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Primer and probe composition and real-time fluorescent quantitative PCR (polymerase chain reaction) kit for para-influenza virus typing detection

A real-time fluorescence quantitative and virus typing technology, applied in the field of biomedicine, can solve the problems of poor primer specificity, low accuracy of detection results, poor primer sensitivity, etc.

Pending Publication Date: 2021-08-20
NINGBO HEALTH GENE TECHNOLOGIES CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The above detection kits have the following deficiencies: (1) The specificity of the primers is poor, which may cause serious missed detection or off-target effects, resulting in false negative or false positive results; (2) the specificity of each parainfluenza virus serotype There is a large difference in the Tm value of the primers, which makes the accuracy of the detection result low; (3) The sensitivity of the primers is poor when typing and detecting each parainfluenza virus

Method used

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  • Primer and probe composition and real-time fluorescent quantitative PCR (polymerase chain reaction) kit for para-influenza virus typing detection
  • Primer and probe composition and real-time fluorescent quantitative PCR (polymerase chain reaction) kit for para-influenza virus typing detection
  • Primer and probe composition and real-time fluorescent quantitative PCR (polymerase chain reaction) kit for para-influenza virus typing detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Design specific primers and probes for parainfluenza virus type 1 HN gene, parainfluenza virus type 2 L gene, parainfluenza virus type 3 L gene and parainfluenza virus type 4 F gene, and commission Sangon Bioengineering (Shanghai) Co., Ltd.; the nucleotide sequence information of the four sets of primers and probes obtained is shown in Table 1.

[0059] Table 1 Primer and probe composition sequence information

[0060]

[0061] Among them, the length of the HN gene amplification product of parainfluenza virus type 1 is 90 bp, the length of the amplification product of parainfluenza virus type 2 L gene is 137 bp, the length of the amplification product of parainfluenza virus type 3 L gene is 128 bp, and the length of the amplification product of parainfluenza virus type 4 F The gene amplification product is 90bp in length.

[0062] The multiplex RT-qPCR kit for the detection of parainfluenza virus typing is prepared by using the primer probe composition shown in Tabl...

Embodiment 2

[0082] Each positive plasmid comprising the HN gene of parainfluenza virus type 1, the L gene of parainfluenza virus type 2, the L gene of parainfluenza virus type 3, and the F gene of parainfluenza virus type 4 after measuring the concentration and purity is made into a 10-fold gradient dilution respectively, obtained from 2.4×10 1 ~2.4×10 6 A total of 6 dilutions of positive plasmids in copies / μL were used as standard templates. Prepare the PCR reaction system according to Table 5 in Example 1, carry out multiple RT-qPCR reactions according to the reaction program shown in Table 6, and obtain the fluorescence amplification curve (such as figure 1 , figure 2 , image 3 and Figure 4 shown), draw a standard curve and determine the lowest detection limit. Depend on figure 1 , figure 2 , image 3 and Figure 4 shown, at a concentration of 2.4 x 10 2 ~2.4×10 6 Within the range of copies / μL, the kit of the present invention amplifies the parainfluenza virus type 1 HN ...

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Abstract

The invention discloses a primer and probe composition and real-time fluorescent quantitative PCR (polymerase chain reaction) kit for para-influenza virus typing detection, which can be used for detecting at least two of para-influenza virus type 1, para-influenza virus type 2, para-influenza virus type 3 and para-influenza virus type 4. In the primer and probe composition, four groups of primers and probes all have excellent specificity and can specifically perform typing detection on corresponding parainfluenza virus serotypes, the first group of primers and probes can specifically amplify a parainfluenza virus type 1 HN gene, the second group of primers and probes can specifically amplify a parainfluenza virus type 2 L gene, the third group of primers and probes can specifically amplify a parainfluenza virus type 3 L gene, the fourth group of primers and probes can specifically amplify a parainfluenza virus type 4 F gene, different groups of primers and probes do not interfere with each other, the typing effect on four serotypes of the parainfluenza virus is excellent, and the detection sensitivity and accuracy are high.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a primer probe composition and a real-time fluorescent quantitative PCR kit for typing and detecting parainfluenza virus. Background technique [0002] Parainfluenza virus is a kind of enveloped single-stranded negative-strand RNA virus belonging to the genus Paramyxovirus. It is a common pathogen of respiratory tract infection. It mainly causes severe lower respiratory tract infection in infants and children. At present, there is no effective treatment. Drugs and virus vaccines. [0003] According to heredity and antigenicity, parainfluenza virus can be divided into type 1, type 2, type 3 and type 4. Type 1-3 parainfluenza virus is an important pathogen that causes acute respiratory infection in infants and young children, and type 4 only Causes respiratory infections in infants with mild symptoms. The symptoms of infection caused by parainfluenza virus are sim...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2563/107C12Q2521/107C12Q2521/531C12Q2545/113
Inventor 张迪骏赵林清徐智钱渊梁宜朱汝南曾县平孙宇余丁陈冬梅吴勇
Owner NINGBO HEALTH GENE TECHNOLOGIES CO LTD
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