Kit for detecting 10 pathogens of respiratory tract infection and method of use thereof

A respiratory and pathogen technology, applied in the field of molecular biology, can solve the problems of delayed treatment, inability to cultivate bacteria, poor cultivation effect, etc., and achieve the effect of reducing pollution interference, reducing the possibility of pollution and result deviation, and avoiding sample contamination.

Active Publication Date: 2020-04-14
NANJING LANSION BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although ordinary fluorescent PCR detection reagents have good sensitivity and specificity, most of them are aimed at single or several pathogens. If you want to detect multiple pathogens, you need to use multiple sets of different detection kits, which is inconvenient to operate and expensive; Although the immunoassay detection reagent can detect multiple pathogens at the same time, because the immunoassay detects the specific antibodies produced by the organism caused by the pathogens in the sample, there is a long window period and the sensitivity is relatively low; and for bacteria For the detection of pathogen-like pathogens, although the bacterial culture method has high accuracy, it takes a long time and the culture effect is poor. Many bacteria cannot be cultured, which delays the best time for treatment

Method used

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  • Kit for detecting 10 pathogens of respiratory tract infection and method of use thereof
  • Kit for detecting 10 pathogens of respiratory tract infection and method of use thereof
  • Kit for detecting 10 pathogens of respiratory tract infection and method of use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] A kit for detecting 10 respiratory infection pathogens, the kit consists of primer-probe mixture, RT-PCR buffer, mixed enzyme solution, positive quality control and RNase-free water; RT-PCR buffer includes PCR buffer, dATP , dUTP, dCTP, dGTP and MgCl 2 ; The mixed enzyme solution includes HotStart Taq enzyme, reverse transcriptase and UNG enzyme; the positive quality control contains plasmids corresponding to the amplified gene sequences of the above 10 respiratory pathogens.

[0054] The final concentration of the primer in the amplification system is 100-1000nM; the final concentration of the probe in the amplification system is 50-500nM.

[0055] The reaction system of the kit is 25 μL, specifically: 2 μL of nucleic acid template, 20 μL of RT-PCR buffer, 1 μL of mixed enzyme solution, and 2 μL of primer-probe mixture.

[0056] Kit operation and result determination

[0057] (1) Add 2 μL each of sample DNA / RNA co-extraction template (extracted from human sputum, thr...

Embodiment 2

[0068] Use this kit to detect the corresponding pathogen plasmid samples respectively, according to the dilution concentration of 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 Sensitivity test was carried out with five gradients of copies / μL and 10copies / μL. Detection was carried out in the same manner as in Example 1.

[0069] The detection result shows that the sensitivity of the kit of the present invention is good, and the Ct value changes in a gradient with concentration reduction, the results are shown in Figure 2-11 .

[0070] The test results show that the kit is effective in the diagnosis of respiratory pathogens Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Bordetella pertussis, rhinovirus, influenza A virus, influenza B virus, respiratory adenovirus, syncytial virus, parainfluenza virus High sensitivity, the detection sensitivity of each item can reach 100copies / μL.

Embodiment 3

[0072] Use this kit to detect Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Bordetella pertussis, rhinovirus, influenza A virus, influenza B virus, respiratory adenovirus, syncytial virus, parainfluenza virus and other samples. Detection was carried out in the same manner as in Example 1.

[0073] The detection result shows that the kit of the present invention can only detect the pathogen corresponding to the detection reagent, and cannot detect other pathogens.

[0074] The test results show that the kit is specific for the detection of respiratory pathogens Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Bordetella pertussis, rhinovirus, influenza A virus, influenza B virus, respiratory adenovirus, syncytial virus, parainfluenza virus Strong, does not cross-react with nucleic acids of other pathogens, see results Figure 12-21 .

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Abstract

The invention provides a kit for detecting 10 respiratory tract infection pathogens and an application method thereof. The kit comprises primers and a TaqMan probe, wherein the primers are used for amplifying the 10 respiratory tract infection pathogens which include mycoplasma pneumoniae, chlamydia pneumoniae, legionella pneumophila, bordetella pertussis, rhinovirus, respiratory tract adenovirus, influenza A virus, influenza B virus, respiratory syncytial virus and parainfluenza virus. The application method includes: mixing 2 microliters of sample DNA / RNA co-extraction template, 20 microliters of RT-PCR buffer, 1 microliter of mixed enzyme liquid and 2 microliter of primer-probe mixed liquid to perform PCR amplification reaction. The kit uses the Taqman probe fluorescent PCR technology to detect the 10 common respiratory tract infection pathogens, a detection result can be obtained within 2 hours, the kit is high in specificity, and the sensitivity of the kit can reach 100copies / microliter.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a kit for detecting 10 kinds of respiratory tract infection pathogens and a use method thereof. Background technique [0002] Respiratory tract infection is one of the most common diseases in the world, and its incidence rate plays a major role 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 infection can lead to symptoms such as rhinitis, runny nose, nasal congestion, cough, mild pharyngitis, general fever, etc., and severe cases can lead to dyspnea and even death. Acute respiratory infection has become one of the important infectious diseases of children at home and abroad, and its incidence rate ranks first among all kinds of diseases in children. The pathogens that cause respiratory tract infections in children mainly i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/689C12Q1/6851C12Q1/04C12Q1/06
CPCC12Q1/6851C12Q1/689C12Q1/701C12Q2600/16C12Q2537/143C12Q2561/101C12Q2521/531C12Q2563/107Y02A50/30
Inventor 许行尚杰弗瑞·陈于沛朱滔张蓉蓉
Owner NANJING LANSION BIOTECH CO LTD
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