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MASA (multi-analyte suspension array) detection kit for influenza virus H5N1 subtype and H7N9 subtype

A technology of H5N1 and influenza virus, which is applied in the field of biomedicine, can solve problems such as the complexity of influenza virus sequences, and achieve good sensitivity, high sensitivity, and good specificity

Active Publication Date: 2015-03-04
INST OF LAB ANIMAL SCI CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these principles are not complicated for general primer design, due to the complexity of the influenza virus sequence, there are no reports on the specificity and accuracy of liquid chip technology primers and probes that can meet the requirements of clinical applications.

Method used

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  • MASA (multi-analyte suspension array) detection kit for influenza virus H5N1 subtype and H7N9 subtype
  • MASA (multi-analyte suspension array) detection kit for influenza virus H5N1 subtype and H7N9 subtype
  • MASA (multi-analyte suspension array) detection kit for influenza virus H5N1 subtype and H7N9 subtype

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1: The present invention provides primer annealing temperature, probe hybridization temperature, and staining temperature screening

[0075] 1. Use the cDNA of influenza virus subtype H5N1 or H7N9 as a template, perform temperature gradient PCR amplification, and set the annealing temperature at 50°C to 66°C, with a gradient of 4°C. The amplified products were subjected to agarose gel electrophoresis, and the results were as follows: figure 1 shown. From the results of electrophoresis, it can be effectively amplified at 54°C to 58°C. Considering the sequence similarity between different virus subtypes and avoiding non-specific amplification, a higher annealing temperature was selected.

[0076] 2. Coat the probe on the microsphere, combine the amplified product obtained under the annealing temperature of 58°C with the microsphere coated with the probe, and perform hybridization at 40°C, 43°C, and 46°C as the hybridization temperature . Then stain. The result...

Embodiment 2

[0080] Embodiment 2 The present invention provides the specificity and repeatability detection of the kit

[0081] Using the kit provided by the invention, different subtypes of influenza virus samples are detected to prove the specificity of the invention. And one of the H5N1 subtype samples and one H7N9 subtype sample were tested three times to identify the repeatability.

[0082] The specific steps of the detection method are as follows:

[0083] a. Acquisition of viral cDNA: Take virus samples, and extract viral RNA according to the instruction of RNeasy Mini Kit (Qiagen Company, Cat. No. 74104). According to SuperScript III First-Strand Synthesis System (Invitrogen Company, product number 18080-051) instructions, the first-strand cDNA of the virus was synthesized.

[0084] b. Perform PCR using the cDNA obtained in step a as a template, and add 20 μL of the following substances to the PCR tube:

[0085]

[0086] The primers are a mixture of 4 primers as shown in SEQ ...

Embodiment 3

[0126] Embodiment 3 The present invention provides the sensitivity detection of kit

[0127] The H5N1 subtype virus liquid, the H7N9 subtype virus liquid, and the lung tissue cDNA of the ferret model infected with H7N9 were diluted 10 times to obtain 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 double diluted samples. Quantification was then carried out by real-time PCR method. At the same time, the kit provided by the invention is used to carry out PCR, molecular hybridization and on-machine detection on the cDNA with gradient dilution, so as to identify the sensitivity of the kit provided by the invention. The usage method of the kit is the same as that in Example 2. The results are shown in Table 3:

[0128] Table 3 Sensitivity detection of the kit provided by the invention

[0129]

[0130] Note: The copy number of the corresponding real-time PCR is in parentheses

[0131] The results show that the diluted sample can be detected effectively with the kit pr...

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Abstract

The invention relates to the field of biological medicines, particularly to an MASA (multi-analyte suspension array) detection kit for influenza virus H5N1 and H7N9 subtypes, and provides a primer pair and a probe for detecting influenza virus surface hemagglutinin antigen H5 and H7 subtypes, a primer pair and a probe for detecting influenza virus neuraminidase antigen N1 and N9 subtypes as well as a kit comprising the primer pairs and the probes and a usage method of the kit. The kit is good in sensitivity, accuracy and specificity and simple, convenient and quick to use, and detection of to-be-detected samples can be finished within four hours. Tests prove that no false positive result exists when the kit is used for detecting negative samples; the detection rate of 100% is realized when the kit is used for detecting samples containing H7N9 subtype viruses and H5N1 subtype viruses only; the H7N9 and H5N1 subtype viruses contained in samples can be completely detected when the kit is used for detecting the samples containing both the H5N1 subtype viruses and the H7N9 subtype viruses. Besides, the kit can be used for effectively detecting to-be-detected samples containing more than 10 copies.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a liquid phase chip detection kit for H5N1 subtype and H7N9 subtype of influenza virus. Background technique [0002] Influenza is an acute respiratory infectious disease caused by influenza virus, which is characterized by rapid spread and wide spread. So far, influenza viruses have caused four world influenza pandemics, causing huge losses to the economy and health of human society. [0003] Influenza virus belongs to the Orthomyxoviridae family, and its genome is segmented negative-strand RNA. According to the difference of viral nucleocapsid protein (NP) and matrix protein (M), it can be divided into three types: A, B, and C, all of which can infect people. Influenza A and B viruses are the main epidemic viruses in the population. Influenza A viruses are divided into 16 HA subtypes according to the difference of the hemagglutinin antigen on the virus surface; they are divided int...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/701C12Q2600/112
Inventor 许黎黎袁静秦川
Owner INST OF LAB ANIMAL SCI CHINESE ACAD OF MEDICAL SCI
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