An ultra-fast thermocycling fluorescence quantitative PCR system for detection of influenza virus: evaluation on its efficacy

Yantong GUO; Zhongming LIU; Haiyan ZHANG; Bao ZHANG

doi:10.16016/j.2097-0927.202203128

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An ultra-fast thermocycling fluorescence quantitative PCR system for detection of influenza virus: evaluation on its efficacy

Laboratory Medicine | 更新时间：2026-01-09

- An ultra-fast thermocycling fluorescence quantitative PCR system for detection of influenza virus: evaluation on its efficacy
- Journal of Army Medical University Vol. 44, Issue 20, Pages: 2113-2119(2022)
- 作者机构：
  
  1.510515 广州，南方医科大学公共卫生学院广东省热带病研究重点实验室BSL-3实验室(广东)
  2.510010 广州，中国人民解放军南部战区总医院
- 作者简介：
- 基金信息：
  
  the Special Project of Biosecurity Research of PLA(19SWAQ24);the Special Fund for Military Laboratory Animals(SYDW[2020]18)
- DOI：10.16016/j.2097-0927.202203128
  CLC： R197.39;R373.13;R446.61
- Received：15 March 2022，
  
  Revised：2022-07-04，
  
  Published：30 October 2022
- 稿件说明：
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Yantong GUO, Zhongming LIU, Haiyan ZHANG, et al. An ultra-fast thermocycling fluorescence quantitative PCR system for detection of influenza virus: evaluation on its efficacy[J]. Journal of Army Medical University, 2022, 44(20): 2113-2119.
DOI：

Yantong GUO, Zhongming LIU, Haiyan ZHANG, et al. An ultra-fast thermocycling fluorescence quantitative PCR system for detection of influenza virus: evaluation on its efficacy[J]. Journal of Army Medical University, 2022, 44(20): 2113-2119. DOI： 10.16016/j.2097-0927.202203128.

摘要

目的

探讨极速热循环荧光定量PCR系统在甲型流感病毒(influenza A virus，IAV)和乙型流感病毒(influenza B virus，IBV)检测中的性能。

方法

构建IAV和IBV的质粒标准品，浓度梯度稀释，每个浓度重复检测20次后，制作浓度标准曲线，评价系统的最低检出限、线性范围和线性相关系数。收集来自南部战区疾病预防控制中心生物安全科2016年至2018年的临床样本278例(IAV阳性样本88例，IBV阳性样本87例，阴性样本103例)，其中男性164人(59%)，女性114人(41%)，平均年龄24岁，通过重复检测相同IAV、IBV临床样本评价其分析重现性；将系统检测278例鼻/咽拭子临床样本与传统实时荧光定量PCR(real-time quantitative PCR，RT-qPCR)结果比较评价其效果。

结果

极速热循环荧光定量PCR系统检测IAV和IBV质粒标准品的最低检出限为1×10

copies/μL；线性检测范围是1×10

~1×10

copies/μL；此系统检测流感临床样本的敏感度为92.57%，特异度均为100%，阴、阳性预测值分别为88.79%和100%，与RT-qPCR结果的符合率为95.32%；IAV与IBV之间以及IAV和IBV与其他多种病原体之间无交叉反应；30 g/dL的血红蛋白、3.2 g/dL的甘油三酯和100 μg/mL头孢曲松对检测结果无干扰。

结论

极速热循环荧光定量PCR系统体积小、重量轻、无需核酸提取步骤、耗时短、检测性能良好，适用于即时检测(point-of-care testing，POCT)。

Abstract

Objective

To evaluate the performance and application efficacies of a ultra-fast thermocycling fluorescence quantitative PCR system for detection of influenza A virus (IAV) and influenza B virus (IBV).

Methods

Our constructed plasmids of IAV and IBV was employed as standards

and then the concentration was diluted gradiently. After each concentration was repeatedly detected with the ultra-fast thermocycling fluorescence quantitative PCR system for 20 times

a standard curve was drawn to verify the minimum detection limit and liner range of the system and correlative coefficient. A total of 278 clinical samples (88 IAV-positive

87 IBV-positive

and 103 negative samples) were collected from the Biosafety Section of Center for Disease Control and Prevention

Southern Theater Command. They were isolated during 2016 and 2018

from the patients of 164 males (59%) and 114 females (41%)

and at an average age of 24 years. The repeatability of the system was analyzed through the system repeatedly detect the identified 88 IAV-positive and 87 IBV-positive samples. The results of the system and traditional real-time q-PCR (RT q-PCR) were compared to evaluate the efficacy of the system.

Results

The results of ultra-fast thermocycling fluorescence quantitative PCR system showed that the minimum detection limit for detection of IAV and IBV plasmids was 1×10

copies/μL

and the liner range ranged from 1×10

to 1×10

copies/μL. In detection of clinical samples

this system obtained a sensitivity of 92.57%

a specificity of 100%

and negative and positive predictive values of 88.79% and 100%

respectively

with a coincidence rate of 95.32% when compared with the results of traditional RT q-PCR. There were no cross-reactions between IAV and IBV or among them with other pat

hogens. No interference was observed in the detection results of influenza in presence of 30 g/dL hemoglobin

3.2 g/dL triglyceride and 100 μg/mL ceftriaxone sodium.

Conclusion

The system shows good detection performance in detection of clinical samples

with advantages of small in size

light in weight

short in time

and no procedure of nucleic acid extraction

and is suitable for point-of-care testing (POCT).

关键词

Keywords

references

MAHONY J B, PETRICH A, SMIEJA M. Molecular diagnosis of respiratory virus infections[J]. Crit Rev Clin Lab Sci, 2011, 48(5/6): 217-249. DOI: 10.3109/10408363.2011.640976.

疾病预防控制局, 2021年全国法定传染病疫情概况[EB/OL]. [2022-04-22]. http://www.nhc.gov.cn/jkj/s3578/202204/4fd88a291d914abf8f7a91f6333567e1.shtml

National Administration of Disease Prevention and Control, National epidemiological profile of statutory infectious diseases in 2021[EB/OL][2022-04-22]. http://www.nhc.gov.cn/jkj/s3578/202204/4fd88a291d914abf8f7a91f6333567e1.shtml

FANG S S, LI J X, CHENG X W, et al . Simultaneous detection of influenza virus type B and influenza A virus subtypes H1N1, H3N2, and H5N1 using multiplex real-time RT-PCR[J ] . Appl Microbiol Biotechnol, 2011, 90(4): 1463-1470. DOI: 10.1007/s00253-011-3192-8.

DIMAIO M A, SAHOO M K, WAGGONER J, et al . Comparison of Xpert Flu rapid nucleic acid testing with rapid antigen testing for the diagnosis of influenza A and B[J ] . J Virol Methods, 2012, 186(1/2): 137-140. DOI: 10.1016/j.jviromet.2012.07.023.

GANGULI A, MOSTAFA A, BERGER J, et al . Rapid isothermal amplification and portable detection system for SARS-CoV-2[J ] . Proc Natl Acad Sci USA, 2020, 117(37): 22727-22735. DOI: 10.1073/pnas.2014739117.

ALAFEEF M, MOITRA P, DIGHE K, et al . RNA-extraction-free nano-amplified colorimetric test for point-of-care clinical diagnosis of COVID-19[J ] . Nat Protoc, 2021, 16(6): 3141-3162. DOI: 10.1038/s41596-021-00546-w.

AZMI I, FAIZAN M I, KUMAR R, et al . A saliva-based RNA extraction-free workflow integrated with Cas13a for SARS-CoV-2 detection[J ] . Front Cell Infect Microbiol, 2021, 11: 632-646. DOI: 10.3389/fcimb.2021.632646.

CHAN K, WEAVER S C, WONG P Y, et al . Rapid, affordable and portable medium-throughput molecular device for zika virus[J ] . Sci Rep, 2016, 6: 38223. DOI: 10.1038/srep38223.

LEE J H, CHEGLAKOV Z, YI J, et al . Plasmonic photothermal gold bipyramid nanoreactors for ultrafast real-time bioassays[J ] . J Am Chem Soc, 2017, 139(24): 8054-8057. DOI: 10.1021/jacs.7b01779.

LEE S H, PARK S M, KIM B N, et al . Emerging ultrafast nucleic acid amplification technologies for next-generation molecular diagnostics[J ] . Biosens Bioelectron, 2019, 141: 111448. DOI: 10.1016/j.bios.2019.111448.

MENDOZA-GALLEGOS R A, RIOS A, GARCIA-CORDERO J L. An affordable and portable thermocycler for real-time PCR made of 3D-printed parts and off-the-shelf electronics[J]. Anal Chem, 2018, 90(9): 5563-5568. DOI: 10.1021/acs.analchem.7b04843.

LI Z Y, WANG P, TONG L M, et al . Gold nanorod-facilitated localized heating of droplets in microfluidic chips[J ] . Opt Express, 2013, 21(1): 1281-1286. DOI: 10.1364/OE.21.001281.

LI T J, CHANG C M, CHANG P Y, et al . Handheld energy-efficient magneto-optical real-time quantitative PCR device for target DNA enrichment and quantification[J ] . NPG Asia Mater, 2016, 8(6): e277. DOI: 10.1038/am.2016.70.

军队急性呼吸道传染病病原监测方案(2019修订版)[S], 2019.

Army acute respiratory infectious disease pathogen surveillance program (2019)[S], 2019.

王伟. 介绍一种评价临床检查结果一致性的新指标——Kappa值[J]. 天津医药, 1991, 19(10): 639-640.

WANG W. Introduction of a new index to evaluate the consistency of clinical examination results—Kappa value[J]. Tianjin Med J, 1991, 19(10): 639-640.

AZAR M M, LANDRY M L. Detection of influenza A and B viruses and respiratory syncytial virus by use of clinical laboratory improvement amendments of 1988 (CLIA)-waived point-of-care assays: a paradigm shift to molecular tests[J]. J Clin Microbiol, 2018, 56(7): e00367-e00318. DOI: 10.1128/JCM.00367-18.

SLOMSKI A. Molecular point-of-care test improved influenza care[J]. JAMA, 2021, 325(12): 1135. DOI: 10.1001/jama.2021.3574.

WESTGARD S A, GOLDSCHMIDT H M J, EHRMEYER S S. POCT analysts' perspective: practices and wants for improvement[J]. J Appl Lab Med, 2020, 5(3): 480-493. DOI: 10.1093/jalm/jfaa037.

MAUTNER L, BAILLIE C K, HEROLD H M, et al . Rapid point-of-care detection of SARS-CoV-2 using reverse transcription loop-mediated isothermal amplification (RT-LAMP)[J ] . Virol J, 2020, 17(1): 160. DOI: 10.1186/s12985-020-01435-6.

覃岭, 代艳超, 孙坚萍, 等. 流感患者TNF-α和IL-10水平以及与病毒载量相关性研究[J]. 传染病信息, 2019, 32(5): 415-417, 421.

QIN L, DAI Y C, SUN J P, et al . Levels of TNF-α and IL-10 and their correlation with influenza virus load in influenza patients[J ] . Infect Dis Inf, 2019, 32(5): 415-417, 421.

陈韵颖, 叶先飞, 周俊, 等. 2357例流感样患儿甲乙型流感病毒检测结果及病毒载量分析[J]. 中华医院感染学杂志, 2020, 30(2): 278-282.

CHEN Y Y, YE X F, ZHOU J, et al . Detection of influenza A and B viruses and viral loads in 2 357 children with influenza-like illness[J ] . Chin J Nosocomiology, 2020, 30(2): 278-282.

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