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Multiplex PCR detection kit for nucleic acids of twelve respiratory viruses

A technology for detecting kits and viral nucleic acids, applied in the measurement/inspection of microorganisms, resistance to vector-borne diseases, biochemical equipment and methods, etc., can solve the problems affecting the efficiency of PCR amplification and the wide application of multiple PCR, and achieve Good specificity, low requirements for detection platform and manual technical level, and guaranteed reliability

Active Publication Date: 2016-07-06
NANJING MOKOBIO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since multiplex PCR is a PCR reaction in which multiple pairs of primers are added to the same PCR reaction system to amplify multiple DNA fragments, multiple pairs of primers in the same reaction system are prone to interactions, such as forming hairpin structures, dimer structures, etc. etc., the more primer pairs and the amount of primers, the more obvious the interaction between primers, which affects the efficiency of PCR amplification, which in turn affects the wide application of multiplex PCR

Method used

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  • Multiplex PCR detection kit for nucleic acids of twelve respiratory viruses
  • Multiplex PCR detection kit for nucleic acids of twelve respiratory viruses
  • Multiplex PCR detection kit for nucleic acids of twelve respiratory viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 112

[0041] Example 112 Respiratory Virus Nucleic Acid Multiplex PCR Detection Kit

[0042] 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×onestepRT-PCRBuffer (Tris-HClpH8.5100mM, KCl500nM, 2 15nM), dNTPs(10mMeach), 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 / μLPMD19-T clone DNA composition.

[0043] 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.

[0044] The reaction system of the kit is 25ul, and the details are as follows: 2.0μL of enzyme system, 18.0μL of PCR reaction solution, 2~5μL of nucleic acid template, and supplem...

Embodiment 2

[0045] Operation and result determination of embodiment 2 kit

[0046] (1) Extraction of viral genomic DNA

[0047] Use the Viral Genome DNA / RNA Extraction Kit (Product No.: DP315) from Tiangen Biochemical Technology Co., Ltd. to extract nucleic acid in the sample processing area according to the instructions.

[0048] (2) Preparation of reaction system

[0049] 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.

[0050] (3) PCR amplification

[0051] 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, 7...

Embodiment 3

[0057] Embodiment 3 kit specific experiment

[0058] 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.

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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.

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. About 10% of the residents suffer from respiratory tract infection at the peak of the epidemic each year. 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),...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143C12Q2545/113Y02A50/30
Inventor 李伯安金鑫刘文干郭桐生
Owner NANJING MOKOBIO BIOTECH
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