A novel multiplex PCR for virus detection by melting curve analysis

被引:3
作者
Liu, Pengcheng [1 ]
Lu, Lijuan [1 ]
Xu, Menghua [1 ]
Zhong, Huaqing [1 ]
Jia, Ran [1 ]
Su, Liyun [1 ]
Cao, Lingfeng [1 ]
Dong, Zuoquan [1 ]
Dong, Niuniu [1 ]
Zhou, Linfu [2 ]
Xu, Jin [1 ]
机构
[1] Fudan Univ, Childrens Hosp, Dept Clin Lab, 399 Wanyuan Rd, Shanghai 201102, Peoples R China
[2] Zhejiang Univ, Med Biotechnol Lab, 866 Yuhangtang Rd, Hangzhou 310058, Zhejiang, Peoples R China
关键词
Detection; Melting curve; Multiplex PCR; TaqMan probe; Virus; ALPHA-THALASSEMIA; RAPID DETECTION; NONDELETIONAL MUTATIONS; ONE-STEP;
D O I
10.1016/j.jviromet.2018.09.010
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Background: Rapid and accurate laboratory diagnoses of viral infections are crucial for the management and treatment of patients with viral infections. Conventional methods for virus detection are labourious, time consuming, and only a single virus can be analysed in one assay. Objectives: The objective of this study was to develop a novel real-time PCR method for multiple virus detection by melting curve analysis using Taqman probes in a single reaction. Study design: As a model, six respiratory viruses were detected in one tube using three fluorophores. The specificity was assessed by cross-reaction tests with other common respiratory pathogens. The analytical sensitivity was assessed by testing the limit of detection of the assay using artificial plasmids as the positive template. The clinical evaluation of the established assay was evaluated for the detection of respiratory viruses in clinical samples, and the results were compared with direct fluorescent antibody testing (DFA). Results: The six respiratory viruses were clearly distinguished by their respective melting temperature values in the corresponding fluorescence detection channels. No cross reactions were observed by cross reaction tests. The detection limits of this assay were 2 to 2 x 10(3) copies per reaction for each virus. The clinical evaluation of this assay was demonstrated by analysing 352 clinical samples, and 67(19.0%) samples were positive for at least one virus. The accordance rate between the established PCR and DFA testing was high, and ranged from 94.57% to 100%. Conclusions: Taqman probe-based melting curve analysis is well suited for detection of multiple viruses in clinical and research laboratories because of its high throughput, reliability, and cost savings.
引用
收藏
页码:56 / 60
页数:5
相关论文
共 20 条
  • [1] Botezatu I V, 2017, Mol Biol (Mosk), V51, P50, DOI 10.7868/S0026898417010062
  • [2] Optimization of melting analysis with TaqMan probes for detection of KRAS, NRAS, and BRAF mutations
    Botezatu, Irina V.
    Nechaeva, Irina O.
    Stroganova, Anna M.
    Senderovich, Anastasia I.
    Kondratova, Valentina N.
    Shelepov, Valery P.
    Lichtenstein, Anatoly V.
    [J]. ANALYTICAL BIOCHEMISTRY, 2015, 491 : 75 - 83
  • [3] Development of multiplexed bead arrays for the simultaneous detection of nucleic acid from multiple viruses in bat samples
    Boyd, Victoria
    Smith, Ina
    Crameri, Gary
    Burroughs, Amy L.
    Durr, Peter A.
    White, John
    Cowled, Christopher
    Marsh, Glenn A.
    Wang, Lin-Fa
    [J]. JOURNAL OF VIROLOGICAL METHODS, 2015, 223 : 5 - 12
  • [4] Development of a multiplex one step RT-PCR that detects eighteen respiratory viruses in clinical specimens and comparison with real time RT-PCR
    Choudhary, Manohar L.
    Anand, Siddharth P.
    Heydari, Mostafa
    Rane, Grishma
    Potdar, Varsha A.
    Chadha, Mandeep S.
    Mishra, Akhilesh C.
    [J]. JOURNAL OF VIROLOGICAL METHODS, 2013, 189 (01) : 15 - 19
  • [5] Rapid detection of α-thalassaemia alleles of _SEA/, -α3.7/ and -α4.2/ using a dual labelling, self-quenching hybridization probe/melting curve analysis
    Gao, Lan
    Liu, Yanhui
    Sun, Manna
    Zhao, Ying
    Xie, Rungui
    He, Yi
    Xu, Wanfang
    Liu, Jianxin
    Lin, Yangyang
    Lou, Jiwu
    [J]. MOLECULAR AND CELLULAR PROBES, 2015, 29 (06) : 438 - 441
  • [6] Simultaneous Genotyping of α-Thalassemia Deletional and Nondeletional Mutations by Real-Time PCR-Based Multicolor Melting Curve Analysis
    Huang, Qiuying
    Wang, Xudong
    Tang, Ning
    Yan, Tizhen
    Chen, Ping
    Li, Qingge
    [J]. JOURNAL OF MOLECULAR DIAGNOSTICS, 2017, 19 (04) : 567 - 574
  • [7] Rapid detection of non-deletional mutations causing α-thalassemia by multicolor melting curve analysis
    Huang, Qiuying
    Wang, Xudong
    Tang, Ning
    Zhu, Chunjiang
    Yan, Tizhen
    Chen, Ping
    Li, Qingge
    [J]. CLINICAL CHEMISTRY AND LABORATORY MEDICINE, 2016, 54 (03) : 397 - 402
  • [8] Multiplex Fluorescence Melting Curve Analysis for Mutation Detection with Dual-Labeled, Self-Quenched Probes
    Huang, Qiuying
    Liu, Zanzan
    Liao, Yiqun
    Chen, Xiaoyun
    Zhang, Yi
    Li, Qingge
    [J]. PLOS ONE, 2011, 6 (04):
  • [9] Development of a one-run real-time PCR detection system for pathogens associated with bovine respiratory disease complex
    Kishimoto, Mai
    Tsuchiaka, Shinobu
    Rahpaya, Sayed Samim
    Hasebe, Ayako
    Otsu, Keiko
    Sugimura, Satoshi
    Kobayashi, Suguru
    Komatsu, Natsumi
    Nagai, Makoto
    Omatsu, Tsutomu
    Naoi, Yuki
    Sano, Kaori
    Okazaki-Terashima, Sachiko
    Oba, Mami
    Katayama, Yukie
    Sato, Reiichiro
    Asai, Tetsuo
    Mizutani, Tetsuya
    [J]. JOURNAL OF VETERINARY MEDICAL SCIENCE, 2017, 79 (03) : 517 - 523
  • [10] Combination of fluorescence color and melting temperature as a two-dimensional label for homogeneous multiplex PCR detection
    Liao, Yiqun
    Wang, Xiaobo
    Sha, Chao
    Xia, Zhongmin
    Huang, Qiuying
    Li, Qingge
    [J]. NUCLEIC ACIDS RESEARCH, 2013, 41 (07) : e76