Primer set for real-time fluorescent quantitative PCR detection of swine influenza virus

A swine influenza virus and primer set technology, applied in the biological field, can solve the problems of high price, cumbersome probe design and synthesis, and achieve good specificity

Active Publication Date: 2018-04-03
兆丰华生物科技(南京)有限公司 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The primers in the TaqMan probe method include a pair of oligonucleotide primers and a fluorescent-labeled specific probe. The design and synthesis of fluorescent-labeled probes are more cumbersome and expensive than ordinary primers.

Method used

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  • Primer set for real-time fluorescent quantitative PCR detection of swine influenza virus
  • Primer set for real-time fluorescent quantitative PCR detection of swine influenza virus
  • Primer set for real-time fluorescent quantitative PCR detection of swine influenza virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Experimental operation steps of the present invention are as follows:

[0021] 1. Extraction of viral RNA: Trizol reagent from Invitrogen Company was selected, and viral RNA was extracted according to the instructions of the reagent.

[0022] 2. Preparation of cDNA: Use the RNA extracted in step 1 to prepare cDNA using the TIANScript cDNA First Strand Synthesis Kit from Tiangen Company.

[0023] 3. Real-time PCR: The selected Real-time PCR kit is from TAKARA company Premix Ex Taq TM II (Perfect Real Time), each sample set 3 replicates. The reaction system and its components are shown in Table 2.

[0024] Table 2 Real-time PCR reaction system and its components

[0025]

[0026] Amplification conditions used Premix Ex Taq TM Standard amplification conditions provided by the II (Perfect Real Time) kit, such as 95°C for 30 seconds; 95°C for 15 seconds; 60°C for 30 seconds for 40 cycles; melting.

[0027] When performing Real-time PCR detection, when the CT va...

Embodiment 2

[0029] Example 2 Primer Sensitivity Detection

[0030] 1. Sample: H1N1SIV (10 8 EID 50 / ml) or H3N2SIV (10 8 EID 50 / ml) as a reference, and nuclease-free water as a negative control. H1N1 SIV is used to detect the sensitivity of SIV-M, SIV-H1 and SIV-N1 primers. H3N2 SIV is used to detect the sensitivity of SIV-M, SIV-H3 and SIV-N2 primers. Both H1N1 SIV and H3N2 SIV were isolated from pigs that clinically showed the main symptoms of SIV infection, and their subtypes were determined by the current general RT-PCR typing detection method.

[0031] For SIV-Taq primers, see "A Real-time Fluorescent RT-PCR Detection Kit for Swine Influenza Virus" (Patent No.: 200910039407), which is a general primer for detecting swine influenza virus and a reference primer for the general primer SIV-M in this example , which is used to compare the sensitivity difference between the universal primer provided in the present invention and the universal primer in the existing invention.

[003...

Embodiment 3

[0044] Example 3 Primer Specific Detection

[0045] 1. Samples: H9N2 avian influenza virus; common seasonal H1N1 influenza virus cDNA; 2009 pandemic H1N1 influenza virus cDNA. The subtypes of the above influenza virus samples were all determined by the current general RT-PCR typing detection method. H1N1 SIV and H3N2 SIV are the same as in Example 2.

[0046] H1N1 SIV is the positive control for the specific detection of SIV-H1 and SIV-N1 primers; H3N2 SIV is the positive control for the specific detection of SIV-H3 and SIV-N2 primers. The negative control was nuclease-free water.

[0047] 2. Operation steps:

[0048] 1) Extraction of viral RNA.

[0049] 2) Preparation of cDNA.

[0050] 3) Real-time PCR.

[0051] 3. Test results

[0052] Utilize the SIV-H1 and SIV-N1 primers provided by the present invention, and use the cDNA of common seasonal H1N1 influenza virus, 2009 pandemic H1N1 influenza virus, H9N2 avian influenza virus and H3N2 SIV as templates to detect the sp...

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Abstract

The invention discloses a set of primers for swine influenza virus SYBGREEN I Real-time PCR detection method, including swine influenza virus universal M gene, H1 subtype, N1 subtype, H3 subtype and N2 subtype primers. The present invention provides a group of Real-time PCR primers for swine influenza virus detection and typing, the primers are suitable for SYBR Green I dye method, to reduce the detection cost of SIV, and realize the rapid typing detection of SIV, for Provide technical support for clinical monitoring of SIV epidemic dynamics and epidemic trends.

Description

technical field [0001] The invention relates to nucleotide fragment amplification primers of H1 and H3 subtype swine influenza viruses, which are used for Real-time PCR detection and typing of swine influenza viruses, and belong to the field of biotechnology. Background technique [0002] Swine Influenza (SI) is an acute and contagious porcine respiratory disease caused by Swine influenza virus (SIV). It can also cause reproductive failure in pregnant sows. SIV belongs to the genus Influenza A of the family Orthomyxoviridae. There are radially arranged protruding glycoproteins on the envelope, which are hemagglutinin (HA), neuraminidase (Neuraminidase, NA) and M2 protein (Matrix 2protein). ). According to the differences in the antigenicity of viral hemagglutinins (HA) and neuraminidase (Neuraminidase, NA), influenza A viruses can be divided into 16 different HA subtypes (H1~H16) and 10 NA subtypes. Type (N1~N9). [0003] Since there are both α2,6 sialic acid receptors a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
Inventor 孟珊珊王丹娜杜金玲王吉玮石磊王贵华赵亚荣
Owner 兆丰华生物科技(南京)有限公司
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