CN109371174A - Middle East respiration syndrome coronavirus fluorescence PCR detection reagent kit - Google Patents

Middle East respiration syndrome coronavirus fluorescence PCR detection reagent kit Download PDF

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Publication number
CN109371174A
CN109371174A CN201811566975.5A CN201811566975A CN109371174A CN 109371174 A CN109371174 A CN 109371174A CN 201811566975 A CN201811566975 A CN 201811566975A CN 109371174 A CN109371174 A CN 109371174A
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China
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middle east
kit
syndrome coronavirus
respiration syndrome
pcr
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CN201811566975.5A
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不公告发明人
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JIANGSU UNINOVO BIOLOGICAL TECHNOLOGY Co Ltd
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JIANGSU UNINOVO BIOLOGICAL TECHNOLOGY Co Ltd
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Priority to CN201811566975.5A priority Critical patent/CN109371174A/en
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    • 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]
    • 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 present invention relates to a kind of using kit existing for Middle East respiration syndrome coronavirus nucleic acid in Fluorescence PCR assay specific detection sample.Utilize kit of the invention, Middle East respiration syndrome coronavirus nucleic acid can be whether there is in quick test sample, the kit can be detected delicately and identify respiration syndrome coronavirus in the Middle East present in sample, its Monitoring lower-cut is the 10 every reaction systems of copy, has important application value in fields such as disease surveillance, clinical diagnosises.

Description

Middle East respiration syndrome coronavirus fluorescence PCR detection reagent kit
Technical field
The present invention relates to a kind of using Middle East respiration syndrome coronavirus core in Fluorescence PCR assay specific detection sample Kit existing for acid belongs to the in-vitro diagnosis field of Middle East respiration syndrome coronavirus.
Background technique
Middle East respiration syndrome coronavirus (Middle East Respiratory Syndrome, MERS) is a kind of new The coronavirus of type, this virus have been named as Middle East breathing syndrome coronavirus, most of MERS virus infection diseases Example occurs in Saudi Arabia.The intracorporal coronavirus of people is most separated earlier than generation nineteen sixty in Britain, and virus is because of its surface imperial crown The bump of shape and gain the name.It may be related to the infection in respiratory system of people, pig, cat, dog, mouse and chicken.China occurs within 2003 SARS virus belong to coronavirus.MERS is found in Saudi Arabia for the first time in September, 2012, is then gone out successively in the Middle East, Europe Existing case report, the symptom and SARS after infecting Middle East novel coronavirus are somewhat like, and the infected will appear acute, serious breathing Tract disease, with fever, cough, shortness of breath and expiratory dyspnea, serious case will appear renal failure and death.MRES is Human coronary virus known to six kinds, and be separated in 10 years in the past the third.
In laboratory testing, PCR method to substitute since its fast and convenient feature gradually shows with Virus culture and The trend of traditional detection method based on Serologic detection.And for PCR method, the specificity of primer is the spy of its detection Anisotropic and sensibility basis.
The present invention target sequence design primer and MGB probe special for Middle East respiration syndrome coronavirus, utilize The method of real-time RT-PCR can be quick for identifying respiration syndrome coronavirus nucleic acid in the Middle East in test sample Obtain specific detection result.
Summary of the invention
The present invention solves the technical problem of whether there is Middle East respiration syndrome in rapidly and accurately test sample Coronavirus provides a kind of fluorescent PCR kit of specific detection Middle East respiration syndrome coronavirus.
The technical problem to be solved by the present invention is to what is be achieved through the following technical solutions:
The kit of Middle East respiration syndrome coronavirus in a kind of specific detection sample, component include that PCR reaction solution, enzyme are mixed Close liquid, negative quality-control product and positive quality control product.Wherein PCR reaction solution mainly contain relevant primer and probe, reaction buffer, Mg2+ and dNTP etc., enzyme mixation mainly contain reverse transcriptase and hot start Taq polymerase, and negative quality-control product is no RNase and DNase Water, positive quality control product are that the RNA of target sequence is detected containing Middle East respiration syndrome coronavirus.
Primer in PCR reaction solution for nucleic acid amplification is that P1 and P2, P1 and P2 are coronal for Middle East respiration syndrome The specific primer that viral genome group specificity conserved sequence is designed and filtered out through preliminary experiment；For glimmering in PCR reaction solution The oligonucleotide probe of optical monitoring signal is Probel, and Probel is for Middle East respiration syndrome coronavirus gene group spy The specific probe that anisotropic conserved sequence is designed and filtered out through preliminary experiment, wherein fluorescent reporter group X1 is FAM, fluorescent quenching Group Y1 is MGB (being shown in Table 1).
The primer and probe sequence of the detection Middle East respiration syndrome coronavirus of table 1
Title Sequence
P1 5’-TGCTAAGAATAGAGCTCGCACTGTT-3’
P2 5’-TAGTACCAATGACGCAAGTCGCT-3’
Probe1 5’-X1-TCGCCAGTACCATCAG-Y1-3’
It is a further object to provide this fluorescence PCR detection reagent kit eastern respiration syndrome coronavirus in the detection Application method, comprising:
The PCR reaction system that kit is selected is 20 μ l, including 2 × PCR buffer, 10 μ l, 1.0 μ l of 10mmol/L dNTP, 25 μm each 0.5 μ l of ol/L primer, 10 μm of 0.2 μ l of ol/L probe, 0.8 μ l of reverse transcriptase and hot start Taq polymerase mixed liquor, sample RNA 2 μ l add aqua sterilisa to whole 20 μ l of system.
The PCR reaction cycle parameter of kit is reverse transcription step: 42 DEG C, 10min；The initial denaturation stage: 94 DEG C, 10sec； Pre-amplification phase: 94 DEG C of denaturation, 5sec；50 DEG C of annealing, 20sec；Extend 72 DEG C, 20sec；Totally 5 circulations.Pay attention to this stage not Acquire fluorescence signal.Detection-phase: 94 DEG C of denaturation, 5sec；56 DEG C of annealing, 50sec；Extend 72 DEG C, 15sec；Totally 40 circulations. Fluorescence signal is detected in annealing steps.
Quality control: experiment should set up positive and negative control every time, and negative control is without Ct value (or Ct value is 0), positive control Value≤30 Ct, otherwise experimental result is invalid.
As a result interpretation:
It is positive: " S " type amplification curve, value≤35 Ct occur；It is suspicious: " S " type amplification curve, but Ct value > 35 occur；It is negative: not have It occurs " S " type amplification curve or although curve has been more than threshold value, but be not in " S " type；To suspect results, experiment should be repeated, If repeating experiment or " S " type amplification curve occur, negative control is not polluted, be can determine whether as the positive.
Sample requirement: clinical sample type includes throat swab and viral cultures；It should be transported with ice after clinical sample acquisition Defeated, -20 DEG C of preservations are unable to multigelation；RNA is extracted from clinical sample, it is proposed that uses commercial reagents stable and reliable for performance Box, specific method should detect immediately referring to corresponding commodity kit specification, the RNA of extraction, otherwise, after RNA should be dispensed- 80 DEG C~-20 DEG C preservations.
Kit provided by the invention has specificity well, can detect the core of Middle East respiration syndrome coronavirus Acid, but the nucleic acid of non-Middle East respiration syndrome coronavirus pathogen cannot be detected；To Middle East respiration syndrome coronavirus core The Monitoring lower-cut of acid is the 10 every reaction systems of copy；Only need detection can be completed within 2 hours, it can be coronal for Middle East respiration syndrome The disease surveillance of virus and clinical diagnosis provide experimental basis.
Detailed description of the invention
Fig. 1 is the amplification curve diagram of substance real-time fluorescence PCR detection Middle East respiration syndrome coronavirus nucleic acid sensitivity.From a left side It is respectively that Middle East respiration syndrome coronavirus is transcribed in vitro (2 × 10 after rna ladder degree dilutes to 5 right curves6-2× 102Copies/ μ l) amplification.Abscissa is reaction cycle number, and ordinate is the △ Rn of different recurring number fluorescent assay signals Value.
Fig. 2 is that substance real-time fluorescence PCR detection system expands Middle East respiration syndrome coronavirus detection of nucleic acids specificity Increase curve graph.There is S type amplification curve in Middle East respiration syndrome coronavirus, and other pathogenic microorganism Quality Control strains do not go out Existing S type amplification curve.Abscissa is reaction cycle number, and ordinate is the △ Rn value of different recurring number fluorescent assay signals.
Specific embodiment
Detailed description of the preferred embodiments of the present invention combined with specific embodiments below.It should be pointed out that the reality listed here Apply example only
The purpose being merely illustrative, and it should not be constructed as any limitation on the scope of the present invention.It is tried used in it The reagents such as agent box, buffer are only the reagent being specifically chosen in the specific embodiment, it should be appreciated that those skilled in the art can root According to need to select the corresponding reagent of other companies to achieve the object of the present invention.
1, the design of primer and Taqman probe and synthesis utilize Blast tool to institute in Genbank and domestic and foreign literature Some Middle East respiration syndrome coronavirus genome sequences are analyzed, and the conservative region for selecting its stable respectively is as detection Target sequence, and for this detection target sequence design and synthesis primer and probe (being shown in Table 1).Primer and probe is by Japanese TaKaRa Dalian treasured biotech firm synthesizes, wherein the end of detection probe 5 ' the flag F AM fluorophor of Middle East respiration syndrome coronavirus, and 3 ' End label MGB fluorescent quenching group.
2, it detects Middle East respiration syndrome coronavirus used in preparation the present embodiment of strain and other is negative Compare strain (coronavirus, adenovirus type III, 7 types, enterovirus, influenza virus A H1N1, AH3N2, AH7N9, parainfluenza virus 3 types, bocavirus) buy in Nat'l Pharmaceutical & Biological Products Control Institute.
3, the nucleic acid extraction or purified reagent QIAamp DSP of QIAGEN company of Germany production are selected in the extracting of bacterial strain RNA Virus Spin Kit (state's tool is for 20140008QIAGEN GmbH) extracts strain RNA.Specific steps are said with reference to kit operation Bright book.
4, the screening of primer and probe is preced with using the Middle East respiration syndrome of the primer and probe difference Detection and Extraction of design The geneome RNA of shape virus and negative control strain is tested repeatedly, and it is optimal to filter out sensitivity, specificity and repeatability Primer combination of probe is (see sequence table, Middle East respiration syndrome coronavirus forward primer P1, reverse primer P2 and probe Probe1)。
5, the building and preparation of standard items are utilized respectively P1, P2 primer, and RT-PCR expands the coronal disease of Middle East respiration syndrome Malicious specific gene segment, and it is cloned into the plasmid (such as pGEM-T easy vector) containing T7 promoter sequence, using big The RNA Polymerase of Lian Bao biotech firm synthesizes RNA, the pancreas DNA enzymatic I without RNA enzyme is added with responsive transcription outside cessative aspect, By with phenol: chloroform purifying RNA.It is measured respectively using ultraviolet-uisible spectrophotometer at wavelength 260nm, at 280nm Absorptance, then calculate RNA concentration and purity, then 10 times of gradient dilutions to 200 every microlitre of copies.
6, reaction condition optimization optimizes the elements such as primer, probe, enzyme one by one, determining reaction system are as follows: 2 × PCR is slow 10 μ l of fliud flushing, 1.0 μ l of 10mmol/L dNTP, each 0.5 μ l of 25 μm of ol/L primers, 10 μm of 0.2 μ l of ol/L probe, reverse transcriptase and 0.8 μ l of hot start Taq polymerase mixed liquor, template 2ul add aqua sterilisa to whole 20 μ l of system.
According to amplified fragments length, the annealing temperature of primer and probe and enzyme viability, mainly to the annealing temperature of reaction Degree and extension of time are optimized, and finally determine loop parameter are as follows: reverse transcription step: 42 DEG C, 10min；The initial denaturation stage: 94 DEG C, 10sec；Pre-amplification phase: 94 DEG C of denaturation, 5sec；50 DEG C of annealing, 20sec；Extend 72 DEG C, 20sec；Totally 5 circulations.Note This stage of anticipating does not acquire fluorescence signal.Detection-phase: 94 DEG C of denaturation, 5sec；56 DEG C of annealing, 50sec；Extend 72 DEG C, 15sec； Totally 40 circulations.Fluorescence signal is detected in annealing steps.
Data are analyzed by identical conditions after amplification, determine the Ct value of each sample.
7, the detection limit of kit provided by the invention, sun are evaluated in the evaluation of detection limit with the positive criteria product in above-mentioned 5 Property standard concentration are as follows: 2 × 106copies/μl、2×105copies/μl、2×104copies/μl、2×103copies/μ l、2×102Copies/ μ l, kit provided by the invention are to the Monitoring lower-cut of Middle East respiration syndrome coronavirus nucleic acid The 10 every reaction systems of copy.
8, the evaluation of detection specificity has rated the specificity of this kit using the strain RNA in above-mentioned 2 as template.Centering Eastern respiration syndrome coronavirus RNA visible specific amplification curve when being detected, to above-mentioned 9 kinds other common respiratory tracts When common pathogenic microorganisms RNA is detected, positive amplification curve is not generated, illustrates probe that we use and primer and we are selected Other strains between be not present cross reaction.
Although above in a preferred manner, illustrating certain embodiment party of the invention by specific embodiment Formula, but it will be understood by a person skilled in the art that, the present invention is not limited to embodiments disclosed above, but can be according to this The knowledge of technical field that the present invention belongs to modifies to it, makes an amendment without departing from the scope of protection of present invention.For example, Fluorescent real time PCR used in the present invention also can according to need using the fluorescence pointed out in embodiment listed in specification Mark substance other than reporter group and fluorescent quenching group, such as Tet, HEX, TAMRA, ROX, Cy3, TxRd, JOE label Object；Or using other label systems except Taqman technology, for example, it is MGB probe, molecular beacon MB probe, scorpion shape probe, glimmering The fluorescence probes labelling techniques such as light double cross probe；Or the unsaturated dyestuff such as method such as SYBR Green I and LC are fitted into using dyestuff The saturable dyes such as Green, as long as it uses specific primer sequence of the present invention, it can qualitative or quantitative testing goal The presence of gene, and then specifically detect the presence of Middle East respiration syndrome coronavirus.So these those skilled in the art The replacement of change and customary means that member is understood is also fallen within the scope of the present invention.Protection scope of the present invention should be by institute Attached claims are limited.
Sequence table
<110>Jiangsu and wound Biotechnology Co., Ltd
<120>Middle East respiration syndrome coronavirus fluorescence PCR detection reagent kit
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA/RNA
<213>Middle East respiration syndrome coronavirus (Middle East Respiratory Syndrome)
<400> 1
tgctaagaat agagctcgca ctgtt 25
<210> 2
<211> 23
<212> DNA/RNA
<213>Middle East respiration syndrome coronavirus (Middle East Respiratory Syndrome)
<400> 2
tagtaccaat gacgcaagtc gct 23
<210> 3
<211> 16
<212> DNA/RNA
<213>Middle East respiration syndrome coronavirus (Middle East Respiratory Syndrome)
<400> 3
tcgccagtac catcag 16

Claims (3)

1. a kind of kit for Middle East respiration syndrome coronavirus in specific detection sample is reacted including PCR Liquid, enzyme mixation, negative quality-control product and positive quality control product, the primer sequence in PCR reaction solution for nucleic acid amplification reaction are as follows:
P1:5`-TGCTAAGAATAGAGCTCGCACTGTT-3`,
P2:5`-TAGTACCAATGACGCAAGTCGCT-3`,
Sequence in PCR reaction solution for the oligonucleotide probe of fluorescence signal monitoring is as follows:
Probe1:5`-X1-TCGCCAGTACCATCAG-Y1-3`,
Probe1 fluorescent reporter group X1 is FAM, and fluorescent quenching group Y1 is MGB.
2. kit as described in claim 1, it is further characterized in that PCR reaction system is 20 μ l, including 2 × PCR buffer 10 μ l, 1.0 μ l of 10mmol/L dNTP, each 0.5 μ l of 25 μm of ol/L primers, 10 μm of 0.2 μ l of ol/L probe, reverse transcriptase and heat open Dynamic 0.8 μ l of Taq enzyme mixed liquor, 2 μ l of sample RNA, add aqua sterilisa to whole 20 μ l of system.
3. kit as described in claim 1, it is further characterized in that PCR reaction cycle parameter are as follows:
Reverse transcription step: 42 DEG C, 10min；The initial denaturation stage: 94 DEG C, 10sec；Pre-amplification phase: 94 DEG C of denaturation, 5sec；Annealing 50 DEG C, 20sec；Extend 72 DEG C, 20sec；Totally 5 circulations；Notice that this stage does not acquire fluorescence signal；
Detection-phase: 94 DEG C of denaturation, 5sec；56 DEG C of annealing, 50sec；Extend 72 DEG C, 15sec；Totally 40 circulations；It is walked in annealing Rapid detection fluorescence signal.
CN201811566975.5A 2018-12-20 2018-12-20 Middle East respiration syndrome coronavirus fluorescence PCR detection reagent kit Pending CN109371174A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111206122A (en) * 2020-04-06 2020-05-29 山西医科大学 Novel coronavirus nucleic acid detection kit
CN111455098A (en) * 2020-03-24 2020-07-28 德必碁生物科技（厦门）有限公司 Coronavirus nucleic acid detection kit and detection method thereof
WO2021189546A1 (en) * 2020-03-27 2021-09-30 中国科学院合肥物质科学研究院 Kit for detecting sars-cov-2 coronavirus, and specific primer and probe thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111455098A (en) * 2020-03-24 2020-07-28 德必碁生物科技（厦门）有限公司 Coronavirus nucleic acid detection kit and detection method thereof
WO2021189546A1 (en) * 2020-03-27 2021-09-30 中国科学院合肥物质科学研究院 Kit for detecting sars-cov-2 coronavirus, and specific primer and probe thereof
CN111206122A (en) * 2020-04-06 2020-05-29 山西医科大学 Novel coronavirus nucleic acid detection kit

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