实时荧光核酸恒温扩增检测技术在乙型肝炎病毒感染者低病毒载量样本检测中的优势分析及临床验证

黄群芳; 谢如冰; 兰艳平; 荀振; 欧启水; 刘灿

doi:10.12182/20260360201

实时荧光核酸恒温扩增检测技术在乙型肝炎病毒感染者低病毒载量样本检测中的优势分析及临床验证

Analysis of the Advantages and Clinical Validation of Real-Time Fluorescence Nucleic Acid Isothermal Amplification Detection Technology in Detecting Low Viral Load Samples From HBV-Infected Patients

摘要

摘要:
目的探讨实时荧光核酸恒温扩增检测技术（simultaneous amplification and testing, SAT）在乙型肝炎病毒（HBV）感染者低病毒载量样本检测中的优势及其临床应用价值。
方法以逆转录实时荧光定量PCR法（RT-qPCR）检测为参照，评估实时荧光核酸恒温扩增检测技术（SAT）的检测性能（包括线性范围、精密度和检出限）。两种方法检测样品均为HBV核糖核酸（RNA）国家标准品。共纳入170例慢性HBV感染者进行方法学比较，根据血清HBV DNA水平将其分为高水平组（>100 IU/mL，n=111）和低水平组（≤100 IU/mL，n=59），验证两种方法检测结果的相关性和一致性。进一步基于1006例慢性HBV感染患者数据分析HBV RNA的分布特征及其与HBV标志物的相关性。
结果 SAT与RT-qPCR法检测HBV RNA相比，线性范围更宽（10²～10⁸ copies/mL vs. 10³～10⁸ copies/mL），低浓度样本精密度更高（批内变异系数4.23% vs. 12.82%），检出限更低（50 copies/mL vs. 500 copies/mL）。在临床样本检测中，SAT法总体检出率高于RT-qPCR法（72.35% vs. 57.64%，P<0.01），在HBV DNA低水平组中SAT法检出率亦高于RT-qPCR法（50.85% vs. 28.81%，P=0.007）。大样本分析显示，在HBV DNA<20 IU/mL的患者中，仍有40.4%可检出HBV RNA，且HBsAg≥100 IU/mL者阳性率达55.5%。相关性分析显示，HBV RNA与HBsAg（r=0.506）及HBeAg（r=0.454）均呈中等强度正相关，与ALT（r=－0.098）及AST（r=－0.082）呈微弱负相关（P均<0.05）。
结论 SAT法在检测低水平HBV RNA时具有更高的灵敏度与稳定性。HBV RNA可作为评估病毒转录活性的血清学标志物，在HBV DNA阴性或低水平患者的临床管理中具有应用价值。

Abstract:
Objective This paper examines the advantages and clinical application value of simultaneous amplification and testing (SAT) real-time fluorescent nucleic acid isothermal amplification detection technology for detecting low viral load samples in individuals infected with hepatitis B virus (HBV).
Methods Using reverse transcription real-time fluorescence quantitative PCR (RT-qPCR) as the reference, the detection performance of real-time fluorescence nucleic acid isothermal amplification detection technology (SAT) was evaluated, including linear range, precision, and detection limit. Both methods were used to detect samples, which were the national standard substances of hepatitis B virus ribonucleic acid (HBV RNA). A total of 170 patients with chronic HBV infection were included for methodological comparison. They were divided into a high-level group (serum HBV DNA > 100 IU/mL, n = 111) and a low-level group (serum HBV DNA ≤ 100 IU/mL, n = 59) based on serum HBV DNA levels. The correlation and consistency of the test results from the two methods were evaluated. Additionally, the distribution characteristics of HBV RNA and its correlation with HBV markers were analyzed using data from 1006 patients with chronic HBV infection.
Results Compared with the RT-qPCR method for detecting HBV RNA, the SAT method demonstrated a wider linear range (10²-10⁸ copies/mL vs. 10³-10⁸ copies/mL), higher precision for low-concentration samples (intra-assay coefficient of variation: 4.23% vs. 12.82%), and a lower detection limit (50 copies/mL vs. 500 copies/mL). In clinical sample testing, the overall detection rate of the SAT method was higher than that of the RT-qPCR method (72.35% vs. 57.64%, P < 0.01), and the detection rate of the SAT method was also higher in the HBV DNA low-level group (50.85% vs. 28.81%, P = 0.007). Large-sample analysis showed that among patients with HBV DNA < 20 IU/mL, 40.4% still had detectable HBV RNA, and the positive rate for HBsAg ≥ 100 IU/mL was 55.5%. Correlation analysis indicated that HBV RNA was moderately positively correlated with HBsAg (r = 0.506) and HBeAg (r = 0.454), and weakly negatively correlated with ALT (r = －0.098) and AST (r = －0.082) (all P < 0.05).
Conclusion The SAT method offers higher sensitivity and stability in detecting low-level HBV RNA. HBV RNA can serve as a serological marker for evaluating viral transcription activity and has clinical application value in managing patients who are HBV DNA-negative or have low HBV DNA levels.

HTML全文

参考文献(31)

施引文献

资源附件(0)