A multiplex PCR detection kit for 12 kinds of respiratory virus nucleic acids

A technology for detecting kits and viral nucleic acids, which can be applied in the direction of resistance to vector-borne diseases, measurement/testing of microorganisms, biochemical equipment and methods, etc., and can solve problems that affect the wide application of multiplex PCR and affect the efficiency of PCR amplification, etc., and achieve The detection platform and artificial technology level requirements are low, the specificity is good, and the effect of large detection throughput

CN105734168BActive Publication Date: 2019-05-17NANJING MOKOBIO BIOTECH
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Publication Date
2019-05-17

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Abstract

The invention discloses a Multiplex PCR detection kit for nucleic acids of twelve respiratory viruses. The kit includes primers for amplifying the specific gene loci of the following twelve respiratory viruses: influenza A virus (Inf A), influenza A H1N1 (2009), influenza A H3N2, human parainfluenza virus (HPIV1), human parainfluenza virus (HPIV2), human parainfluenza virus (HPIV3), human parainfluenza virus (HPIV4), human metapneumovirus (hMPV), respiratory adenovirus (AdV), respiratory syncytial virus (RSV), bocavirus (BoV) and coronavirus (CoV). The kit allows multiplex detection, has high sensitivity and is convenient and quick to use. Specific primer sequences ensure reliability of detection results. A detection method is simple in operation, save time and labor intensity, has high detection throughput, is low in cost of reagent and consumable, and can directly detect the nucleic acids extracted from a respiratory pathogen sample, is low in requirement on detection platform and operation technology, and can be widely promoted in common detection.
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Description

technical field

[0001] The invention belongs to the field of gene detection, and relates to a kit for detecting 12 kinds of respiratory virus nucleic acids by a multiplex PCR amplification method mediated by universal primers. Background technique

[0002] Respiratory tract infection is one of the most common diseases in the world, and the incidence rate occupies a major position in the overall structure of the morbidity rate of residents in various countries. At the peak of the epidemic each year, about 10% of the residents suffer from respiratory tract infection. Respiratory tract infections are mainly caused by various respiratory viruses and some bacteria, mycoplasma and chlamydia. Common viruses are influenza A virus (InfA), influenza A H1N1 (2009), influenza A virus H3N2, human parainfluenza virus (HPIV1), human parainfluenza virus (HPIV2), human parainfluenza virus (HPIV3) , human parainfluenza virus (HPIV4), human metapneumovirus (hMPV), respiratory adenovirus (AdV)...

Examples

Embodiment 112

[0040] Example 1 12 kinds of respiratory virus nucleic acid multiplex PCR detection kits

[0041] The kit is composed of PCR reaction solution, enzyme system, positive control substance, negative control substance and DEPC water. The PCR reaction solution is 5×one step RT-PCR Buffer (Tris-HCl pH8.5 100mM, KCl 500nM, MgCl 2 15nM), dNTPs(10mM each), Mg 2+ , 12 pairs of chimeric primers and 1 pair of universal primers; the enzyme system consists of reverse transcriptase, RNase inhibitor, hot-start DNA polymerase and activators of the above enzymes; the positive control substance consists of 10 of 12 targets 7 copy / μL PMD19-T clone DNA composition.

[0042] The final concentration of the universal primer in the amplification system is 800nM; the final concentration of the chimeric primer in the amplification system is 60nM.

[0043] The reaction system of the kit is 25ul, specifically as follows: 2.0 μL of enzyme system, 18.0 μL of PCR reaction solution, 2-5 μL of nucleic acid ...

Embodiment 2

[0044] Operation and result determination of embodiment 2 kit

[0045] (1) Extraction of viral genomic DNA

[0046] Use the viral genome DNA / RNA extraction kit (product number: DP315) from Tiangen Biochemical Technology Co., Ltd. to extract nucleic acid in the sample processing area according to the instructions.

[0047] (2) Preparation of reaction system

[0048] Use the kit of Example 1 to carry out the following experiments. After the PCR reaction solution of the kit is completely dissolved at room temperature, shake and mix quickly. The 25 μL PCR reaction system is: 18 μL of PCR reaction solution, 2 μL of enzyme system, 3 μL of template, and supplemented with DEPC water to 25 μL.

[0049] (3) PCR amplification

[0050] Put the PCR tube into an ordinary PCR machine, open the hot cover, and set the reaction program of the PCR machine according to the following requirements: program 1, 50°C, 25min, 1 cycle; program 2, 95°C, 10min, 1 cycle; program 3, 95°C, 15s, 56°C, 30s...

Embodiment 3

[0056] Embodiment 3 kit specific experiment

[0057] The kit described in Example 1 is used for rhinovirus, influenza virus H1N1, influenza B virus, enterovirus, poliovirus, Mycoplasma pneumoniae, Chlamydia pneumoniae, Streptococcus pneumoniae, Klebsiella pneumoniae, oligomaltophilia Trotrophomonas, Citrobacter, Bacillus cloacae, Acinetobacter baumannii, Legionella pneumophila, and Bordetella pertussis did not have any fragment amplification after detection, and when the target strain was detected, there was nothing except the target fragment The generation of specific fragments shows that the kit of the present invention has good specificity.