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Kit for jointly detecting respiratory tract pathogen through multiple fluorescent PCR method

A combined detection and multiple fluorescence technology, applied in the field of fluorescence quantitative PCR, can solve the problems of time-consuming and labor-intensive separation and culture, low sensitivity of immune detection, etc., and achieve the effect of improving the accuracy of medication, shortening the detection process, and clarifying the cause of the disease.

Active Publication Date: 2017-08-18
DEBIQI BIOTECH XIAMEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Isolation and culture are time-consuming and labor-intensive, require a strict laboratory environment, and usually take 3 days to confirm the diagnosis; while immunoassays have low sensitivity and there is a "window period", and the corresponding antigen or antibody can only be detected 3 to 7 days after infection

Method used

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  • Kit for jointly detecting respiratory tract pathogen through multiple fluorescent PCR method
  • Kit for jointly detecting respiratory tract pathogen through multiple fluorescent PCR method
  • Kit for jointly detecting respiratory tract pathogen through multiple fluorescent PCR method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 1. Preparation of the reaction solution

[0033] (1) Preparation of reaction solution A

[0034] Take a 10mL volumetric flask and add 0.5mol / L Trizma ® HCl 64μL, 0.5mol / L Trizma ® Base 336μL, 1 mol / L MgCl 2 25μL, 1mol / L KCl 600μL, 0.5mol / L EDTA·2Na 2μL, DMSO 100μL, dNTPs 90μL, 50μmol / L Influenza A virus upstream and downstream primers each 83.4μL, 50μmol / L Influenza A virus probe 25μL, 50μmol / 83.4µL of influenza B upstream and downstream primers each, 50µmol / L influenza B virus probe 25µL, 50µmol / L adenovirus upstream and downstream primers 83.4µL each, 50µmol / L adenovirus probe 25µL, 50µmol / L adenovirus Respiratory syncytial virus upstream and downstream primers are 83.4µL each, and 50µmol / L respiratory syncytial virus probe is 25µL. Make up the volume to 10mL with double distilled water, invert to mix thoroughly, transfer the liquid to a 10mL beaker, distribute 1mL / branch into centrifuge tubes, and store at -20°C for later use.

[0035] (2) Preparation of reac...

Embodiment 2

[0062] 1. Reagent specificity verification

[0063] (1) Experimental sample

[0064] 10 specific samples were taken to verify the specificity of the reagents, of which 4 were saline, 1 was human metapneumovirus, 1 was rubella virus, 1 was measles virus, and 1 was Klebsi pneumoniae Escherichia coli samples, 1 sample for Escherichia coli, and 1 sample for Staphylococcus aureus.

[0065] (2) Experimental process

[0066] Use reaction solution A, reaction solution B, and reaction solution C to detect the above 10 specific samples respectively, analyze the detection results, and verify the specificity of the reagents.

[0067] (3) Experimental results

[0068] All 10 specific samples tested by the three reaction solutions were negative, indicating that the reagents had good specificity and no cross-reaction. The specific results are shown in the table below:

[0069] Reaction solution A specific test results

[0070]

[0071] Reaction solution B specific test results

[0...

Embodiment 3

[0103] Example 3: Results of testing 120 clinical samples

[0104] 1. According to the preparation method shown in Example 1, the relevant components of the kit were prepared and stored at -20°C for later use.

[0105]2. The Wuhan Maternal and Child Health Hospital collected throat swab samples and sputum samples from 120 patients with clinical symptoms of respiratory tract infection, and used nucleic acid extraction reagents (viral type) produced by DoBeek Biotechnology (Xiamen) Co., Ltd. for nucleic acid extraction. , The purity of DNA samples was detected by UV spectrophotometer, and the OD260 / OD280 of 120 samples were all between 1.6 and 2.0.

[0106] 3. According to the steps shown in Example 1, add DNA samples and use a fluorescence quantitative PCR instrument for detection. The instrument used this time is ABI7500.

[0107] 4. According to the judgment criteria shown in Example 1, the results are interpreted and counted, and the results are as follows:

[0108]

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Abstract

The invention provides a kit for jointly detecting respiratory tract pathogen through a multiple fluorescent PCR method. The kit comprises six components: reaction liquid A, reaction liquid B, reaction liquid C, enzyme mixed liquid, positive control and negative control, and comprises 11 common respiratory tract pathogen detections (general type of influenza virus A, influenza virus B, respiratory syncytial virus, 1 / 2 / 3 type of human parainfluenza virus, adenovirus, mycoplasma pneumoniae, chlamydia pneumonia, legionella pneumophila, streptococcus pneumonia, haemophilus influenza, A streptococcal); the amplification is performed through three reaction buffers, and each reaction buffer contains four fluorescent channels, 90% pathogen infection on the clinic can be checked.

Description

technical field [0001] The invention belongs to the field of fluorescence quantitative PCR, in particular to a kit for combined detection of respiratory pathogens by multiplex fluorescence PCR. Background technique [0002] Acute respiratory tract infections have high morbidity and mortality, especially lower respiratory tract infections cause a significant disease burden, 20% of preschool children die globally from lower respiratory tract infections (SARI), and 90% of deaths are attributed to pneumonia. Pneumonia, or acute pneumonia, is the predominant form of infection in febrile respiratory syndrome and a major cause of morbidity and mortality in children. [0003] The incidence rate of pneumonia in children under 1 year old is 0.01-0.68 times / person-year, and the incidence rate of pneumonia in children under 5 years old is 0.14-0.66 times / person-year; the mortality rate of pneumonia in children under 1 year old is 485 / 100,000-890 / 100,000 , 184 / 100,000 to 1 223 / 100,000 ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12Q1/04
CPCC12Q1/686C12Q1/689C12Q1/701C12Q2600/16C12Q2537/143C12Q2563/107C12Q2561/113Y02A50/30
Inventor 童超邱一帆
Owner DEBIQI BIOTECH XIAMEN
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