CN109207639A - Respiratory pathogen rapid fluorescence PCR detection kit and its primer combination of probe - Google Patents

Respiratory pathogen rapid fluorescence PCR detection kit and its primer combination of probe Download PDF

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Publication number
CN109207639A
CN109207639A CN201811231737.9A CN201811231737A CN109207639A CN 109207639 A CN109207639 A CN 109207639A CN 201811231737 A CN201811231737 A CN 201811231737A CN 109207639 A CN109207639 A CN 109207639A
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Prior art keywords
seq
probe
primer
group
virus
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Chinese (zh)
Inventor
陈�峰
吴海磊
杨庆贵
杨国平
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Nantong International Travel Health Care Clinic
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Nantong International Travel Health Care Clinic
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Priority to CN201811231737.9A priority Critical patent/CN109207639A/en
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    • 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
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    • 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
    • C12Q1/6851—Quantitative amplification
    • 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

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  • General Health & Medical Sciences (AREA)
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  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Virology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a kind of respiratory pathogen rapid fluorescence PCR detection kit and its primer combination of probe, the kit includes amplification chip and contrast agents, and the amplification chip by dispensing and the detection reagent being lyophilized in micro-fluidic chip hole is constituted in advance；The contrast agents include negative control and positive control；The detection reagent includes that ingredient is RT-PCR reaction solution, ingredient Buffer, 2.0mM dNTPs, 1U/ μ L Taq archaeal dna polymerase, 2U/ μ L M-MLV reverse transcriptase, 16 type detection primer probes of 0.3U/ μ LRRI and 9 kinds of respiratory pathogens.Kit is lyophilized pre- method for filling using reagent and 16 type detection primer probe mixed liquors of 9 kinds of respiratory pathogens is sub-packed in advance in 16 duct microfluids amplification pipe, augmentation detection is carried out in conjunction with quickly micro-fluidic real-time fluorescent PCR amplification instrument, it can disposable screening 9 kinds of infectious diseases, 16 types relevant to respiratory tract heating paresthesia.

Description

Respiratory pathogen rapid fluorescence PCR detection kit and its primer combination of probe
Technical field
The present invention discloses a kind of respiratory pathogen rapid fluorescence PCR detection kit and its primer combination of probe, belongs to Biological technology application.
Background technique
Respiratory tract infection is one of most common disease in world wide, and disease incidence is in various countries resident disease incidence overall structure In occupy main status, the annual epidemic peak phase, there are about 10% residents to suffer from respiratory tract infection.Cause respiratory tract infection Mainly by various Respirovirus and some bacteriums, mycoplasma, Chlamydia.Commonly influenza A virus, second in virus Type influenza virus, parainfluenza virus, coronavirus, Respiratory Syncytial Virus(RSV), human metapneumovirus, human bocavirus, respiratory tract gland Virus, rhinovirus etc..These Respirovirus mostly have very strong appeal and propagation is fast, incubation period is short, morbidity is anxious.And And there are more and more new pathogenic Respirovirus to be found, it is caused greatly to Check and Examination of Port quarantine mechanism prevention and control Difficulty.And since in recent years, the epidemic situation that Respirovirus is caused constantly occurs, some Respirovirus such as Flu-A Virus, Respiratory Tract Adenovirus and the caused epidemic situation broken out of new virus constantly to make a variation become what field of public health faced One important problem, brings very huge economic loss and social danger.
It also largely relies on the laboratory testing of Respirovirus tradition to be separately cultured and biochemical identification, sensibility It is low, time-consuming and laborious, it needs one week or so to complete, it is difficult to the requirement quickly detected when reaching outbreak of epidemic, and the disease having There is presently no suitable cell systems and animal model to be cultivated for substance.Certainly, it is also wide that there are also immunological methods The general detection for being applied to Respirovirus, but its sensibility is low, and false negative rate is high, be easy to cause missing inspection, it is difficult to reach quick The requirement of detection.
Summary of the invention
The object of the present invention is to provide a kind of respiratory pathogen rapid fluorescence PCR detection kit and its primed probe groups It closes, to solve the problems, such as that respiratory tract heating pathogen body detection sensitivity is low, detection cycle is long in the prior art, detection flux is low.
To achieve the goals above, the technical solution of invention is as follows: the invention discloses a kind of respiratory pathogen is quick Fluorescence PCR detection reagent kit, including amplification chip and contrast agents, the amplification chip is by dispensing in advance and being lyophilized micro-fluidic Detection reagent in chip hole is constituted；The contrast agents include negative control and positive control；
The detection reagent includes that ingredient is RT-PCR reaction solution, ingredient Buffer, 2.0mM dNTPs, 1U/ μ L Taq archaeal dna polymerase, 2U/ μ L M-MLV reverse transcriptase, 16 type detection primers of 0.3U/ μ LRRI and 9 kinds of respiratory pathogens Probe.
The present invention has the advantage that compared with prior art
1, rapid and convenient, pollution-free, kit dispense technology using reagent in advance, and the PCR of every kind of pathogen detection is reacted MIX is sub-packed in advance in micro fluid reaction hole, when amplification only need to be added it is nucleic acid-templated, according to whether there is or not glimmering in corresponding aperture after amplification Optical signal judges whether there is corresponding pathogenic infection.
2, accuracy is good, high sensitivity, using taqman sonde method fluorescence quantitative PCR detection, all pathogenic bacteria all in accordance with Its specific gene design primer probe, guarantees the reliability of testing result；Chemical thermal starting archaeal dna polymerase presses down with low temperature System, high temperature resistant amplification ability, detection sensitivity is high, and lowest detection is limited to 103Copies/mL。
3, flux is big, and primary amplification can screen 16 types of 9 kinds of respiratory pathogens, and detection flux is big.
4, the Taq archaeal dna polymerase quickly, extended using the reverse transcriptase and rapid polymerization of quick cDNA synthesis, it is mating micro- The quick real-time fluorescence PCR instrument of fluid can be realized in 30min pathogen augmentation detection parainfluenza virus (HPIV 1/2/3/4), Coronavirus (NL63/OC43/229E/HKU1/MERS-CoV), Respiratory Syncytial Virus(RSV), Respiratory Tract Adenovirus, the inclined tuberculosis of people Poison, human bocavirus and rhinovirus.
16 type detection primers of the pre- method for filling by 9 kinds of respiratory pathogens are lyophilized using reagent in kit of the present invention Probe mixed liquor is sub-packed in advance in 16 duct microfluids amplification pipe, is expanded in conjunction with quick micro-fluidic real-time fluorescent PCR amplification instrument Increase detection, can disposable screening 9 kinds of infectious diseases, 16 types relevant to respiratory tract heating paresthesia, solve port burst and breathe To the timely control and elimination of infectious disease in road transmission disease contingency procedure, prevents burst respiratory infectious disease from further spreading, have Effect carries out burst infectious health prevention and control, escorts safely for border.
Detailed description of the invention
Fig. 1 is that kit detects influenza A virus sensitivity results；
Fig. 2 is that kit detects influenza B virus sensitivity results；
Fig. 3 micro-fluidic chip PCR instrument runs program interface figure.
Specific embodiment
The present invention disclose a kind of 9 kinds of respiratory pathogen rapid fluorescence PCR detection kits that port is often sent out and its primer, Probe combinations cover the respiratory pathogen classification often sent out at port.9 kinds of respiratory pathogens include influenza A virus, B-mode Influenza virus, parainfluenza virus (HPIV 1/2/3/4), coronavirus (NL63/OC43/229E/HKU1/MERS-CoV), breathing Road syncytial virus, Respiratory Tract Adenovirus, human metapneumovirus, human bocavirus and rhinovirus；Kit is by micro-fluid chip and RT- PCR (amplification) reaction solution composition, kit are lyophilized after being dispensed in advance using amplifing reagent.Wherein, micro-fluid chip contains 16 instead Ying Kong, every hole can detect a kind of pathogen type, by corresponding pathogen detection primed probe and reference gene primed probe (or Person is internal standard gene detection primer probe) it constitutes, RT-PCR (amplification) reaction solution is by Buffer, Mg2+, dNTPs, taqDNA polymerization Enzyme, reverse transcriptase M-MLV, RNase inhibitor RRI and primed probe are constituted.The Taq archaeal dna polymerase and reverse transcriptase M- MLV is respectively provided with the function of rapid polymerization extension and quick reverse transcription；The pathogen and the end of internal standard detection probe 5 ' label Fluorescent reporter group, 3 ' end mark fluorescent quenching groups.It can be doubtful to port immigration fever personnel using kit of the invention The quick detection for infecting respiratory pathogen infection, with flux is big, detection cycle is short, high sensitivity, testing result accurately may be used By the advantages that.
Below in conjunction with specific embodiment, the present invention is further described.It should be understood that these embodiments be only used for the present invention and It is not used in and limits the scope of the invention.Unless otherwise defined or described herein, scientific term described in this patent and the common skill in this field Art personnel, which understand, to be had the same meaning.
1 amplification system of embodiment optimization, by taking influenza A virus as an example
One, primer concentration optimizes
In PCR system, primer concentration is excessively high may to cause mispairing, lead to non-specific amplification, and when concentration is too low, it can shadow Ring the generation of PCR product, it is therefore necessary to optimize to primer concentration.In an experiment, it is dense to be provided with 3 different primers for we Degree, with plasmid bacterial (1 × 105Copies/mL and 1 × 103Copies/mL) and negative control is as detection sample, and template consumption is 3 μ L, each reaction system final volume are 10 μ L, detect the amplification difference of various concentration.When primer concentration is low, amplification efficiency It is slightly worse, and fluorescence response intensity is relatively low.When fluA gene primer (10 μm of ol/L) dosage is 1.0 μ L or more, amplification Efficiency is without significant difference.Comprehensively consider, selects 1000nmol/L as primer final concentration.
The determination of table 2-1 SS gene primer concentration
Two, concentration and probe concentration optimizes
Fluorescence probe is the core of entire quantitative PCR system, directly affects the quality of fluorescent PCR testing result.In reality Three concentration are provided in testing altogether to be compared.With fluA plasmid bacterial (1 × 105Copies/ml and 1 × 103Copies/ml) and Negative control is 3 μ L as detection sample, template consumption, and each reaction system final volume is 10 μ L.Experimental result see the table below 3-1, different concentration and probe concentrations has an impact to Ct value and fluorescence height, when fluA gene by fluorescence probe (10 μm of ol/L) dosage is Ideal test effect can integrally be reached when 0.5 μ L.
The influence that the concentration of table 2-2 EV71 probe detects fluorescent PCR
Three, the optimization of hot start Taq polymerase dosage
Hot start Taq polymerase is the important composition in PCR reaction, and how much dosage directly influences the amplification efficiency of PCR, because The PCR reaction solution of 4 kinds of this this experimental formula different hot start Taq polymerase dosages, hot start Taq polymerase dosage is 1.5U/ respectively Person-portion, 2.0U/ person-portion, 2.5U/ person-portion, 3.0U/ person-portion；With fluA gene plasmid bacterium (1 × 105Copies/mL and 1 × 103Copies/mL) and negative control is as detection sample, and template consumption is 5 μ L, and each reaction system final volume is 25 μ L. The experimental results showed that the dosage for suitably increasing hot start Taq polymerase is conducive to PCR amplification detection, 1.0U/ people is added in each system Part system hot start Taq polymerase can reach ideal expanding effect, be shown in Table 2-3.
Influence of the table 2-3 hot start Taq polymerase dosage to SS amplification efficiency
Four, reverse transcriptase M-MLV dosage optimization
How much reverse transcriptase (M-MLV) dosage directly influences transcriptional efficiency.Therefore 4 kinds of difference M- of this experimental formula The PCR reaction solution of MLV dosage, M-MLV dosage are 10U/ person-portion, 15U/ person-portion, 20U/ person-portion, 25U/ person-portion respectively；Use fluA Plasmid bacterial (1 × 105Copies/ml and 1 × 103Copies/ml) and negative control is as detection sample, and template consumption is 3 μ l, Each reaction system final volume is 10 μ l.The experimental results showed that the dosage for suitably increasing M-MLV is conducive to PCR amplification detection, 15U/ person-portion system M-MLV is added in each system can reach ideal expanding effect, be shown in Table 2-4.
Influence of the table 2-4 M-MLV dosage to fluA amplification efficiency
2 respiratory infectious disease of embodiment detects working specification
One, nucleic acid extraction
1.1 nucleic acid extractions: reagent is extracted in mating mono- step of the DNA/RNA cracking of kit, using 200 μ L serum or throat swab 50ul lysate is added in sample, and 5000RPM is centrifuged 2min after 95 DEG C of 2min, takes 50 μ L supernatants as template, specific extraction step Please refer to corresponding extracts kit specification.
Two, reagent prepares
2.1, according to measuring samples quantity (n), take out 16 hole chip versions of respective number, yin and yang attribute contrast agents, in room temperature Brief centrifugation after thawing；
Above-mentioned 16 hole chip version is moved to sample process area by 2.2.
Three, it is loaded
Processed 50 μ L of sample supernatant is taken to be added separately to 16 holes equipped with PCR reaction solution respectively with the suction nozzle with filter core PCR reacts in the well of microwell plate, and every hole is moved automatically into 3 μ L templates；
Four, augmentation detection
PCR reaction tube is put into fluorescent PCR amplification instrument and carries out augmentation detection by 4.1,；
The setting of 4.2 loop parameters: (V280):
50℃5min；95℃8s；95 DEG C of 7S, 60 DEG C of 14S, 40 circulations；
The 4.3 selection channels FAM and CY5 carry out fluorescence detection
The advantages of basic principles and main features and invention of invention have been shown and described above.The technical staff of the industry It should be appreciated that invention is not restricted to the described embodiments, what is described in the above embodiment and the description is only the principle of invention, Inventing under the premise of not departing from spirit and range will also have various changes and improvements, these changes and improvements both fall within requirement In the range of the invention of protection.The protection scope that invention requires is defined by appended claims and its equivalent.
Sequence table
<110>Nantong international travel health care clinic
<120>9 kinds of respiratory pathogen rapid fluorescence PCR detection kits and its primer combination of probe
<140>
<141>
<160>51
<210>1
<211>20
<212>DNA
<213>artificial sequence
<220>
<223>for detecting Flu-A disease forward primer
<400>1
CGAGGTCGAAACGTAYGTTCT
<210>2
<211>22
<212>DNA
<213>artificial sequence
<220>
<223>for detecting Flu-A disease reverse primer
<400>2
GGTCTTGTCTTTAGCCATTCCA
<210>3
<211>21
<212>DNA
<213>artificial sequence
<220>
<223>for detecting Flu-A disease oligonucleotide probe
<400>3
CCCCCTCAAAGCCGAGATCGC
<210>4
<211>22
<212>DNA
<213>artificial sequence
<220>
<223>for detecting influenza B virus forward primer
<400>4
CTTGTTGCCACTGATGATCTTA
<210>5
<211>19
<212>DNA
<213>artificial sequence
<220>
<223>for detecting influenza B virus reverse primer
<400>5
ACTCCCACCGCAGTTTCAG
<210>6
<211>25
<212>DNA
<213>artificial sequence
<220>
<223>for detecting influenza B virus oligonucleotide probe
<400>6
CCATCGGATCCTCAACTCACTCTTC
<210>7
<211>23
<212>DNA
<213>artificial sequence
<220>
<223>for detecting haemadsorption virus 2 forward primer
<400>7
ACTAGGTGTGACAGACACAGCAA
<210>8
<211>19
<212>DNA
<213>artificial sequence
<220>
<223>for detecting haemadsorption virus 2 reverse primer
<400>8
TCCCCTATCAGCRGTGTCC
<210>9
<211>22
<212>DNA
<213>artificial sequence
<220>
<223>for detecting haemadsorption virus 2 oligonucleotide probe
<400>9
TGCCACTTCTATRGCTCCACCA
<210>10
<211>19
<212>DNA
<213>artificial sequence
<220>
<223>for detecting acute laryngo-tracheo-bronchitis virus forward primer
<400>10
ATGGGCCACAATCAATCCT
<210>11
<211>20
<212>DNA
<213>artificial sequence
<220>
<223>for detecting acute laryngo-tracheo-bronchitis virus reverse primer
<400>11
GCGACCACCATATACAGGAA
<210>12
<211>23
<212>DNA
<213>artificial sequence
<220>
<223>for detecting acute laryngo-tracheo-bronchitis virus oligonucleotide probe
<400>12
CCTAGATGATAGATCCCGCTTCC
<210>13
<211>22
<212>DNA
<213>artificial sequence
<220>
<223>for detecting haemadsorption virus 1 forward primer
<400>13
GGACCACGCGCTCCATTCATCT
<210>14
<211>21
<212>DNA
<213>artificial sequence
<220>
<223>for detecting haemadsorption virus 1 reverse primer
<400>14
CCACTCCCATTGCATAACTCC
<210>15
<211>25
<212>DNA
<213>artificial sequence
<220>
<223>for detecting haemadsorption virus 1 oligonucleotide probe
<400>15
ATAGTTGCCTGGTGCGAACTCACCA
<210>16
<211>20
<212>DNA
<213>artificial sequence
<220>
<223>for detecting parainfluenza virus type 4 forward primer
<400>16
CCCACTGGATCTGAAGAGAG
<210>17
<211>21
<212>DNA
<213>artificial sequence
<220>
<223>for detecting parainfluenza virus type 4 reverse primer
<400>17
AATTTTCCTKGCATCTGTTTG
<210>18
<211>25
<212>DNA
<213>artificial sequence
<220>
<223>for detecting parainfluenza virus type 4 oligonucleotide probe
<400>18
TCYAACATCCTRCCCTGCTGCTTGT
<210>19
<211>22
<212>DNA
<213>artificial sequence
<220>
<223>for detecting coronavirus N L63 forward primer
<400>19
ATTCCCAGGAATCTTGTCCCTA
<210>20
<211>22
<212>DNA
<213>artificial sequence
<220>
<223>for detecting coronavirus N L63 reverse primer
<400>20
CTTTAGGAGGCAAATCAACACG
<210>21
<211>22
<212>DNA
<213>artificial sequence
<220>
<223>for detecting coronavirus N L63 oligonucleotide probe
<400>21
CGCCAACGCTCTTGAACATTCC
<210>22
<211>19
<212>DNA
<213>artificial sequence
<220>
<223>for detecting Coronavirus OC43 forward primer
<400>22
ACCGTATGCTAAAGACCAG
<210>23
<211>20
<212>DNA
<213>artificial sequence
<220>
<223>for detecting Coronavirus OC43 reverse primer
<400>23
CCTCATCGCTACTTGGGTCC
<210>24
<211>21
<212>DNA
<213>artificial sequence
<220>
<223>for detecting Coronavirus OC43 oligonucleotide probe
<400>24
CTGATGTCAATACCCCGGCTG
<210>25
<211>22
<212>DNA
<213>artificial sequence
<220>
<223>for detecting coronavirus 229E forward primer
<400>25
TGAAGGTGTTGTCTGGGTTGCT
<210>21
<211>20
<212>DNA
<213>artificial sequence
<220>
<223>for detecting coronavirus 229E reverse primer
<400>26
GATTGAAGTGTGGTATCTCTG
<210>27
<211>21
<212>DNA
<213>artificial sequence
<220>
<223>for detecting coronavirus 229E oligonucleotide probe
<400>27
CTTGCGCCTAACACCGTAACC
<210>28
<211>21
<212>DNA
<213>artificial sequence
<220>
<223>for detecting Coronavirus HKU1 forward primer
<400>28
ACAGCTGATGGTCAACAAAAG
<210>29
<211>19
<212>DNA
<213>artificial sequence
<220>
<223>for detecting Coronavirus HKU1 reverse primer
<400>29
CGTGGGCATCACCATAGGA
<210>30
<211>25
<212>DNA
<213>artificial sequence
<220>
<223>for detecting golden Coronavirus HKU1 oligonucleotide probe
<400>30
CTATCTCGGTACCGGYCCATATGCC
<210>31
<211>21
<212>DNA
<213>artificial sequence
<220>
<223>for detecting Middle East breathing syndrome MERS-CoV forward primer
<400>31
ACTAATCGCCAGTACCATCAG
<210>32
<211>20
<212>DNA
<213>artificial sequence
<220>
<223>for detecting Middle East breathing syndrome MERS-CoV reverse primer
<400>32
GTAGTACCAATGACGCAAGT
<210>33
<211>21
<212>DNA
<213>artificial sequence
<220>
<223>for detecting Middle East breathing syndrome MERS-CoV oligonucleotide probe
<400>33
AGTCCATGGCTGCAACTCGTG
<210>34
<211>20
<212>DNA
<213>artificial sequence
<220>
<223>for detecting Respiratory Syncytial Virus(RSV) forward primer
<400>34
ATGGGGCAAATATGGAAACA
<210>35
<211>22
<212>DNA
<213>artificial sequence
<220>
<223>for detecting Respiratory Syncytial Virus(RSV) reverse primer
<400>35
GCACCCATATTGTTAGTGATGC
<210>36
<211>24
<212>DNA
<213>artificial sequence
<220>
<223>for detecting Respiratory Syncytial Virus(RSV) oligonucleotide probe
<400>36
CTTCACGAGGGCTCCACATACACA
<210>37
<211>17
<212>DNA
<213>artificial sequence
<220>
<223>for detecting Respiratory Tract Adenovirus forward primer
<400>37
CACCCCCTCGATGATGC
<210>38
<211>22
<212>DNA
<213>artificial sequence
<220>
<223>for detecting Respiratory Tract Adenovirus reverse primer
<400>38
CTCAGGTACTCCGAAGCATCCT
<210>39
<211>17
<212>DNA
<213>artificial sequence
<220>
<223>for detecting Respiratory Tract Adenovirus oligonucleotide probe
<400>39
TACATGCACATCGCCGG
<210>40
<211>25
<212>DNA
<213>artificial sequence
<220>
<223>for detecting human metapneumovirus forward primer
<400>40
AARCATGCTATATTAAAAGAGTCTC
<210>41
<211>26
<212>DNA
<213>artificial sequence
<220>
<223>for detecting human metapneumovirus reverse primer
<400>41
CCTATYTCTGCAGCATATTTGTAATC
<210>42
<211>28
<212>DNA
<213>artificial sequence
<220>
<223>for detecting human metapneumovirus oligonucleotide probe
<400>42
CAACHGCAGTGACACCYTCATCATTGCA
<210>43
<211>21
<212>DNA
<213>artificial sequence
<220>
<223>for detecting human bocavirus forward primer
<400>43
GAAGAGACACTGGCAGACAAC
<210>44
<211>17
<212>DNA
<213>artificial sequence
<220>
<223>for detecting human bocavirus reverse primer
<400>44
CCACTGTGTCGGATCGG
<210>45
<211>20
<212>DNA
<213>artificial sequence
<220>
<223>for detecting human bocavirus oligonucleotide probe
<400>45
CTGCGGCTCCTGCTCCTGTG
<210>46
<211>23
<212>DNA
<213>artificial sequence
<220>
<223>for detecting rhinovirus forward primer
<400>46
GACATGGTGYGAAGAGYCTATTG
<210>47
<211>22
<212>DNA
<213>artificial sequence
<220>
<223>for detecting rhinovirus reverse primer
<400>47
AAACACGGACACCCAAAGTAGT
<210>48
<211>22
<212>DNA
<213>artificial sequence
<220>
<223>for detecting rhinovirus oligonucleotide probe
<400>48
CCGGCCCCTGAATGCGGCTAAT
<210>49
<211>17
<212>DNA
<213>artificial sequence
<220>
<223>for detecting people's reference gene β-actin gene forward primer
<400>49
CGAGCGCGGCTACAGCT
<210>50
<211>22
<212>DNA
<213>artificial sequence
<220>
<223>for detecting people's reference gene β-actin gene reverse primer
<400>50
TCCTTAATGTCACGCACGATTT
<210>51
<211>18
<212>DNA
<213>artificial sequence
<220>
<223>for detecting people's reference gene β-actin gene oligonucleotide probe
<400>51
ACCACCACGGCCGAGCGG
Sequence table
<110>Nantong international travel health care clinic
<120>respiratory pathogen rapid fluorescence PCR detection kit and its primer combination of probe
<160> 51
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ccccctcaaa gccgagatcg c 21
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cttgttgcca ctgatgatct ta 22
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<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
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ccactcccat tgcataactc c 21
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<213>artificial sequence (Artificial Sequence)
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atagttgcct ggtgcgaact cacca 25
<210> 16
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
cccactggat ctgaagagag 20
<210> 17
<211> 21
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<213>artificial sequence (Artificial Sequence)
<400> 17
aattttcctk gcatctgttt g 21
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<211> 25
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<213>artificial sequence (Artificial Sequence)
<400> 18
tcyaacatcc trccctgctg cttgt 25
<210> 19
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
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<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
ctttaggagg caaatcaaca cg 22
<210> 21
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
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cgccaacgct cttgaacatt cc 22
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<213>artificial sequence (Artificial Sequence)
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<210> 23
<211> 20
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<213>artificial sequence (Artificial Sequence)
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<213>artificial sequence (Artificial Sequence)
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<213>artificial sequence (Artificial Sequence)
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<213>artificial sequence (Artificial Sequence)
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<213>artificial sequence (Artificial Sequence)
<400> 36
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<213>artificial sequence (Artificial Sequence)
<400> 37
caccccctcg atgatgc 17
<210> 38
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
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<213>artificial sequence (Artificial Sequence)
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<210> 40
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
aarcatgcta tattaaaaga gtctc 25
<210> 41
<211> 26
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<213>artificial sequence (Artificial Sequence)
<400> 41
cctatytctg cagcatattt gtaatc 26
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<213>artificial sequence (Artificial Sequence)
<400> 42
caachgcagt gacaccytca tcattgca 28
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<211> 21
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<213>artificial sequence (Artificial Sequence)
<400> 43
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<213>artificial sequence (Artificial Sequence)
<400> 44
ccactgtgtc ggatcgg 17
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<211> 20
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<213>artificial sequence (Artificial Sequence)
<400> 45
ctgcggctcc tgctcctgtg 20
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gacatggtgy gaagagycta ttg 23
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Claims (10)

1. a kind of respiratory pathogen rapid fluorescence PCR detection kit, including amplification chip and contrast agents, the amplification Chip by dispensing and the detection reagent being lyophilized in micro-fluidic chip hole is constituted in advance；The contrast agents include negative control and Positive control；
The detection reagent includes that ingredient is RT-PCR reaction solution, ingredient Buffer, 2.0mM dNTPs, 1U/ μ L Taq Archaeal dna polymerase, 2U/ μ L M-MLV reverse transcriptase, 16 type detection primers of 0.3U/ μ LRRI and 9 kinds of respiratory pathogens are visited Needle.
2. respiratory pathogen rapid fluorescence PCR detection kit according to claim 1, which is characterized in that described Taq archaeal dna polymerase is selected from Ex Taq HS, Platinum II Taq HS DNA polymerase, AmpliTaq Gold Any one in DNA polymerase；The reverse transcriptase be selected from PrimerScript II RTase, Any one in PrimerScript RTase, Superscript IV.
3. respiratory pathogen rapid fluorescence PCR detection kit according to claim 1, which is characterized in that described Buffer includes 10mM Tris-HCl, 15mM KCl, 3.5mM MgCl2。
4. a kind of respiratory pathogen rapid fluorescence PCR detection kit described in any one of -3 according to claim 1 Primer combination of probe, which is characterized in that micro-fluid chip contains 16 reacting holes, and every hole can detect a kind of pathogen type, by Corresponding pathogen detection primed probe and reference gene primed probe are constituted；
9 kinds of respiratory pathogens include influenza A virus, influenza B virus, parainfluenza virus, coronavirus, exhale Inhale road syncytial virus, Respiratory Tract Adenovirus, human metapneumovirus, human bocavirus and rhinovirus；
The particular sequence and serial number of described 9 kinds of respiratory pathogens, 16 type detection primers and probe are as follows:
Influenza A virus upstream primer F:CGAGGTCGAAACGTAYGTTCT SEQ ID NO.1
Influenza A virus downstream primer R:GGTCTTGTCTTTAGCCATTCCA SEQ ID NO.2
Influenza A probe P:5 '-fluorescent reporter group-CCCCCTCAAAGCCGAGATCGC- fluorescence is quenched the SEQ of group -3 ' ID NO.3
Influenza B virus upstream primer F:CTTGTTGCCACTGATGATCTTA SEQ ID NO.4
Influenza B virus downstream primer R:ACTCCCACCGCAGTTTCAG SEQ ID NO.5
Influenza B virus probe P:5 '-fluorescent reporter group-CCATCGGATCCTCAACTCACTCTTC- fluorescence is quenched group -3 ' SEQ ID NO.6
Haemadsorption virus 2 upstream primer F:ACTAGGTGTGACAGACACAGCAA SEQ ID NO.7
Haemadsorption virus 2 downstream primer R:TCCCCTATCAGCRGTGTCC SEQ ID NO.8
Haemadsorption virus 2 probe P:5 '-fluorescent reporter group-TGCCACTTCTATRGCTCCACCA- fluorescence is quenched group -3 ' SEQ ID NO.9
Acute laryngo-tracheo-bronchitis virus upstream primer F:ATGGGCCACAATCAATCCT SEQ ID NO.10
Acute laryngo-tracheo-bronchitis virus downstream primer R:GCGACCACCATATACAGGAA SEQ ID NO.11
Acute laryngo-tracheo-bronchitis virus probe P:5 '-fluorescent reporter group-CCTAGATGATAGATCCCGCTTCC- fluorescence is quenched group -3 ' SEQ ID NO.12
Haemadsorption virus 1 upstream primer F:GGACCACGCGCTCCATTCATCT SEQ ID NO.13
Haemadsorption virus 1 downstream primer R:CCACTCCCATTGCATAACTCC SEQ ID NO.14
Haemadsorption virus 1 probe P:5 '-fluorescent reporter group-ATAGTTGCCTGGTGCGAACTCACCA- fluorescence is quenched group- 3’SEQ ID NO.15
Parainfluenza virus type 4 upstream primer F:CCCACTGGATCTGAAGAGAG SEQ ID NO.16
Parainfluenza virus type 4 downstream primer R:AATTTTCCTKGCATCTGTTTG SEQ ID NO.17
Parainfluenza virus type 4 probe P:5 '-fluorescent reporter group-TCYAACATCCTRCCCTGCTGCTTGT- fluorescence is quenched group- 3’SEQ ID NO.18
Coronavirus N L63 upstream primer F:ATTCCCAGGAATCTTGTCCCTA SEQ ID NO.19
Coronavirus N L63 downstream primer R:CTTTAGGAGGCAAATCAACACG SEQ ID NO.20
Coronavirus N L63 probe P:5 '-fluorescent reporter group-CGCCAACGCTCTTGAACATTCC- fluorescence is quenched the SEQ of group -3 ' ID NO.21
Coronavirus OC43 upstream primer F:ACCGTATGCTAAAGACCAG SEQ ID NO.22
Coronavirus OC43 downstream primer R:CCTCATCGCTACTTGGGTCC SEQ ID NO.23
Coronavirus OC43 probe P:5 '-fluorescent reporter group-CTGATGTCAATACCCCGGCTG- fluorescence is quenched the SEQ of group -3 ' ID NO.24
Coronavirus 229E upstream primer F:TGAAGGTGTTGTCTGGGTTGCT SEQ ID NO.25
Coronavirus 229E downstream primer R:GATTGAAGTGTGGTATCTCTG SEQ ID NO.26
Coronavirus 229E probe P:5 '-fluorescent reporter group-CTTGCGCCTAACACCGTAACC- fluorescence is quenched the SEQ of group -3 ' ID NO.27
Coronavirus HKU1 upstream primer F:ACAGCTGATGGTCAACAAAAG SEQ ID NO.28
Coronavirus HKU1 downstream primer R:CGTGGGCATCACCATAGGA SEQ ID NO.29
Coronavirus HKU1 probe P:5 '-fluorescent reporter group-CTATCTCGGTACCGGYCCATATGCC- fluorescence is quenched group -3 ' SEQ ID NO.30
Middle East breathing syndrome MERS-CoV upstream primer F:ACTAATCGCCAGTACCATCAG SEQ ID NO.31
Middle East breathing syndrome MERS-CoV downstream primer R:GTAGTACCAATGACGCAAGT SEQ ID NO.32
Middle East breathing syndrome MERS-CoV probe P:5 '-fluorescent reporter group-AGTCCATGGCTGCAACTCGTG- fluorescence is quenched The SEQ ID of group -3 ' NO.33
Respiratory Syncytial Virus(RSV) upstream primer F:ATGGGGCAAATATGGAAACA SEQ ID NO.34
Respiratory Syncytial Virus(RSV) downstream primer R:GCACCCATATTGTTAGTGATGC SEQ ID NO.35
Respiratory Syncytial Virus(RSV) gene probe P:5 '-fluorescent reporter group-CTTCACGAGGGCTCCACATACACA- fluorescence is quenched base - 3 ' SEQ ID NO.36 of group
Respiratory Tract Adenovirus upstream primer F:CACCCCCTCGATGATGC SEQ ID NO.37
Respiratory Tract Adenovirus downstream primer R:CTCAGGTACTCCGAAGCATCCT SEQ ID NO.38
Respiratory Tract Adenovirus probe P:5 '-fluorescent reporter group-TACATGCACATCGCCGG- fluorescence is quenched the SEQ of group -3 ' ID NO.39
Human metapneumovirus upstream primer F:AARCATGCTATATTAAAAGAGTCTC SEQ ID NO.40
Human metapneumovirus downstream primer R:CCTATYTCTGCAGCATATTTGTAATC SEQ ID NO.41
Human metapneumovirus probe P:5 '-fluorescent reporter group-CAACHGCAGTGACACCYTCATCATTGCA- fluorescence is quenched group- 3’SEQ ID NO.42
Human bocavirus upstream primer F:GAAGAGACACTGGCAGACAAC SEQ ID NO.43
Human bocavirus downstream primer R:CCACTGTGTCGGATCGG SEQ ID NO.44
Human bocavirus probe P:5 '-fluorescent reporter group-CTGCGGCTCCTGCTCCTGTG- fluorescence is quenched the SEQ of group -3 ' ID NO.45
Rhinovirus upstream primer F:GACATGGTGYGAAGAGYCTATTG SEQ ID NO.46
Rhinovirus downstream primer R:AAACACGGACACCCAAAGTAGT SEQ ID NO.47
Rhinovirus probe P:5 '-fluorescent reporter group-CCGGCCCCTGAATGCGGCTAAT- fluorescence is quenched the SEQ of group -3 ' ID NO.48
People's reference gene β-actin upstream region of gene primers F: CGAGCGCGGCTACAGCT SEQ ID NO.49
People's reference gene β-actin downstream of gene primer R:TCCTTAATGTCACGCACGATTT SEQ ID NO.50 people's internal reference Gene β-actin gene probe P:5 '-fluorescent reporter group-ACCACCACGGCCGAGCGG- fluorescence is quenched the SEQ of group -3 ' ID NO.51。
5. the primer combination of probe of respiratory pathogen rapid fluorescence PCR detection kit according to claim 4, special Sign is that the parainfluenza virus is 1/2/3/4 parainfluenza virus of HPIV.
6. the primer combination of probe of respiratory pathogen rapid fluorescence PCR detection kit according to claim 4, special Sign is that the coronavirus is NL63/OC43/229E/HKU1/MERS-CoV coronavirus.
7. the primer combination of probe of respiratory pathogen rapid fluorescence PCR detection kit according to claim 4, special Sign is, the fluorescent reporter group is selected from FAM, HEX, ROX, CY3, CY5 fluorescent reporter group, and the fluorescence is quenched group Selected from BHQ1, BHQ2, TAMRA, MGB fluorescent quenching group.
8. the primer combination of probe of respiratory pathogen rapid fluorescence PCR detection kit according to claim 4, special Sign is, in each chip reacting hole target pathogen detection primer concentration and probe concentration ratio be 500nM~1200nM:300nM~ 600nM, reference gene primed probe concentration ratio are 200nM~500nM:100nM~300nM.
9. the primer combination of probe of respiratory pathogen rapid fluorescence PCR detection kit according to claim 8, special Sign is that target pathogen detection primer concentration and probe concentration ratio is 800nM:400nM in each chip reacting hole.
10. the primer combination of probe of respiratory pathogen rapid fluorescence PCR detection kit according to claim 8, It is characterized in that, reference gene primed probe concentration ratio is 400nM:200nM.
CN201811231737.9A 2018-10-22 2018-10-22 Respiratory pathogen rapid fluorescence PCR detection kit and its primer combination of probe Pending CN109207639A (en)

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CN110408614A (en) * 2019-07-22 2019-11-05 深圳市人民医院 The kit detected for five kinds of Respirovirus and its application
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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
CN111088408A (en) * 2020-03-20 2020-05-01 广州凯普医药科技有限公司 Detection kit for new coronavirus, influenza A and influenza B and respiratory syncytial virus
CN111808989A (en) * 2020-06-18 2020-10-23 重庆浦洛通基因医学研究院有限公司 Coronavirus/influenza virus/rhinovirus nucleic acid combined detection kit and use method thereof
CN111876526A (en) * 2020-08-07 2020-11-03 福州大学 Microfluidic chip for detecting HPV (human papillomavirus) virus and typing
CN112553380A (en) * 2020-12-31 2021-03-26 哈尔滨星云医学检验所有限公司 Method for rapidly detecting 12 respiratory viruses by utilizing multiplex PCR (polymerase chain reaction) technology and application thereof
CN115992270A (en) * 2022-08-30 2023-04-21 四川省亚中基因科技有限责任公司 Primer probe composition, reagent and kit for detecting respiratory bacterial pathogens

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