应用NASBA定量检测HCV RNA的研究进展

NASBA（nucleic acid sequence-based amplification)即核酸序列依赖性扩增法，是由一对引物介导的、连续均一的、体外特异性核苷酸序列等温扩增的RNA新技术。反应在42℃进行，可在2h内将RNA模板扩增约10^9倍。本文综述NASBA在HCV RNA定量检测中的应用。     

实时荧光RT-PCR法和NASBA方法在呼吸道多病原检测中的应用评价

目的 评价实时荧光RT-PCR和核酸依赖性序列扩增法（NASBA）两种方法在呼吸道多病原检测中的应用价值。方法 收集北京地区急性呼吸道感染病例标本140份,分别采用实时荧光RT-PCR方法和NASBA方法对样本进行平行检测并对两种方法的病原检出限、重复性、检测一致性及检测时间等指标进行比较。结果 两种检测方法批内重复CV值均<5%,重复性较好;实时荧光RT-PCR方法对脊灰毒株核酸和H1N1病毒核酸最低检出限分别为5.62 CCID50 /0.1 ml（107 倍稀释）和10³倍稀释度,NASBA方法最低检出限分别为56.2 CCID50 /0.1 ml（106 倍稀释）和10²倍稀释。实时荧光RT-PCR灵敏度略优于NASBA方法。针对两种方法共同检测呼吸道病原体种类,实时荧光RT-PCR和NASBA方法检测阳性率分别为89.28%（125/140）、70%（98/140）。两种方法对甲型H1N1病毒、肠道病毒及新型冠状病毒的检测结果一致性达100%,对甲型流感病毒、甲型H3N2、副流感病毒1-4型的检测结果基本一致, Kappa 值范围为0.81~1;对冠状病毒229E/HKU1/OC43/NL63、鼻病毒、呼吸道合胞病毒和乙型流感病毒的检测结果一致性较好, Kappa 值范围为0.61~0.80;对人偏肺病毒的检测结果一致性差, Kappa 值仅为0.55。实时荧光RT-PCR方法检测时间为90 min,NASBA方法仅需40 min即可完成。 结论 实时荧光RT-PCR方法灵敏度较高,适合对大批量临床样本进行筛查;NASBA方法检测时间短,不易污染,适用于时限性强但对灵敏度要求不太高的应用场景,如临床上重症呼吸道感染者的快速检测。建议根据不同需求和应用场景,选择合适的检测方法和产品。     

Accordance and concordance of PCR and NASBA followed by oligochromatography for the molecular diagnosis of Trypanosoma brucei and Leishmania

Objective To evaluate the repeatability and reproducibility of four simplified molecular assays for the diagnosis of Trypanosoma brucei spp. or Leishmania ssp. in a multicentre ring trial with seven participating laboratories. Methods The tests are based on PCR or NASBA amplification of the parasites nucleic acids followed by rapid read‐out by oligochromatographic dipstick (PCR‐OC and NASBA‐OC). Results On purified nucleic acid specimens, the repeatability and reproducibility of the tests were Tryp‐PRC‐OC, 91.7% and 95.5%; Tryp‐NASBA‐OC, 95.8% and 100%; Leish‐PCR‐OC, 95.9% and 98.1%; Leish‐NASBA‐OC, 92.3% and 98.2%. On blood specimens spiked with parasites, the repeatability and reproducibility of the tests were Tryp‐PRC‐OC, 78.4% and 86.6%; Tryp‐NASBA‐OC, 81.5% and 89.0%; Leish‐PCR‐OC, 87.1% and 91.7%; Leish‐NASBA‐OC, 74.8% and 86.2%. Conclusion As repeatability and reproducibility of the tests were satisfactory, further phase II and III evaluations in clinical and population specimens from disease endemic countries are justified.     

Development and evaluation of a real-time nucleic acid sequence based amplification assay for rapid detection of influenza A

The development and introduction of effective treatment for influenza A in the form of neuraminidase inhibitors have made the rapid diagnosis of infection important especially in high-risk populations. The aim of this study was to develop a real-time nucleic acid sequenced based amplification (NASBA) using a molecular beacon that could detect a wide range of influenza A subtypes and strains in a single reaction by targeting a conserved region of the influenza genome, and to evaluate its sensitivity and specificity against traditional laboratory techniques on a range of clinical samples usefulness during the 2003/2004 influenza season. The results demonstrated the assay to be highly sensitive and specific, detecting <0.1 TCID50 of virus stock. Three hundred eighty-nine clinical samples were tested in total from two patient groups. Overall, the real-time NASBA assay detected 64% (66/103) more influenza positive samples than cell culture and direct immunofluorescence (IF) and, therefore, was shown to be more sensitive in detecting influenza A in a wide range of respiratory samples than traditional methods. In conclusion, the real-time influenza A assay demonstrated clinical usefulness in both hospital and community populations.     

Sensitivity studies for quantitative assays: use of censored data analysis

Probit regression analysis is frequently used to study the relation between the concentration of an analyte in a sample and the probability that the assay used yields a positive test result. For these analyses only the qualitative classification 'positive' or 'negative' is used, whereas, particularly in the case where the assay is quantitative in nature, the results contain more information. In the current paper, we propose an alternative method, in which the negative test results are treated as being (left) censored. As such, more efficient use is made of the information in the data. The procedures are illustrated using two generations of NucliSens assays (BioMérieux), which are designed to quantify the viral load of HIV-1 in blood samples. Computer simulations are used to illustrate some properties of the estimated parameters.     

Plasma HIV-1 load and disease progression in HIV-Infected patients in Hungary

Nucleic Acid Sequence Based Amplification (NASBA) is a suitable method for the quantification of HIV-1 RNA in plasma and serum samples. Since determination of the viral load appears to be a valuable marker for the prediction of disease progression and for monitoring the efficiency of antiretroviral therapy, the National AIDS Committee initiated the introduction of NASBA in Hungary at the end of 1996. We obtained plasma samples from patients with ARC and AIDS of the Szt. László Hospital, Budapest. We found an increased viral burden in untreated AIDS (CDC group C) patients compared to untreated ARC (CDC group B) patients. In plasma samples of clinically stable ARC and AIDS patients treated with antiretroviral drugs we detected relatively low HIV-1 RNA copy levels while similarly treated ARC and AIDS patients with progressive disease had high HIV-1 RNA copy numbers. The CD4⁺ T-cell count was lower in AIDS patients compared to ARC patients, as expected. In general, there was an inverse correlation (r = -0.487, P < 0.0001) between CD4⁺ T-cell counts and HIV-1 RNA levels. We concluded that measurement of HIV-1 RNA plasma level has an important role in assessing prognosis and effects of antiretroviral therapy in HIV-infected patients.     

一种基于NASBA技术的新型快速肠道病毒检测方法的应用研究

目的: 基于核酸等温扩增技术(nucleic acid sequence-based amplification,NASBA)荧光分子信标探针检测技术,进行肠道病毒的快速检测与辅助诊断?方法:根据Genebank上肠道病毒5′端非编码区序列,设计特异性引物与捕获探针,应用纳米技术对商品化磁珠进行偶联,NASBA方法进行扩增,NucliSens读数仪检测,荧光定量RT-PCR方法进行验证,同时验证NASBA方法的特异性?灵敏度和重复性?结果:该方法对肠道病毒具有高度特异性,与RSV等呼吸道相关病毒均无交叉反应,反应体系具有很高的稳定性?结论:本研究应用的肠道病毒NASBA检测方法特异?灵敏?快速简便,适用于肠道病毒的日常监测和爆发疫情的应急诊断?     

Predicting CIN2+ when detecting HPV mRNA and DNA by PreTect HPV-proofer and consensus PCR: A 2-year follow-up of women with ASCUS or LSIL Pap smear

It has been suggested that human papillomavirus (HPV) testing improves follow-up of atypical cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL) in cervical cancer screening programs. To evaluate the prognostic value of including HPV testing as an adjunct to cytology, we carried out a 2-year follow-up study of 77 women with ASCUS or LSIL Papanicolaou (Pap) smear in the Norwegian Cervical Cancer Screening Program (NCCSP) for detection of histological cervical intraepithelial neoplasia (CIN) 2+. The study includes a comparison between viral mRNA and DNA detection. PreTect HPV-Proofer was used for HPV E6/E7 mRNA detection from the 5 high-risk types 16, 18, 31, 33 and 45, and Gp5+/6+ consensus PCR was used for HPV DNA detection. Twice as many women were positive for HPV DNA (54.6%) than for HPV mRNA (23.4%). PreTect HPV-Proofer and consensus PCR had a sensitivity of 85.7% (95% confidence interval [CI] = 42.1-99.6) for detecting CIN2+ during follow-up. The specificity was significantly higher for PreTect HPV-Proofer, 84.9% (95% CI = 73.9-92.5), than for consensus PCR, 50.0% (95% CI = 37.4-62.6). PreTect HPV-Proofer positive women were 69.8 times (95% CI = 4.3-1137.3) more likely to be diagnosed with CIN2+ within 2 years than PreTect HPV-Proofer negative women. Consensus PCR-positive women were 5.7 times (95% CI = 0.6-52.0) more likely to be diagnosed with CIN2+ within 2 years than PCR-negative women. With equal sensitivity and higher specificity than consensus PCR, the PreTect HPV-Proofer might offer an improvement for the triage of women with ASCUS or LSIL Pap smear.     

Comparison of nucleic acid sequence-based amplification and loop-mediated isothermal amplification for diagnosis of human African trypanosomiasis

Diagnosis of human African trypanosomiasis (HAT) using molecular tests should ideally achieve high sensitivity without compromising specificity. This study compared 2 simplified tests, nucleic acid sequence-based amplification (NASBA) combined with oligochromatography (OC) and loop-mediated isothermal amplification (LAMP), executed on 181 blood samples from 65 Trypanosoma brucei gambiense HAT patients, 86 controls, and 30 serological suspects from Uganda. Basing on the composite reference standard, the diagnostic sensitivity and specificity of NASBA were 93.9% (95% confidence interval [CI] = 84.9–98.3%) and 100% (95% CI = 94.9–100%), respectively. The same parameters for LAMP were 76.9% (95% CI = 64.8–86.5%) and 100% (95% CI = 91.6–100%), respectively. The level of agreement between LAMP and microscopy was good with a kappa (κ) value of 79.2% (95% CI = 69.4–88.9%), while that of NASBA-OC/microscopy was very good (κ value 94.6%; 95% CI = 89.3–99.8%). The sensitivity of NASBA-OC was significantly higher than that of LAMP (Z = 2.723; P = 0.007). These tests have potential application to HAT surveillance.