Method for detecting H1N1 influenza A virus variant by multiple target point gene and kit

A technology for influenza virus and gene detection, applied in the field of primers for multi-target detection, which can solve the problems of false negatives or false positives

Inactive Publication Date: 2013-07-24
HANGZHOU DIANZI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the fluorescent RT-PCR detection method also has the possibility of false negative or false positive in the detection

Method used

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  • Method for detecting H1N1 influenza A virus variant by multiple target point gene and kit
  • Method for detecting H1N1 influenza A virus variant by multiple target point gene and kit
  • Method for detecting H1N1 influenza A virus variant by multiple target point gene and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Preparation of a multi-gene target detection kit for influenza A (H1N1) 2009 mutant strains.

[0073] 1) Synthesize an oligodeoxynucleic acid primer, the sequence of which is shown in SEQ ID NO: 1-8 above, and the 5' end of the antisense primer is labeled with a reporter fluorescent group FAM (6-carboxy-fluorescein).

[0074] 2) Purchase RT-PCR enzyme and DNA polymerase.

[0075] 3) Configure the reaction solution.

[0076] 4) Configure the positive control and negative control and dilute to the optimum concentration.

[0077] Table 4 kit preparation composition

[0078] Composition (50tests / box)

quantity

RT-PCR reaction solution

1.5ml

RT-PCR enzyme 100U / ul

10ul

PCR reaction solution

1.5ml

Taq DNA polymerase 5U / ul

15ul

Molecular Biological Purity Water

2ml

positive control

1ml

negative control

1ml

[0079] The specific material composition is as described above...

Embodiment 2

[0085] Example 2 Application of Multi-gene Target Detection Kit for Influenza A H1N1 Influenza Virus 2009 Variant Strain

[0086] 1) Sample processing (RNA extraction)

[0087] Use Qiagen's QIAamp Viral RNA Mini Kit, or other corresponding kits, to extract from samples collected from humans. Performed in the sample handling area. Samples were stored at -80°C.

[0088] 2) Amplification reagent preparation and reaction process

[0089] RT-PCR: Take out the RT-PCR reaction solution and RT-PCR enzyme from the kit, melt at room temperature, and centrifuge at 2,000g for 5sec. Assuming that the number of PCR tubes required is n (n=number of samples+1 tube of negative control+1 tube of positive control), each RT-PCR test reaction system is 20 μL. Calculate the amount of each reagent used, add it to a test tube of appropriate volume, mix well, dispense 15 μL into each PCR tube, and transfer to the sample processing area. Add 5 μL of each prepared RNA solution to each set PCR tube,...

Embodiment 3

[0096] Embodiment 3, the specificity test of kit

[0097] 1) Materials, as listed in Table 5, 322 known clinical samples.

[0098] 2) method

[0099] Clinical samples were tested with the diagnostic kit prepared in Example 1 to verify the specificity of the method.

[0100] 3) Results

[0101] As shown in Table 5, the results indicated that the established method had high specificity and produced no false positive or false negative results.

[0102] The test results are shown in Table 5.

[0103] Table 5 Test results of clinical samples

[0104]

[0105] In Table 5, detection gene segment A is the human beta-2-microglobulin gene, segment B is the M gene for all influenza A, segment C is the HA gene for all H1N1 influenza, and segment D is the HA gene for influenza A 2009H1N1.

[0106] As can be seen from the results in Table 5, 322 known samples are detected, and the results show that the established method can detect all of the influenza A H1N1 influenza virus (2009 v...

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Abstract

The invention provides a reliable method for detecting H1N1 influenza A virus variant by a multiple target point gene and a kit. Groups of primer needles are designed to multiple gene target points. The accuracy of each fragment amplified is intuitively determined by displaying size of PCR (Polymerase Chain Reaction) sequence fragment through capillary electrophoresis fluorescent detection. The polygene target points confirm positive results layer by layer, so that the specificity of detection result is ensured. In addition, the kit can detect non-H1N1 influenza A injection and non A influenza injection samples, so as to further provide associated information for diagnosis.

Description

technical field [0001] The invention relates to the field of virus inspection and detection, in particular to primers for multi-target detection, a corresponding kit and a detection method for detecting variant strains of influenza A (H1N1) virus aimed at M gene and HA gene. Background technique [0002] Influenza A (H1N1) began in Mexico in April 2009 and caused a global pandemic. Sequence evolution analysis shows that the virus is a new variant of influenza A, which is significantly different from human seasonal H1N1 influenza virus and classic H1N1 swine influenza virus. Sequence analysis showed that its 8 gene fragments had obvious specificity. However, its HA fragment has the highest homology with the North American swine influenza virus HA fragment, about 95%; NA and M are the most similar to the Eurasian swine virus fragment, with 92% and 97% homology respectively. Therefore, in actual diagnosis and detection, when nucleic acid amplification method is used for ident...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 沈洁厉力华徐铭恩
Owner HANGZHOU DIANZI UNIV
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