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Reverse transcriptase droplet digital PCR vs reverse transcriptase quantitative real‐time PCR for serum HBV RNA quantification
Author(s) -
Limothai Umaporn,
Chuaypen Natthaya,
Poovorawan Kittiyod,
Chotiyaputta Watcharasak,
Tanwandee Tawesak,
Poovorawan Yong,
Tangkijvanich Pisit
Publication year - 2020
Publication title -
journal of medical virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 121
eISSN - 1096-9071
pISSN - 0146-6615
DOI - 10.1002/jmv.25792
Subject(s) - reverse transcriptase , virology , real time polymerase chain reaction , reverse transcription polymerase chain reaction , digital polymerase chain reaction , rna directed dna polymerase , rna , biology , microbiology and biotechnology , messenger rna , polymerase chain reaction , gene , genetics
Serum hepatitis B virus (HBV) RNA is a novel marker reflecting the activity of covalently closed circular DNA. However, the methodology for detecting HBV RNA has been a technical challenge. In this study, the performance of reverse transcription droplet digital polymerase chain reaction (RT‐ddPCR) for quantifying HBV RNA was compared with that of reverse transcription quantitative real‐time PCR (RT‐qPCR) in serum samples collected from treatment‐naïve patients with different phases of chronic hepatitis B (CHB). A total of 417 serum samples, including 136 HBeAg‐positive CHB and 281 HBeAg‐negative CHB were examined. HBV RNA levels measured by RT‐ddPCR and RT‐qPCR showed a high degree of linearity and quantitative correlation. The limit of detections of RT‐ddPCR and RT‐qPCR assays were 10 2 and 10 3 copies/mL, respectively. Our results also demonstrated that RT‐ddPCR was superior to RT‐qPCR in terms of its consistency for quantifying HBV RNA across all concentrations. In the HBeAg‐positive group, serum HBV RNA levels based on RT‐ddPCR were moderately correlated with HBV DNA ( r = 0.591, P < .001) and HBsAg ( r = 0.502, P < .001). Among patients with HBeAg‐negative CHB, serum HBV RNA levels were moderately correlated with HBV DNA ( r = 0.603, P < .001) but had weak correlation with HBsAg ( r = 0.203, P = .001). In summary, RT‐ddPCR could enhance the sensitivity of serum HBV RNA detection, particularly among the HBeAg‐negative group with low viral loads. Thus, RT‐ddPCR could serve as an optimal method for HBV RNA quantification in clinical practice.