CN107058622B - Kit for joint detection of respiratory pathogens by multiple fluorescence PCR method - Google Patents
Kit for joint detection of respiratory pathogens by multiple fluorescence PCR method Download PDFInfo
- Publication number
- CN107058622B CN107058622B CN201710201003.5A CN201710201003A CN107058622B CN 107058622 B CN107058622 B CN 107058622B CN 201710201003 A CN201710201003 A CN 201710201003A CN 107058622 B CN107058622 B CN 107058622B
- Authority
- CN
- China
- Prior art keywords
- specific primers
- probe
- probes
- bhq
- influenza
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a kit for detecting respiratory pathogens by a multiplex fluorescence PCR method, which comprises 6 components: reaction liquid A, reaction liquid B, reaction liquid C, enzyme mixed liquid, positive control and negative control, wherein the reaction liquid comprises 11 common respiratory pathogen detections (influenza A virus universal type, influenza B virus, respiratory syncytial virus, 1/2/3 type of human parainfluenza virus, adenovirus, mycoplasma pneumoniae, chlamydia pneumoniae, legionella pneumophila, streptococcus pneumoniae, haemophilus influenzae and A-group streptococcus), the detection is amplified through 3 reaction buffers, each reaction buffer comprises four fluorescence channels, and 90% of pathogen infection in clinic can be checked.
Description
Technical Field
The invention belongs to the field of fluorescent quantitative PCR, and particularly relates to a kit for jointly detecting respiratory pathogens by a multiple fluorescent PCR method.
Background
Acute respiratory infections have a high morbidity and mortality rate, particularly lower respiratory infections cause a significant disease burden, with 20% of preschool children worldwide dying from lower respiratory infections (SARI) and 90% of deaths due to pneumonia. Pneumonia or acute pneumonia is the main form of infection of febrile respiratory syndrome and also the leading cause of morbidity and mortality in children.
The incidence rate of pneumonia of children within 1 year of age is 0.01-0.68 times per year, and the incidence rate of pneumonia of children within 5 years of age is 0.14-0.66 times per year; the death rate of pneumonia is 485/10-890/10 ten thousand for children under 1 year old, and 184/10-1223/10 ten thousand for children under 5 years old. (the data source refers to the incidence and mortality of pneumonia in mainland China).
In the present stage, respiratory tract pathogens are diagnosed and checked clinically mainly by conventional means such as clinical symptoms, separation culture, immunodetection and the like. The separation and culture are time-consuming and labor-consuming, require strict laboratory environment and usually require 3 days for accurate diagnosis; the immunity detection sensitivity is low, a window period exists, and corresponding antigens or antibodies can be detected 3-7 days after infection.
Disclosure of Invention
The invention aims to provide a kit for detecting respiratory pathogens by combining a multiple fluorescence PCR method, which mainly adopts the multiple real-time fluorescence PCR method to detect the respiratory pathogens.
In order to achieve the purpose, the invention adopts the following technical scheme:
the kit comprises 11 common respiratory pathogen detections (influenza A virus universal type, influenza B virus, respiratory syncytial virus, 1/2/3 types of human parainfluenza virus, adenovirus, mycoplasma pneumoniae, chlamydia pneumoniae, legionella pneumophila, streptococcus pneumoniae, haemophilus influenzae and group A streptococcus), is amplified by 3 reaction buffers, and each reaction buffer comprises four fluorescence channels, so that 90% of pathogen infection in clinic can be detected.
The kit comprises the following components; the reaction solution A comprises specific primers and probes for influenza A, specific primers and probes for influenza B, specific primers and probes for adenovirus and specific primers and probes for respiratory syncytial virus;
the reaction solution B comprises mycoplasma pneumoniae specific primers and probes, chlamydia pneumoniae specific primers and probes, parainfluenza virus specific primers and probes, and internal control specific primers and probes;
the reaction solution C comprises specific primers and probes of legionella pneumophila, specific primers and probes of streptococcus pneumoniae, specific primers and probes of haemophilus influenzae and specific primers and probes of group A streptococcus;
the enzyme mixed solution contains hot start Taq enzyme and M-MLV enzyme;
the positive control comprises specific plasmid and TE water;
the negative control contained TE water.
The primer probe sequence is as follows:
the reagent component formula comprises:
the formula of the reaction solution A is as follows: tris (pH 8.8) 20mmol/L, KCl 60mmol/L, MgCl22.5mmol/L, EDTA 2Na0.1mmol/L, 1% DMSO, dATP 200. mu. mol/L, dGTP 200. mu. mol/L, dCTP 200. mu. mol/L, dTTP 200. mu. mol/L, influenza A virus upstream primer 417nmol/L, influenza A virus downstream primer 417nmol/L, influenza A virus probe 125nmol/L, influenza B virus upstream primer 417nmol/L, influenza B virus downstream primer 417nmol/L, influenza B virus probe 125nmol/L, adenovirus upstream primer 417nmol/L, adenovirus downstream primer 417nmol/L, adenovirus probe 125nmol/L, respiratory syncytial virus upstream primer 417nmol/L, respiratory syncytial virus downstream primer 417nmol/L, respiratory syncytial virus probe 125 nmol/L.
The formula of the reaction liquid B is as follows: tris (pH 8.8) 20mmol/L, KCl 60mmol/L, MgCl22.5mmol/L, EDTA 2Na0.1mmol/L, 1% DMSO, dATP 200. mu. mol/L, dGTP 200. mu. mol/L, dCTP 200. mu. mol/L, dTTP 200. mu. mol/L, Mycoplasma pneumoniae upstream primer 417nmol/L, Mycoplasma pneumoniae downstream primer 417nmol/L, Mycoplasma pneumoniae probe 125nmol/L, Chlamydia pneumoniae upstream primer 417nmol/L, Chlamydia pneumoniae downstream primer 125nmol/L, parainfluenza virus upstream primer 417nmol/L, parainfluenza virus downstream primer 417nmol/L, parainfluenza virus probe 125nmol/L, internal control upstream primer 417nmol/L, internal control downstream primer 417nmol/L, internal control probe 125 nmol/L.
The formula of the reaction solution C is as follows: tris (pH 8.8) 20mmol/L, KCl 60mmol/L, MgCl22.5mmol/L, EDTA.2 Na0.1mmol/L, 1% DMSO, dATP 200. mu. mol/L, dGTP 200. mu. mol/L, dCTP 200. mu. mol/L, dTTP 200. mu. mol/L, Streptococcus pneumoniae forward primer 417nmol/L, Streptococcus pneumoniae reverse primer 417nmol/L, Streptococcus pneumoniae probe 125nmol/L, Legionella pneumophila forward primer 417nmol/L, Legionella pneumophila reverse primer 417nmol/L, Legionella pneumophila forward primer 417nmol/L, group A Streptococcus reverse primer 417nmol/L, group A Streptococcus probe 125nmol/L, Haemophilus influenzae forward primer 417nmol/L, Haemophilus influenzae reverse primer 417nmol/L, Haemophilus influenzae probe 125 nmol/L.
The formula of the enzyme mixed solution is as follows: 4U/mu L Taq enzyme and 10U/mu L M-MLV enzyme.
The invention has the advantages that:
the invention has the advantages that: the invention has the advantages that 1 is achieved, and the sensitivity reaches 500 copies/mL; 2. directly obtaining a result after amplification, and obtaining the result 3 hours after obtaining a sample without subsequent operation; 3. no window period, once infected, can be detected.
The invention has the beneficial effects that: 1. the detection sensitivity is improved, the detection rate is increased, and the disease course of a patient is shortened; 2. the etiology is determined, the medicine taking accuracy is improved, and the abuse of medicines is avoided; 3. manual operation is reduced, the detection flow is shortened, and the detection cost is reduced.
Drawings
FIG. 1 is a graph showing the results of the specificity of reaction solution A.
FIG. 2 is a graph showing the results of the specificity of reaction solution B.
FIG. 3 is a graph showing the results of the specificity of reaction solution C.
FIG. 4 is a graph showing the results of the linear sensitivity of reaction solution A.
FIG. 5 is a graph showing the results of the linear sensitivity of reaction solution B.
FIG. 6 is a graph showing the results of the linear sensitivity of reaction solution C.
Detailed Description
Example 1
1. Preparation of reaction solution
(1) Preparation of reaction solution A
Taking a 10mL volumetric flask, and respectively adding 0.5mol/L Trizma®HCl 64μL，0.5mol/L Trizma®Base336μL，1 mol/L MgCl225 mu L, 1mol/L KCl 600 mu L, 0.5mol/L EDTA 2Na 2 mu L, DMSO 100 mu L, dNTPs 90 mu L, 50 mu mol/L influenza A virus upstream and downstream primers 83.4 mu L, 50 mu mol/L influenza A virus probe 25 mu L, 50 mu mol/L influenza B virus upstream and downstream primers 83.4 mu L, 50 mu mol/L influenza B virus probe 25 mu L, 50 mu mol/L adenovirus upstream and downstream primers 83.4 mu L, 50 mu mol/L adenovirus probe 25 mu L, 50 mu mol/L respiratory syncytial virusThe upstream and downstream primers are 83.4 muL each, and the respiratory syncytial virus probe is 25 muL at 50 mumol/L. The volume is made up to 10mL by double distilled water, the mixture is turned over to be fully mixed, the liquid is transferred into a 10mL beaker and is subpackaged into a centrifuge tube according to 1mL per branch, and the liquid is preserved at the temperature of minus 20 ℃ for standby.
(2) Preparation of reaction solution B
Taking a 10mL volumetric flask, and respectively adding 0.5mol/L Trizma®HCl 64μL，0.5mol/L Trizma®Base336μL，1 mol/L MgCl225 mu L, 1mol/L KCl 600 mu L, 0.5mol/L EDTA 2Na 2 mu L, DMSO 100 mu L, dNTPs 90 mu L, 50 mu mol/L mycoplasma pneumoniae upstream and downstream primers 83.4 mu L each, 50 mu mol/L mycoplasma pneumoniae probe 25 mu L, 50 mu mol/L chlamydia pneumoniae upstream and downstream primers 83.4 mu L each, 50 mu mol/L chlamydia pneumoniae probe 25 mu L, 50 mu mol/L parainfluenza virus upstream and downstream primers 83.4 mu L each, 50 mu mol/L parainfluenza virus probe 25 mu L, 50 mu mol/L internal control upstream and downstream primers 83.4 mu L each, and 50 mu mol/L internal control probe 25 mu L. The volume is made up to 10mL by double distilled water, the mixture is turned over to be fully mixed, the liquid is transferred into a 10mL beaker and is subpackaged into a centrifuge tube according to 1mL per branch, and the liquid is preserved at the temperature of minus 20 ℃ for standby.
(3) Preparation of reaction solution C
Taking a 10mL volumetric flask, and respectively adding 0.5mol/L Trizma®HCl 64μL，0.5mol/L Trizma®Base336μL，1 mol/L MgCl225 mu L, 1mol/L KCl 600 mu L, 0.5mol/L EDTA.2Na2uL, DMSO 100 mu L, dNTPs 90 mu L, 50 mu mol/L83.4 mu L of streptococcus pneumoniae upstream and downstream primers, 50 mu mol/L25 mu L of streptococcus pneumoniae probes, 50 mu mol/L83.4 mu L of legionella pneumophila upstream and downstream primers, 50 mu mol/L25 mu L of legionella pneumophila probes, 50 mu mol/L83.4 mu L of streptococcus A upstream and downstream primers, 50 mu mol/LA streptococcus probe 25 mu L, 50 mu mol/L83.4 mu L of haemophilus influenzae upstream and downstream primers, and 50 mu mol/L haemophilus influenzae probes 25 mu L. The volume is made up to 10mL by double distilled water, the mixture is turned over to be fully mixed, the liquid is transferred into a 10mL beaker and is subpackaged into a centrifuge tube according to 1mL per branch, and the liquid is preserved at the temperature of minus 20 ℃ for standby.
(4) Preparation of enzyme mixture
A10 mL volumetric flask was charged with 10000U/mL of Taq enzyme 4mL and 25000U/mL M-MLV enzyme 4mL, respectively. And (3) complementing the volume to 10mL by double distilled water, turning over to fully mix the solution, transferring the solution to a 10mL beaker, and subpackaging the solution into a centrifuge tube according to 100 muL/piece, and storing the solution at-20 ℃ for later use.
(5) Preparation of negative control
And adding physiological saline into a 100mL volumetric flask, fixing the volume to 100mL, transferring the liquid into a 10mL beaker, and subpackaging the liquid into a centrifuge tube according to 500 muL/piece, and storing the liquid at-20 ℃ for later use.
(6) Preparation of Positive control (concentration 5.0X 10)3copies/mL）
100mL volumetric flasks were each charged with 5.0X 107copies/mL of 10. mu.L each of influenza A, influenza B, adenovirus, respiratory syncytial virus, Mycoplasma pneumoniae, Chlamydia pneumoniae, parainfluenza virus, Streptococcus pneumoniae, Legionella pneumophila, group A streptococcus and Haemophilus influenzae plasmids (each plasmid was synthesized by general biosystems (Anhui) Co., Ltd.). And (4) diluting to 100mL by using normal saline, transferring the liquid into a 100mL beaker, and subpackaging the liquid into a centrifuge tube according to 500 mu L/piece, and storing the liquid at-20 ℃ for later use.
2. Nucleic acid extraction
Extracting nucleic acid by using a well-documented nucleic acid extraction kit, and measuring the purity of the nucleic acid by using a trace ultraviolet spectrophotometer after extraction, wherein the OD260/OD280 of the kit is between 1.6 and 2.0.
3. Sample adding machine
(1) Preparation of the reaction mixture
And taking out the reaction liquid A, the reaction liquid B, the reaction liquid C and the enzyme mixed solution, standing at room temperature to fully dissolve the reaction liquid A, the reaction liquid B, the reaction liquid C and the enzyme mixed solution, preparing a reaction mixture (each test configuration system: 29.5 muL of reaction liquid +0.5 muL of enzyme mixed solution), calculating the reagent dosage as required, and centrifuging for 5 seconds at 3000-5000g after fully mixing uniformly.
(2) Sample application
And adding 30 mu L of prepared reaction mixture into 0.2mL of amplification tube or eight-connected tube, then adding 2 mu L of extracted sample into the amplification tube or eight-connected tube, covering the amplification tube or eight-connected tube with a cover, slightly centrifuging, and placing the sample into a fluorescence PCR amplification instrument.
(3) Detection on machine
1) Loop condition setting
5 minutes at 38 ℃ and 10 minutes at 95 ℃; entering the following cycle: 15 seconds at 95 ℃ and 40 seconds at 58 ℃ (detection signal), 40 cycles; 30 seconds at 25 ℃.
2) Instrument detection channel selection
Fluorescein was designated FAM, ROX, HEX and CY 5.
4. Result judgment
(1) Negative controls should have no Ct value or 0; the Ct value of the positive control is less than or equal to 36; the Ct value of the CY5 channel in the reaction liquid B is less than or equal to 36;
(2) the sample test results should be judged according to the following table
Example 2
1. Reagent specificity verification
(1) Experimental sample
The specificity of the reagent is verified by adopting 10 specific samples, wherein 4 specific samples are physiological saline, 1 specific sample is a human metapneumovirus sample, 1 specific sample is a rubella virus sample, 1 specific sample is a measles virus sample, 1 specific sample is a Klebsiella pneumoniae sample, 1 specific sample is an escherichia coli sample and 1 specific sample is a staphylococcus aureus sample.
(2) Procedure of experiment
And (3) respectively detecting the above 10 specific samples by using the reaction liquid A, the reaction liquid B and the reaction liquid C, analyzing the detection result and verifying the specificity of the reagent.
(3) Results of the experiment
The three reaction solutions are negative in detecting 10 specific samples, which shows that the reagent has good specificity and no cross reaction. The specific results are shown in the following table:
detection result of specificity of reaction solution A
Detection result of specificity of reaction solution B
Detection result of specificity of reaction solution C
2. Reagent precision verification
(1) Experimental sample
1 sample of precision (concentration 5.0X 10)3copies/mL) was checked for reagent accuracy, and samples were prepared by adding 5.0X 10 samples to 100mL volumetric flasks7The volume of the plasmid is 100mL by physiological saline after 10 muL each of copies/mL influenza A virus, influenza B virus, adenovirus, respiratory syncytial virus, mycoplasma pneumoniae, chlamydia pneumoniae, parainfluenza virus, internal control, streptococcus pneumoniae, legionella pneumophila, group A streptococcus and haemophilus influenzae plasmid is fixed.
(2) Procedure of experiment
And (3) repeatedly detecting the precision samples for 10 times by using the reaction liquid A, the reaction liquid B and the reaction liquid C respectively, analyzing the detection result and verifying the precision of the reagent.
(3) Results of the experiment
The intra-batch variation coefficient (CV value) of the three reaction solutions for detecting the precision sample is less than 5 percent, which indicates that the reagent has good repeatability. The specific results are shown in the following table:
precision detection result of reaction solution A
Precision detection result of reaction solution B
Precision detection result of reaction solution C
3. Reagent minimum detection limit validation
(1) Experimental sample
1 sample with the lowest detection limit (concentration of 5.0 × 10) was taken2copies/mL) was checked for the lowest detection limit of the reagent, and the samples were prepared by taking 100mL volumetric flasks and adding 5.0X 10, respectively3The volume of the precision sample of copies/mL is 10mL, and the volume is adjusted to 100mL by using physiological saline.
(2) Procedure of experiment
And (3) repeatedly detecting the samples with the minimum detection limits for 10 times by using the reaction liquid A, the reaction liquid B and the reaction liquid C respectively, analyzing the detection result and verifying the minimum detection limit of the reagent.
(3) Results of the experiment
All samples with the lowest detection limit of the three reaction liquids are positive, and the lowest detection limit of the reagent is 5.0 multiplied by 102copies/mL. The specific results are shown in the following table:
4. reagent linear sensitivity validation
(1) Experimental sample
8 linear sensitivity samples (concentrations of 5.0X 10, respectively) were taken8copies/mL、5.0×107copies/mL、5.0×106copies/mL、5.0×105copies/mL、5.0×104copies/mL、5.0×103copies/mL、5.0×102copies/mL、5.0×101copies/mL) to verify the linearity of the reagent.
(2) Procedure of experiment
And respectively detecting the linear samples by using the reaction liquid A, the reaction liquid B and the reaction liquid C, analyzing the detection result and verifying the linear sensitivity of the reagent.
(3) Results of the experiment
The three reaction solutions are all positive in detection of linear sensitivity samples, and the linearity of the reagent is good. Specific results are shown in FIGS. 4-6 below.
Example 3: results of testing 120 clinical specimens
1. The relevant components of the kit were prepared according to the preparation method shown in example 1 and stored at-20 ℃ until use.
2. 120 pharynx swab samples and sputum samples of patients with clinical respiratory infection symptoms are collected by a women-young health care institute in Wuhan City, nucleic acid extraction is carried out by adopting a nucleic acid extraction reagent (virus type) produced by Debi Acer biotechnology (Xiamen) limited company, the purity of DNA samples is detected by an ultraviolet spectrophotometer, and OD260/OD280 of 120 samples are all between 1.6 and 2.0.
3. DNA was loaded and detected using a fluorescent quantitative PCR instrument according to the procedure described in example 1, using ABI7500 as the instrument.
4. The results were interpreted and counted according to the criteria shown in example 1 and are given in the following table:
the above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Debi Acer Biotech (Xiamen) Co.Ltd
<120> kit for joint detection of respiratory pathogens by multiple fluorescence PCR method
<130>36
<160>36
<170>PatentIn version 3.3
<210>1
<211>20
<212>DNA
<213> Artificial sequence
<400>1
<210>2
<211>20
<212>DNA
<213> Artificial sequence
<400>2
<210>3
<211>26
<212>DNA
<213> Artificial sequence
<400>3
tgggatcttg cacctgatat tgtgga 26
<210>4
<211>20
<212>DNA
<213> Artificial sequence
<400>4
atgcaagggt ttccatgttc 20
<210>5
<211>20
<212>DNA
<213> Artificial sequence
<400>5
accctccgtc tccacctact 20
<210>6
<211>21
<212>DNA
<213> Artificial sequence
<400>6
tccgatgacc agatctgggg g 21
<210>7
<211>20
<212>DNA
<213> Artificial sequence
<400>7
<210>8
<211>20
<212>DNA
<213> Artificial sequence
<400>8
<210>9
<211>21
<212>DNA
<213> Artificial sequence
<400>9
cgggcacttc ttcctcaccc g 21
<210>10
<211>20
<212>DNA
<213> Artificial sequence
<400>10
<210>11
<211>20
<212>DNA
<213> Artificial sequence
<400>11
<210>12
<211>25
<212>DNA
<213> Artificial sequence
<400>12
tgctcagaaa ttgggtggag aagca 25
<210>13
<211>20
<212>DNA
<213> Artificial sequence
<400>13
<210>14
<211>20
<212>DNA
<213> Artificial sequence
<400>14
ttgacgtcat cccttccttc 20
<210>15
<211>20
<212>DNA
<213> Artificial sequence
<400>15
<210>16
<211>20
<212>DNA
<213> Artificial sequence
<400>16
<210>17
<211>20
<212>DNA
<213> Artificial sequence
<400>17
<210>18
<211>20
<212>DNA
<213> Artificial sequence
<400>18
<210>19
<211>20
<212>DNA
<213> Artificial sequence
<400>19
<210>20
<211>20
<212>DNA
<213> Artificial sequence
<400>20
<210>21
<211>23
<212>DNA
<213> Artificial sequence
<400>21
ccaatgcagc agaggcaaaa tcc 23
<210>22
<211>20
<212>DNA
<213> Artificial sequence
<400>22
<210>23
<211>20
<212>DNA
<213> Artificial sequence
<400>23
<210>24
<211>20
<212>DNA
<213> Artificial sequence
<400>24
<210>25
<211>20
<212>DNA
<213> Artificial sequence
<400>25
<210>26
<211>20
<212>DNA
<213> Artificial sequence
<400>26
<210>27
<211>23
<212>DNA
<213> Artificial sequence
<400>27
caaaaacaag ccaggcgttg ttg 23
<210>28
<211>20
<212>DNA
<213> Artificial sequence
<400>28
<210>29
<211>20
<212>DNA
<213> Artificial sequence
<400>29
cgcactaaac ccttcagctc 20
<210>30
<211>20
<212>DNA
<213> Artificial sequence
<400>30
<210>31
<211>20
<212>DNA
<213> Artificial sequence
<400>31
<210>32
<211>20
<212>DNA
<213> Artificial sequence
<400>32
gaacggtctt tgcgtttctc 20
<210>33
<211>21
<212>DNA
<213> Artificial sequence
<400>33
aggggattcg cgctttgcag a 21
<210>34
<211>20
<212>DNA
<213> Artificial sequence
<400>34
<210>35
<211>20
<212>DNA
<213> Artificial sequence
<400>35
acgaccaaat ccgttgactc 20
<210>36
<211>20
<212>DNA
<213> Artificial sequence
<400>36
Claims (1)
1. A kit for detecting respiratory pathogens by a multiple fluorescence PCR method is characterized in that; the kit comprises the following components: the reaction solution A comprises specific primers and probes for influenza A, specific primers and probes for influenza B, specific primers and probes for adenovirus and specific primers and probes for respiratory syncytial virus;
the reaction solution B comprises mycoplasma pneumoniae specific primers and probes, chlamydia pneumoniae specific primers and probes, parainfluenza virus specific primers and probes, and internal control specific primers and probes;
the reaction solution C comprises specific primers and probes of legionella pneumophila, specific primers and probes of streptococcus pneumoniae, specific primers and probes of haemophilus influenzae and specific primers and probes of group A streptococcus;
the kit further comprises the following components: the enzyme mixed solution contains hot start Taq enzyme and M-MLV enzyme;
the positive control comprises specific plasmid and TE water;
negative controls included TE water;
the influenza A specific primers in the reaction solution A are as follows: f: tgatcctctcgtcattgcag, R: ctcaggcactccttccgtag, the probe is FAM-tgggatcttgcacctgatattgtgga-BHQ 1;
the specific primers for influenza B are as follows: f: atgcaagggtttccatgttc, R: accctccgtctccacctact, the probe is HEX-tccgatgaccagatctggggg-BHQ 1;
the adenovirus specific primers are: f: agtcttgcatgagccgttct, R: ggactcacacgtatgcatgg, the probe is ROX-cgggcacttcttcctcacccg-BHQ 2;
the specific primers of the respiratory syncytial virus are as follows: f: gtgcagggcaagtgatgtta, R: tgatgcttttgggttgttca, the probe is CY 5-tgctcagaaaattgggtggagaagcaBHQ 2;
the specific primers of the mycoplasma pneumoniae in the reaction liquid B are as follows: f: cagctcgtgtcgtgagatgt, R: ttgacgtcatcccttccttc, the probe is FAM-agtcccgcaacgagcgcaac-BHQ 1;
the chlamydia pneumoniae specific primers are: f: agccataacgccgtgaatac, R: atcccagtcatcagcctcac, the probe is HEX-cccgggccttgtacacaccg-BHQ 1;
parainfluenza virus specific primers were: f: caggtgtcacggctgttcta, R: ttccgtctgggttttactgg, the probe is ROX-ccaatgcagcagaggcaaaatcc-BHQ 2;
the internal control specific primer is as follows: f: acagtcagccgcatcttctt, R: acgaccaaatccgttgactc, the probe is CY5-cagccgagccacatcgctca-BHQ 2;
the specific primers of the legionella pneumophila in the reaction liquid C are as follows: f: ggctttaaccgaacagcaaa, R: ttgcaaaccacttggcaata, the probe is HEX-caaaaacaagccaggcgttgttg-BHQ 1;
the specific primers of the streptococcus pneumoniae are as follows: f: ttatcactggcggaaagacc, R: tcagttcaaccgctgcatag, the probe is FAM-acgttgggggcggttggaat-BHQ 1;
the specific primers of the haemophilus influenzae are as follows: f: ccgacttgcatttgctctct, R: gaacggtctttgcgtttctc, the probe is CY5-aggggattcgcgctttgcaga-BHQ 2;
the group A streptococcus specific primers are: f: gtgttttcggcacaaaaggt, R: cgcactaaacccttcagctc, the probe is ROX-cagctatgcggcgtgcctca-BHQ 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710201003.5A CN107058622B (en) | 2017-03-30 | 2017-03-30 | Kit for joint detection of respiratory pathogens by multiple fluorescence PCR method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710201003.5A CN107058622B (en) | 2017-03-30 | 2017-03-30 | Kit for joint detection of respiratory pathogens by multiple fluorescence PCR method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107058622A CN107058622A (en) | 2017-08-18 |
| CN107058622B true CN107058622B (en) | 2020-03-27 |
Family
ID=59601744
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710201003.5A Active CN107058622B (en) | 2017-03-30 | 2017-03-30 | Kit for joint detection of respiratory pathogens by multiple fluorescence PCR method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107058622B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP4015655A4 (en) * | 2020-04-16 | 2023-05-24 | Sansure Biotech Inc. | Composition, kit and method for detecting and classifying pathogens causing respiratory tract infections, and application |
Families Citing this family (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107630098B (en) * | 2017-11-13 | 2019-11-08 | 湖南圣湘生物科技有限公司 | Fluorescent PCR detection architecture, kit and detection method for joint-detection various respiratory road bacterium |
| CN107828633B (en) * | 2017-12-14 | 2021-04-27 | 苏州天隆生物科技有限公司 | Reagent device, reagent and method for extracting and detecting nucleic acid |
| CN108374059A (en) * | 2018-03-01 | 2018-08-07 | 绍兴迅敏康生物科技有限公司 | Respiratory Syncytial Virus(RSV) kit for detecting nucleic acid and its detection method |
| CN109609692B (en) * | 2018-12-25 | 2019-09-13 | 北京卓诚惠生生物科技股份有限公司 | For detecting the nucleic acid reagent, kit, system and method for respiratory tract infection pathogen |
| CN109897917A (en) * | 2019-04-01 | 2019-06-18 | 广东和信健康科技有限公司 | A kind of swin flu, second stream and adenovirus multiple nucleic acid detection primer probe groups and its kit |
| CN110273026B (en) * | 2019-06-20 | 2022-07-05 | 广州达安基因股份有限公司 | Multiple detection kit and detection method for respiratory tract infection |
| CN110331232B (en) * | 2019-06-20 | 2022-07-05 | 广州达安基因股份有限公司 | Multiple nucleic acid detection kit for respiratory pathogens |
| CN110408725B (en) * | 2019-06-20 | 2022-07-05 | 广州达安基因股份有限公司 | Kit for multiple detection of respiratory pathogens |
| CN110607379A (en) * | 2019-07-25 | 2019-12-24 | 廊坊诺道中科医学检验实验室有限公司 | Multiplex PCR detection method for simultaneously detecting multiple bacteria and mycoplasma and application thereof |
| CN110358865A (en) * | 2019-07-25 | 2019-10-22 | 廊坊诺道中科医学检验实验室有限公司 | For detecting kit and its application of Respirovirus |
| CN110468234A (en) * | 2019-08-09 | 2019-11-19 | 厦门安普利生物工程有限公司 | Multiple fluorescence quantitative PCR kit for 19 kinds of human respiratory viral diagnosis |
| CN111172324A (en) * | 2020-02-17 | 2020-05-19 | 深圳麦科田生物医疗技术有限公司 | Probe, primer, kit and detection method for detecting viruses A and B based on double loop-mediated isothermal amplification technology |
| CN111349720A (en) * | 2020-03-19 | 2020-06-30 | 中国医学科学院病原生物学研究所 | Nucleic acid reagent, kit, system and method for detecting respiratory tract infection virus |
| CN111549183B (en) * | 2020-06-08 | 2021-09-17 | 国药集团武汉血液制品有限公司 | Method for simultaneously detecting SARS-CoV-2 virus and other pathogens in plasma of convalescent coronary pneumonia convalescent patient |
| CN111808997A (en) * | 2020-08-27 | 2020-10-23 | 圣湘生物科技股份有限公司 | Composition, kit, application and method for detecting and typing 10 respiratory tract-related viruses |
| CN113249450A (en) * | 2021-06-29 | 2021-08-13 | 天津国际生物医药联合研究院 | Real-time fluorescent quantitative PCR method for detecting group A streptococcus nucleic acid |
| CN114540544A (en) * | 2021-12-24 | 2022-05-27 | 廊坊诺道中科医学检验实验室有限公司 | Primer-probe combination for detecting respiratory viruses, kit and application thereof |
| CN114058742B (en) * | 2022-01-18 | 2022-04-22 | 广州科方生物技术股份有限公司 | Primer probe composition, kit containing same and detection method thereof |
| CN116334265A (en) * | 2023-05-23 | 2023-06-27 | 爱科睿特生物医疗科技(南京)有限公司 | Multiplex RT-PCR method for simultaneously detecting four human respiratory tract infection bacteria, kit and application |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006073436A2 (en) * | 2004-04-29 | 2006-07-13 | The Trustees Of Columbia University In The City Of New York | Mass tag pcr for multiplex diagnostics |
| CN102212618A (en) * | 2011-05-22 | 2011-10-12 | 浙江大学 | Fourfold fluorescence quantitative PCR (Polymerase Chain Reaction) kit and application |
| CN102851391A (en) * | 2012-07-20 | 2013-01-02 | 宁波检验检疫科学技术研究院 | Human enterovirus four-color fluorescence RT-PCR detection kit and detection method |
| CN103993102A (en) * | 2014-03-07 | 2014-08-20 | 崔淑娟 | Multiple fluorescent PCR method and kit for simultaneous detection of human adenovirus, human mycoplasma pneumonia and bocavirus |
| CN104561377A (en) * | 2014-12-24 | 2015-04-29 | 华美生物工程有限公司 | Real-time fluorescent multiplex PCR (polymerase chain reaction) based kit for rapidly detecting common respiratory pathogens |
| CN105463129A (en) * | 2015-12-09 | 2016-04-06 | 深圳国际旅行卫生保健中心 | Double-fluorescent PCR detection primer, probe, reaction liquid and kit capable of detecting pathogens of respiratory tract |
-
2017
- 2017-03-30 CN CN201710201003.5A patent/CN107058622B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006073436A2 (en) * | 2004-04-29 | 2006-07-13 | The Trustees Of Columbia University In The City Of New York | Mass tag pcr for multiplex diagnostics |
| CN102212618A (en) * | 2011-05-22 | 2011-10-12 | 浙江大学 | Fourfold fluorescence quantitative PCR (Polymerase Chain Reaction) kit and application |
| CN102851391A (en) * | 2012-07-20 | 2013-01-02 | 宁波检验检疫科学技术研究院 | Human enterovirus four-color fluorescence RT-PCR detection kit and detection method |
| CN103993102A (en) * | 2014-03-07 | 2014-08-20 | 崔淑娟 | Multiple fluorescent PCR method and kit for simultaneous detection of human adenovirus, human mycoplasma pneumonia and bocavirus |
| CN104561377A (en) * | 2014-12-24 | 2015-04-29 | 华美生物工程有限公司 | Real-time fluorescent multiplex PCR (polymerase chain reaction) based kit for rapidly detecting common respiratory pathogens |
| CN105463129A (en) * | 2015-12-09 | 2016-04-06 | 深圳国际旅行卫生保健中心 | Double-fluorescent PCR detection primer, probe, reaction liquid and kit capable of detecting pathogens of respiratory tract |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP4015655A4 (en) * | 2020-04-16 | 2023-05-24 | Sansure Biotech Inc. | Composition, kit and method for detecting and classifying pathogens causing respiratory tract infections, and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107058622A (en) | 2017-08-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107058622B (en) | Kit for joint detection of respiratory pathogens by multiple fluorescence PCR method | |
| US20230287524A1 (en) | Composition, kit and method for detecting and classifying pathogens causing respiratory tract infection, and application | |
| CN102732638B (en) | Method for single-tube multiplex fluorescent polymerase chain reaction (PCR) detection of human coronavirus OC43, 229E, NL63, HKU1 and SARS, and primers, probes and kit adopted by the method | |
| TWI329742B (en) | ||
| Michaud et al. | Long-term storage at tropical temperature of dried-blood filter papers for detection and genotyping of RNA and DNA viruses by direct PCR | |
| CN110468234A (en) | Multiple fluorescence quantitative PCR kit for 19 kinds of human respiratory viral diagnosis | |
| CN110283940A (en) | Nucleic acid compositions, the detection kit of influenza virus and micro-fluidic chip | |
| CN105695631B (en) | Human immunodeficiency virus, hepatitis type B virus, Hepatitis C Virus Quick joint inspection kit and its preparation and application | |
| CN102277454A (en) | Primers, probes and assay kit for detecting transmissible gastroenteritis viruses and porcine epidemic diarrhea viruses | |
| CN111321253A (en) | Composition, kit, application and method for detecting and typing respiratory tract related viruses | |
| WO2023087868A1 (en) | Compositions, kits, methods for detecting and identifying pathogens that cause respiratory tract infections and use thereof | |
| CN111926111A (en) | High-flux identification and detection method for fifty-five common respiratory pathogens | |
| CN105886667A (en) | Detection kit for porcine epidemic diarrhea virus and detection method thereof | |
| CN107475389B (en) | Primer group, kit and method for detecting mycoplasma pneumoniae | |
| CN101550454B (en) | Real-time fluorescence PCR reagent kit for human metapneumovirus | |
| CN102321769B (en) | Primer pair for identifying newcastle disease virus and multi-subtype avian influenza virus and application thereof | |
| CN101575649B (en) | Quick detection technology for H9 type avian influenza virus | |
| CN100567504C (en) | A kind of actinobacillus pleuropneumoniae detection kit and application thereof | |
| WO2023109031A1 (en) | Respiratory pathogen detection kit, and preparation method therefor and use thereof | |
| CN100582240C (en) | Real time fluorescence quantitative determination method for pneumonia chlamydia and kit thereof | |
| CN116334305A (en) | Primer probe combination for detecting African swine fever virus and application thereof | |
| CN206768111U (en) | A kind of nest type PCR detection reagent for detecting calicivirus | |
| KÜÇÜK et al. | Antigenic and histopathologic evaluation with molecular characterization and identification of BPIV3 in cattle with respiratory system infections | |
| CN102605100B (en) | Primer, detection kit and detection method of LAMP (Loop-Mediated Isothermal Amplification) avian infectious bronchitis virus | |
| EP4056717A1 (en) | Composition for diagnosis of q fever and method for diagnosing q fever using real-time pcr |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |