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Single-tube multiplex fluorescence PCR (Polymerase Chain Reaction) detection method and kit for 1,2,3-type parainfluenza viruses

A detection method and multiple fluorescence technology, applied in the field of molecular biology detection, can solve the problems of time-consuming and labor-consuming, cumbersome operation, and inability to detect human parainfluenza virus at the same time, and achieve the effect of simple and convenient operation, high sensitivity and low cost

Inactive Publication Date: 2011-01-19
GUANGZHOU INST OF RESPIRATORY DISEASE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, the currently developed parainfluenza virus typing reagents are basically single-fold fluorescent quantitative PCR detection reagents, which cannot simultaneously detect human parainfluenza virus types 1, 2, and 3 (PIV-1, 2, and 3); The detection also needs to be carried out in three tubes respectively, and the operation is cumbersome, time-consuming and labor-intensive

Method used

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  • Single-tube multiplex fluorescence PCR (Polymerase Chain Reaction) detection method and kit for 1,2,3-type parainfluenza viruses
  • Single-tube multiplex fluorescence PCR (Polymerase Chain Reaction) detection method and kit for 1,2,3-type parainfluenza viruses
  • Single-tube multiplex fluorescence PCR (Polymerase Chain Reaction) detection method and kit for 1,2,3-type parainfluenza viruses

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1: the specific experiment of primer, probe

[0060] After using bioinformatics and biological software for designing primers and probes to compare and analyze the sequence data of human parainfluenza virus in the existing database, the primers and probes shown in Table 1 were designed to achieve type 1, 2, and 3 One tube of multiplex fluorescent PCR detection of human parainfluenza virus. Table 1 shows the sequences of primers and probes for human parainfluenza virus types 1, 2, and 3.

[0061] Table 1

[0062]

[0063] In order to verify the specificity of the designed primers and probes, a single-plex fluorescent PCR reaction was designed first, and the primers and probes were evaluated, that is, the three sets of primers and probes of PIV-1, PIV-2 and PIV-3 were detected separately. specificity. The experiment was carried out with positive strains isolated from culture, and the positive strains came from Guangzhou Institute of Respiratory Diseases a...

Embodiment 2

[0069] Embodiment 2: the specificity experiment of PIV multiple detection reagent

[0070] The multiple detection reagents are prepared using the RT-PCR buffer in TAKARA'PrimeScript One Step RT-PCR Kit (Ver.2)' (TAKARA'PrimeScript One Step RT-PCR Kit Ver.2'), and the reagents in Table 1 The primers of PIV-1, 2, and 3 and three kinds of probes were prepared into a tube of reaction solution (M-PIV123). The volume of the prepared reagents was 18 μl / reaction. 0.5-5 pmol; reserve 2 μl for adding enzyme mixture, reserve 5 μl for adding template; total reaction volume is 25 μl / reaction.

[0071] Use single positive template (parainfluenza 1, parainfluenza 2 or parainfluenza 3); double positive template (parainfluenza 1 and 2 mixed; parainfluenza 1 and 3 mixed or parainfluenza 2 and 3 mixed); triple positive template (parainfluenza 2 and 3 mixed); Influenza 1, 2, and 3 mixed) experiments were performed on the multiplex detection reagents, while comparisons were performed using the si...

Embodiment 3

[0075] Embodiment 3: the sensitivity experiment of PIV multiple detection reagent

[0076] In order to further test the sensitivity of the reagent, a 10-fold gradient dilution was performed on the positive samples of parainfluenza 1, 2, and 3, and the dilution was 1 to 6, which respectively indicated that the PIV-1, PIV-2, and PIV-3 viruses were diluted 10 to 106 times , were compared with multiple parainfluenza virus detection reagents and single detection, and virus culture detection was carried out at the same time. The results of quantitative PCR experiments are shown in Table 4. The identifiable range of virus culture was between dilution 3 and dilution 4, and the sensitivity was much lower than singleplex or multiplex PIV quantitative PCR detection (data not shown). Table 4 is a data table of comparative experimental results of multiplex and singlex parainfluenza quantitative PCR detection reagents on 10-fold serially diluted viruses.

[0077] Table 4

[0078]

[0...

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Abstract

The invention provides a single-tube multiplex fluorescence PCR detection method for 1,2,3-type human parainfluenza viruses. In the method, a primer, the sequence of which is shown as SEQ ID NO:1-6 is adopted, and a probe the sequence of which is shown by SEQ ID NO:7-9 is also adopted. The invention also provides a single-tube multiplex fluorescence PCR detection kit for the 1,2,3-type parainfluenza viruses, comprising the primer and the probe. In the invention, the single-tube multiplex detection of the 1,2,3-type parainfluenza viruses is realized by adopting the self-designing PIV-1 / PIV-2 / PIV-3 specific primer and the Taqman probe and using an FAM / JOE / TAMRA multiplex fluorescein sign. The invention has the advantages of strong specificity, high sensitivity, rapidness, simple and convenient operation, low cost and the like, can be used as a reagent for detecting the 1,2,3-type human parainfluenza viruses and is used for scientific research and clinical application.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection, in particular to a one-tube multiple fluorescence quantitative PCR detection method and kit for parainfluenza viruses of types 1, 2, and 3. Background technique [0002] Human parainfluenza viruses (HPIVs) are single-stranded RNA viruses belonging to the Paramyxoviridae family. The surface of the virus contains glycoprotein spines of lysozyme and hemagglutinin-neuraminidase. Serologically, human parainfluenza viruses can be divided into 4 types (type I to type IV), with different sizes of virus particles (the average diameter is between 150 nanometers and 300 nanometers) and different shapes. HPIVs often cause respiratory tract infections in children, and the incubation period is generally about 1 to 7 days. Its pathogenicity is second only to respiratory syncytial virus (respiratory syncytial virus pneumonia, RSV). Like RSV, human parainfluenza viruses can cause recurrent u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12N15/11
Inventor 周荣刘文宽高文娟苏晓波
Owner GUANGZHOU INST OF RESPIRATORY DISEASE
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