.98%) rtA181V mutations, five (5.61%) N236T mutations. Complete concordance between LNA-PCR and sequencing were observed with all samples. Conclusion: LNA-PCR test is a simple, fast, and sensitive method for monitoring ADV resistant mutations of HBV in patients with chronic hepatitis B.

Key words： Hepatitis B virus Adefovir dipivoxil Locked nucleic acid Mutation Drug resistance

Received: 28 March 2018 Published: 12 October 2018

Corresponding Authors: Qian-cheng LUO E-mail: luoqiancheng19@163.com

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Cite this article:

Xiao-yong ZHANG,Qian-cheng LUO. Establishment and Clinical Application of LNA-PCR Assay Detecting Hepatitis B Virus Adefovir Dipivoxil Resistance. China Biotechnology, 2018, 38(9): 48-54.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180907 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I9/48

Table 1 Primers and probe sequences of ADV-resistant rtA181V and rtN236T sites

Fig.1 Identification of recombinant plasmid rt181A/rt181V/rt236N/rt236T

Fig. 2 Mutant plasmid sequencingThe box indicates the mutation site

Fig.3 LNA-PCR detection ability test1-6: Indicate the amplification curve of the HBV ADV mutant plasmid with 10⁷ to 10²copies/ml;7:The PCR negative control;8:The 10⁷copies/ml wild-type plasmid amplification curve

Table 2 Repeatability test results of LNA-PCR method

Fig. 4 Clinical sample LNA-PCR test results0 for PCR negative control


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