A kit for combined detection of respiratory pathogens by multiplex fluorescent PCR

A joint detection and multiple fluorescence technology, applied in the field of fluorescent quantitative PCR, can solve the problems of low sensitivity of immune detection, time-consuming and labor-consuming separation and culture, etc., to shorten the detection process, improve the accuracy of medication, and shorten the course of disease

Active Publication Date: 2020-03-27
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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  • A kit for combined detection of respiratory pathogens by multiplex fluorescent PCR
  • A kit for combined detection of respiratory pathogens by multiplex fluorescent PCR
  • A kit for combined detection of respiratory pathogens by multiplex fluorescent PCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 1. Preparation of reaction solution

[0033] (1) Preparation of reaction solution A

[0034] Take a 10mL volumetric flask, add 0.5mol / L Trizma ® HCl 64μL, 0.5mol / L Trizma ® Base336μL, 1mol / L MgCl 2 25 μL, 1mol / L KCl 600 μL, 0.5mol / L EDTA·2Na 2 μL, DMSO 100 μL, dNTPs 90 μL, 50 μmol / L influenza A virus upstream and downstream primers each 83.4 μL, 50 μmol / L Influenza A virus probe 25 μL, 50 μmol 83.4 µL each of upstream and downstream primers of influenza B virus per L, 25 µL of 50 μmol / L influenza B virus probe, 83.4 µL of 50 μmol / L upstream and downstream primers of adenovirus, 25 μL of 50 μmol / L adenovirus probe, 50 μmol / L Respiratory syncytial virus upstream and downstream primers were 83.4 μL each, and 50 μmol / L RSV probe was 25 μL. Make up the volume to 10mL with double distilled water, turn it over to mix well, transfer the liquid to a 10mL beaker, divide into centrifuge tubes at 1mL per branch, and store at -20°C for later use.

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

Embodiment 2

[0062] 1. Reagent specificity verification

[0063] (1) Experimental samples

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

[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 test results, and verify the specificity of the reagents.

[0067] (3) Experimental results

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

[0069] Specific test results of reaction solution A

[0070]

[0071] Reaction solution B specifi...

Embodiment 3

[0103] Embodiment 3: Detect the result of 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. 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 the nucleic acid extraction reagent (viral type) produced by Dobe Biotech (Xiamen) Co., Ltd. for nucleic acid extraction , the purity of the DNA samples was tested with an ultraviolet 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 perform detection on a fluorescent quantitative PCR instrument. The instrument used this time is ABI7500.

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

...

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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 fluorescent quantitative PCR, in particular to a kit for combined detection of respiratory pathogens by multiple fluorescent PCR methods. Background technique [0002] Acute respiratory tract infection has high morbidity and mortality, especially lower respiratory tract infection causes a huge disease burden. Globally, 20% of preschool children die of lower respiratory tract infection (SARI), and 90% of the deaths are attributed to pneumonia. Pneumonia, or acute pneumonia, is the predominant form of infection in febrile respiratory syndrome and a leading 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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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/689C12Q1/686C12Q1/04
CPCC12Q1/686C12Q1/689C12Q1/701C12Q2600/16C12Q2537/143C12Q2563/107C12Q2561/113Y02A50/30
Inventor 童超邱一帆
Owner DEBIQI BIOTECH XIAMEN
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