CN103484565B - The test kit of a kind of real-time fluorescence RT-PCR detection coronavirus and application thereof - Google Patents
The test kit of a kind of real-time fluorescence RT-PCR detection coronavirus and application thereof Download PDFInfo
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Abstract
The test kit of real-time fluorescence RT-PCR detection coronavirus and an application thereof, belong to field of gene detection. The test kit sensitivity of the present invention and specificity are very high. The quick early detection to the coronavirus in the samples such as phlegm liquid, Nasopharyngeal swabs and quantitative analysis is achieved by test kit of the present invention. Sense cycle of the present invention is short, efficiency height; Detection virus-specific is strong, accuracy rate height; Can also quantitative analysis while virus qualitative analysis; The minimum concentration of detectable virus is 1.0 �� 102Copies/mL, remolding sensitivity regular-PCR and immunological detection method height; Simple to operate, be easy to promote; Experimental result repeatability is good.
Description
Technical field
The invention belongs to field of gene detection, it relates to the test kit of a kind of real-time fluorescence RT-PCR detection coronavirus and application thereof. Including screen and obtain one in the test kit of the present invention to Oligonucleolide primers and an oligonucleotide probe, test kit of the present invention has early stage, quick, sensitive, special feature, also can be used for the quantitative analysis of coronavirus.
Background technology
Coronavirus belongs to coronaviridae (Coronaviridae) coronavirus genus (Coronavirus) in phylogenetic systematics. A mutation of coronavirus is the pathogenic agent causing atypical pneumonia, is separate with it from chicken nineteen thirty-seven at first, and the diameter 60-200nm of virion, mean diameter is 100nm, spherical in shape or oval, has polymorphism. Virus has coating, coating exists spine and dashes forward, and whole virus is as corona, and the spine of different coronavirus is prominent obvious difference. Sometimes the inclusion body of tubulose can be seen in coronavirus infection cell.
There will be a known two kinds of coronavirus and can affect the mankind, be OC43 strain and 229E strain. Wherein the common cold of 2/3 premature infant and respiratory tract infection are by caused by this two-strain. Known at present, coronaviridae only infects vertebrates, relevant with the numerous disease of humans and animals. This viroid has the preferendum of gi tract, respiratory tract and neural system. Coronavirus is one of main pathogen of adult's common cold, can cause upper respiratory tract infection children, and general coronavirus seldom involves lower respiratory tract. The latent period of coronavirus infection is generally 2 to 5 days, is on average 3 days. Typical coronavirus infection is the cold symptoms such as runny nose, discomfort. The virulence difference that different shaped is not viral, the clinical manifestation caused also is not quite similar, and the symptom that OC43 strain causes is generally serious than 229E virus. There is report coronavirus infection that the symptoms such as heating, shiver with cold, vomiting can occur. The course of disease is generally about 1 week, and clinical course is slight, it does not have sequela. Coronavirus can also cause baby, newborn infant's acute gastroenteritis, and main symptom is water sample stool, heating, vomiting, and every day more than 10 times, blood watery stool can occur in severe patient. Coronavirus is excreted by respiratory secretions, through oral fluid, jet, contagion.On clinical, most coronavirus causes slight and self-healing property disease, but minority can have neurological complication.
The method of conventional detection coronavirus infection mainly contains four kinds:
(1) tissue culture virus purification: isolated viral is inoculated in primary human embryonic kidney's cell, human embryonic lung fibroblast or monkey-kidney cells and HeLa cell etc. from nasopharyngeal secretions and pharynx swab, after cultivating 2��3d, make neutralization test and plaque test test of neutralizing antibody, carry out special serotype qualification.
(2) Serologic detection: comprise complement fixation test (CFT), hemagglutination-inhibition test, indirect immunofluorescence and enzyme linked immune assay etc., but owing to lacking suitable cross-reacting antigen to cover numerous CoV serotype so that the application of these methods is very limited.
(3) molecular Biological Detection: the method for RT-PCR is extensively for the detection of CoV recently, it is more responsive compared with tissue culture faster to CoV detection.
(4) fluorescent quantitative PCR technique (FQ-PCR) is the detection technique of a kind of direct nucleic acid fast that development in recent years is got up. Fluorescent quantitative PCR technique is the real-time accounting quantitative measurement technology grown up on the basis of regular-PCR, refer to and add fluorogene in PCR reaction system, utilize the whole PCR process of fluorescent signal accumulation Real-Time Monitoring, finally by typical curve, unknown template is carried out the method for quantitative analysis. One-step method real-time fluorescent RT-PCR technology is that the one of real-time fluorescence quantitative PCR technology is also referred to as reverse transcription PCR in real time, this is a kind of method directly detecting RNA fast, the difference of it and fluorescent quantitative PCR technique has added reversed transcriptive enzyme, has added the step of a reverse transcription reaction simultaneously. The maximum advantage of quantitative real-time PCR be overcome terminal PCR method enter plateau or the period of saturation of crying after quantitative relatively big error, it is achieved the accurate quantification of DNA/RNA. It is quantitative that this technology not only achieves DNA/RNA template, and there is sensitivity and specificity height, multiple reaction can be realized, level of automation height, the feature such as pollution-free, real-time and accurate, this technology has great significance in clinical medicine inspection and clinic study.
Summary of the invention
It is an object of the invention to provide a kind of fluorescent real-time RT-PCR detection kit, particularly relate to test kit early stage, quick, sensitive with one-step method real-time fluorescent reverse transcription polymerase chain reaction (RT-PCR) technology, specific diagnostic coronavirus infection.
The ultimate principle of this test kit detection is the Oligonucleolide primers and the specific oligonucleotide probe that utilize one pair of specificity, at the deoxyribonucleoside triphosphate (dNTPs) of reversed transcriptive enzyme, hot resistant DNA polymerase, high-quality, RNase inhibitor and Mg2+Deng RT-PCR reaction buffer, realized the amplification of target nucleotide by fluorescent PCR amplification instrument, thus realize distinguishing object quick, efficient, special, Real_time quantitative detection coronavirus oligonucleotide.
In order to efficient, special, sensitive detection goes out coronavirus, the present invention's all existing coronavirus gene orders in GeneBank carry out bioinformatic analysis, have found the specific conservative district of coronavirus, and this conservative region is devised multipair primer, probe. by the detection to coronavirus standard strain, filter out highly sensitive, specificity is good, and the one of for coronary virus to primer (for the coronavirus target polynucleotide that increases) and a probe, that is: can with the oligonucleotide forward primer CoV-F of the Article 1 chain specific binding of double-strand target polynucleotide, can with the oligonucleotide reverse primer CoV-R of the Article 2 chain specific binding of double-strand target polynucleotide, can with target polynucleotide specific binding and two ends are combined with the oligonucleotide probe CoV-P of fluorescence report group and quenching of fluorescence group respectively, wherein fluorescent reporter group is optionally from FAM, TET, JOE, HEX, VIC,Quenching of fluorescence group is certainly optional: TAMRA, DABCYL, BHQ.
Wherein: the sequence of forward primer CoV-F is: 5'-GGGAAGGCCAGGACTTAAGC-3'(SEQIDNO:1); The sequence of reverse primer CoV-R is: 5'-CGGTTAAGAGCTTCCGTACT-3'(SEQIDNO:2); The sequence of oligonucleotide probe CoV-P is: 5'-GGCTAAAGGCTTGGATTAGCCATT-3'(SEQIDNO:3), preferably, this oligonucleotide probe 5 ' end is connected with FAM (5 ' Fluoresceincarboxylic acid), 3 ' end is connected with TAMRA (N, N, N ', N '-tetramethyl--6-carboxyrhodamine).
The present invention provides a kind of oligonucleotide composition for detecting coronavirus, and described composition comprises 1)-4) in any one:
1) oligonucleotide forward primer CoV-F; 2) oligonucleotide reverse primer CoV-R; 3) oligonucleotide probe CoV-P; 4) combination of one or more sequence in 1)-3), or include the sequence extended to 5 ' end and/or 3 ' end of above-mentioned sequence; Or the sequence being greater than 85% with above-mentioned sequence homology; Or the complementary base sequences thereof of above-mentioned sequence; Or use any one or more than one the combination in above-mentioned all sequences. Present invention also offers the application of above-mentioned oligonucleotide composition in the reagent of preparation detection coronavirus, wherein said reagent is the reagent for real-time fluorescence PCR.
An object of the present invention is to provide the real-time fluorescence PCR assay kit of a kind of coronavirus, and this test kit comprises 1)��4) in any one: 1) can with the oligonucleotide forward primer CoV-F of the Article 1 chain specific binding of coronavirus double-strand target polynucleotide; 2) can with the oligonucleotide reverse primer CoV-R of the Article 2 chain specific binding of coronavirus double-strand target polynucleotide; 3) can with coronavirus target polynucleotide specific binding and two ends are combined with the oligonucleotide probe CoV-P of fluorescence report group and quenching of fluorescence group respectively; 4) 1)��3) in the combination of one or more sequence, or include the sequence extended to 5 ' end and/or 3 ' end of above-mentioned sequence; Or the sequence being greater than 85% with above-mentioned sequence homology; Or the complementary base sequences thereof of above-mentioned sequence; Or use any one or more than one the combination in above-mentioned all sequences.
An object of the present invention is to provide the real-time fluorescence PCR assay kit of a kind of coronavirus, and this test kit comprises: RNA extracting solution, RT-PCR amplification reaction solution, negative quality control product, positive quality control product, coronavirus positive criteria product etc. Wherein, described RT-PCR amplification reaction solution also comprises archaeal dna polymerase, reversed transcriptive enzyme, RNase inhibitor, dNTPs and PCRbuffer; Described RT-PCR amplification reaction solution also include can with the oligonucleotide forward primer CoV-F of the Article 1 chain specific binding of double-strand target polynucleotide, can with the oligonucleotide reverse primer CoV-R of the Article 2 chain specific binding of double-strand target polynucleotide, can with target polynucleotide specific binding and two ends are combined with in the oligonucleotide probe CoV-P of fluorescence report group and quenching of fluorescence group respectively any one or more than one combination.
Wherein: the sequence of described forward primer CoV-F is: 5'-GGGAAGGCCAGGACTTAAGC-3'(SEQIDNO:1); The sequence of described reverse primer CoV-R is: 5'-CGGTTAAGAGCTTCCGTACT-3'(SEQIDNO:2); The sequence of described oligonucleotide probe CoV-P is: 5'-GGCTAAAGGCTTGGATTAGCCATT-3'(SEQIDNO:3), preferably, this oligonucleotide probe 5 ' end is connected with FAM (5 ' Fluoresceincarboxylic acid), 3 ' end is connected with TAMRA (N, N, N ', N '-tetramethyl--6-carboxyrhodamine);Or include the sequence extended to 5 ' end and/or 3 ' end of above-mentioned sequence; Or the sequence being greater than 85% with above-mentioned sequence homology; Or the complementary base sequences thereof of above-mentioned sequence; Or use any one or more than one the combination in above-mentioned all sequences.
Test kit provided by the present invention also comprises: (1) is equipped with RNA extracting solution, RT-PCR amplification reaction solution respectively, negative quality control product, positive quality control product, the multiple reagent bottle adding lid sealing of coronavirus positive criteria product or pipe, and (2) separates and concentrates the packing box packing these reagent bottles or pipe.
A preferred implementation method according to the present invention is: in described test kit, forward primer concentration be 0.5-1umol/L, reverse primer concentration be 0.5-1umol/L, oligonucleotide probe concentration be 0.25-0.5umol/L; Be preferably: forward primer concentration be 1umol/L, reverse primer concentration be 1umol/L, oligonucleotide probe concentration be 0.5umol/L.
A preferred implementation method according to the present invention is: in described test kit, and the concentration of Taq DNA polymerase is 1-8U/ reaction; It is preferably: the concentration of Taq DNA polymerase is 5U/ reaction.
Another preferred embodiment according to the present invention, wherein for Mg in RT-PCR amplification reaction solution2+Deoxyribonucleoside triphosphate (dNTPs) optimum concn of optimum concn to be 2.0mmol/L, Taq DNA polymerase optimum amount be 5U/ reaction, RT enzyme optimum amount to be 100U/ reaction, RNasin optimum amount be 20U/ reaction, high-quality is 0.2mmol/L.
In detection sample provided by the present invention, the minimum concentration of the CoV that the test kit of human corona virus can detect out is 1.0 �� 102Copies/mL, illustrates that this test kit has extraordinary sensitivity.
Another preferred embodiment according to the present invention is, reverse transcription condition is: 37 DEG C of 60min, 94 DEG C of 5min; PCR reaction conditions is 94 DEG C of 5min; 94 DEG C of 10s, 60 DEG C of 30s, 72 DEG C of 1s totally 30 circulations.
Another preferred embodiment according to the present invention, wherein negative quality control product is physiological saline.
Another preferred embodiment according to the present invention, wherein positive quality control product is CoV in-vitro transcription RNA. Wherein, the technological line setting up CoV in-vitro transcription RNA is as follows: use the qualitative amplicon virus nucleic acid of the forward and reverse primer of CoV specificity, amplified production purifying rear clone enters in pGEM-T carrier, through order-checking, positive colony determines whether object fragment is correctly inserted and determined direction of insertion. Extracting recombinant plasmid pGEM-T-CoV plasmid and carry out endonuclease reaction, rubber tapping obtains its template after reclaiming, for in-vitro transcription. DNA digestion template after in-vitro transcription, carries out RNA purifying, obtains CoV-RNA. Wherein the concentration of positive quality control product is 103copies/ml��
Another preferred embodiment according to the present invention, in test kit of the present invention, quantitative reference material is 104-107Copies/mlCoV in-vitro transcription RNA. Wherein in-vitro transcription step is the same, and the RNA ultraviolet spectrophotometer after in-vitro transcription measures A260 and carries out quantitatively, according to quantitative result, processes water with DEPC and the CoVRNA of in-vitro transcription is diluted to 10 respectively4-107Copies/ml is as reference material quantitative in this test kit. The RNA extracted in extracting in all quantitative reference materials and sample increases simultaneously, and quantitative real time PCR Instrument can draw out typical curve according to quantitative reference material, and the infection amount of human corona virus in detection sample is automatically measured according to this.
A preferred implementation method according to the present invention is: during test kit described in use, detection sample be selected from phlegm liquid, Nasopharyngeal swabs, containing the extract of phlegm liquid or Nasopharyngeal swabs or culture supernatant.
In detection sample provided by the present invention, the test kit of coronavirus is for coronary viral genome conservative gene fragment design special primer and probe, coronavirus can be detected out, but non-coronary viral pathogen can not be detected out, such as rhinovirus, respiratory syncytial virus, adenovirus etc., illustrate that this test kit has good specificity.
In detection sample provided by the present invention, the test kit of coronavirus can detect the coronavirus in the samples such as phlegm liquid, Nasopharyngeal swabs; Can be early diagnosis coronavirus infection sensitive, quick, special and reliable experimental evidence is provided, and can be accurately quantitative, so curative effect effectively can be detected.
The present invention is compared with prior art, have the following advantages: (1) detects coronavirus infection amount in sample to be detected simultaneously, height and the duplication situation of pathogen type, copy number in patient's body can be reflected really, contribute to judging disease, select treatment plan and monitoring therapeuticing effect; (2) compared with elisa technique, there is higher susceptibility, it is applicable to the detection of the multiple samples such as phlegm liquid, Nasopharyngeal swabs; (3) design primer and probe for virus-specific conserved sequence, there is higher specificity, avoid with other such as the cross reaction of the respiratory tract infection viruses such as rhinovirus, respiratory syncytial virus, adenovirus; (4) susceptibility of PCR is combined with the specificity of probe hybridization, change the defect of regular-PCR to a great extent, reduce the reaction times, simplify operation steps; (5) stopped pipe detection does not need PCR aftertreatment, the false positive that the crossed contamination between avoiding due to sample causes and environmental pollution; Real-time detection technique can continuous print detection PCR react in the change of fluorescent signal, avoid " the plateau effect " of regular-PCR, and template quantitatively by end product, but have Ct value to calculate, accuracy and susceptibility all improve a lot.
Accompanying drawing explanation
Fig. 1 is CoV virus standard substance amplification curve;
Fig. 2 is CoV virus standard substance concentration standard curve;
Fig. 3 is 4 example CoV positive sample amplification curves.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention. Experimental technique in following embodiment, if no special instructions, is ordinary method. Test materials used in following embodiment, if no special instructions, is to buy from routine biochemistry reagent shop and obtains. Quantitative test in following examples, all arranges three times or more and to repeat experiment, results averaged.
Embodiment 1: the development of coronavirus one-step method real-time fluorescent quantitative PCR reagent
1, the design of primer and probe: by utilizing DNAman software to carry out sequence alignment analysis coronavirus nucleotide sequence existing in Genebank database, taking the conservative fragments of human corona virus's genome as amplified target site, fundamental principle according to primed probe design, utilizes the multipair primer of software engineer and probe.
2, the selection of sample: show according to document relevant both at home and abroad report, it is possible to select the samples such as phlegm liquid, Nasopharyngeal swabs.
3, the Establishment and optimization of reaction system
The preparation of sample: the 10 parts of samples taking viruses indentification as the coronavirus positive, as the positive reference material of CoV, are respectively CoV-1, CoV-2, CoV-3, CoV-4, CoV-5, CoV-6, CoV-7, CoV-8, CoV-9, CoV-10; Take viruses indentification as 10 parts of non-CoV samples of feminine gender it is negative reference product, it is respectively 3 kinds of viral sample (influenza virus, respiratory syncytial virus, adenovirus) and 7 parts of normal phlegm liquid, Nasopharyngeal swabs samples.Extract the RNA of above-mentioned positive reference material and negative reference product respectively, stand-by.
The screening of primed probe: detect above-mentioned positive reference material and the RNA of negative reference product respectively with many groups primer of design in above-mentioned 1 and probe, through repeated tests, filters out the best primed probe combination that specificity is good, highly sensitive and repeatability is good.
The optimization of primed probe concentration: when other reactive components are constant in reaction system, the primer of concentration gradient of 0.5umol/L to 1umol/L and the probe of the concentration gradient of 0.25umol/L to 0.5umol/L is used to carry out PCR reaction respectively, through repeated multiple times revision test, finally determine that best primer concentration be 1umol/L, concentration and probe concentration is 0.5umol/L.
The optimization of Taq DNA polymerase consumption: other reactive components are constant in reaction system, use respectively from 1U(unit of enzyme) to the enzyme dosage/reaction of 8U concentration gradient, carry out RT-PCR reaction, through repeatedly revision test, finally determine that best Taq enzyme consumption is 5U/ reaction.
The optimization of RT enzyme dosage: other reactive components are constant in reaction system, use the enzyme dosage/reaction from 50U (unit of enzyme) to 400U concentration gradient respectively, carry out RT-PCR reaction, through repeatedly revision test, finally determine that best RT enzyme dosage is 100U/ reaction.
The optimization of RNasin consumption: other reactive components are constant in reaction system, use the enzyme dosage/reaction from 5U (unit of enzyme) to 40U concentration gradient respectively, carry out RT-PCR reaction, through repeatedly revision test, finally determine that best RNasin consumption is 20U/ reaction.
The optimization of dNTPs degree: other reactive components are constant in reaction system, use the dNTPs consumption/reaction of the concentration gradient from 0.1mmol/L to 0.25mmol/L respectively, carry out RT-PCR reaction, through repeatedly revision test, finally determine that best dNTPs concentration is 0.2mmol/L.
The optimization of temperature of reaction, time: according to the length of the activity of enzyme and few polynucleotide, mainly annealing temperature and extension time are optimized, through repeatedly revision test, finally determines that best temperature of reaction and time is: 37 DEG C of 60min, 94 DEG C of 5min; PCR reaction conditions is 94 DEG C of 5min; 94 DEG C of 10s, 60 DEG C of 30s, 72 DEG C of 1s totally 30 circulations.
4. Samples detection: using phlegm liquid, Nasopharyngeal swabs etc. as sample to be checked, after extracting the RNA of sample respectively, through the nucleic acid amplification system detection that above-mentioned optimization is set up, result shows: test kit of the present invention sensitive detection can go out the coronavirus in clinical samples (CoV).
Embodiment 2: human corona virus's single stage method fluorescence real-time quantitative RT-PCR detection kit and use thereof
1, preparation comprises the test kit of following component composition: RNA extracting solution, RT-PCR amplification reaction solution, negative quality control product, positive quality control product, quantitatively reference material, DEPC process water.
2. the collection of sample, transport and preservation
2.1 are suitable for sample type: phlegm liquid, Nasopharyngeal swabs etc.
2.2 collections of specimens and pre-treatment (attention aseptic technique)
2.2.1 sputum specimen collecting: taking morning phlegm as good, application clear water before collect specimen, cold boiling water are gargled or with toothbrush (without toothpaste) cleaning oral cavity and tooth, person should take off artificial tooth (polluting sample for reducing Oral normal flora) artificial tooth, the firmly phlegm (non-post nasal portion secretion, non-saliva) in expectoration respiratory tract deep, phlegm liquid directly spits in aseptic Sputum cup, and specimen amount answers >=1ml.Few or can heat to the 10%NaCl aqueous solution (thick sputum difficulty is coughed, obstructing airway, it is necessary to use Chymetin salt brine solution) of 45 DEG C with Neulized inhalation without phlegm or dys-expectoration person for phlegm amount, make phlegm liquid be easy to discharge rear expectoration.
2.2.2 Nasopharyngeal swabs collection of specimens: pharynx swab: pharynx swab is fully infiltrated in sample solution, upwards mentions and leave liquid level, tube wall extrudes repeatedly several under; Allowing patient head is micro-faces upward, mouth magnifies, and concurrent " " sound, exposes both sides pharyngeal tonsil, and hand-held swab is in a little firmly wiping at least 3 times back and forth of patient both sides pharyngeal tonsil, and then the upper and lower wiping of wall at least 3 times after pharynx; Swab head is immersed in sample solution, swab head contact with tube wall several under, make sample try one's best more than be kept in sample solution, abandon swab hand and pinch tail portion.
Pharynx swab is fully infiltrated in sample solution, upwards mentions and leave liquid level, tube wall extrudes repeatedly several under;
Allowing patient head natural relaxation, swab pastes nostril wall and slowly rotates and enter in patient one nostril, to nose palate place, then wiping limit in limit rotates and slowly takes out. With same swab, with same another nostril of method wiping; Nose swab is put into the sampling tube collecting pharynx swab, and method is with, in step, like this, just having pharynx swab, a nose swab, i.e. a so-called Nasopharyngeal swabs pipe in a sampling tube.
2.3 sample transports and preservation: the sample gathering or processing preserves under 4 DEG C of conditions and should be no more than 48h; If needing long-term preservation ,-80 DEG C of cryogenic refrigerators must be placed, but multigelation (maximum freeze thawing 3 times) should be avoided. After the sample gathered seals, adopt insulation can sealing on the rocks, and it is transported to laboratory as early as possible.
3 detecting steps
(1) RNA extracts
A. get the 1.5ml centrifuge tube of N number of (the negative quality control product+number of awaiting test sample of N=1 pipe) sterilizing, and perform mark.
B. each centrifuge tube adds 600ulTrizol reagent, then adds 200ul testing sample supernatant liquor or negative quality control product respectively, fully the mixed even 15s of concussion, and room temperature leaves standstill 3��5min;
C. each centrifuge tube adds 200ul chloroform, up and down the mixed centrifugal 5min of even 10s, 12,000rpm of concussion;
D. carefully drawing upper colorless layer liquid, be transferred to the 1.5ml centrifuge tube of new sterilizing, then add 10ulRNA extracting solution, fully suction is beaten mixed even, and centrifugal 1 minute of 8,000rpm, then carefully abandons all liquid;
E. adding solution C (confirming to have added dehydrated alcohol) 800ul, fully suction is beaten mixed even, the centrifugal 1min of 8,000rpm, is removed by liquid clean as far as possible;
F. the lid of centrifuge tube is opened and puts into the air-dry 15min of stink cupboard, it is possible to use well heater in 60 DEG C of dry 5min, (mainly removing dehydrated alcohol), then add 30ulDEPC and process water, inhale and beat precipitation in mixed even pipe, obtain liquid, detection can be directly used in, it is possible to be stored in-80 DEG C for subsequent use.
(2) RT-PCR reaction and result analysis, judgement
Get negative quality control product, positive quality control product, quantitatively reference material, each 3ul of sample to be measured respectively, add and PCR reaction tubes carries out RT-PCR amplified reaction. The condition of RT-PCR amplified reaction is: 37 DEG C of reverse transcription 60min; 94 DEG C of 5min; PCR reaction conditions is 94 DEG C of 5min; 94 DEG C of 10s, 60 DEG C of 30s, 72 DEG C of 1s totally 30 circulations.
Result is analyzed: the Value value arranging the Start value of Baseline, Stop value and Threshold according to the amplification curve of quantitative reference material, makes the canonical plotting under Stdcurve window reach the best, and namely correlation numerical value is between-1.0��-0.97.Finally in Analysis menu, select the automatic analytical results of Analyze.
Result judges: positive sample amplification curve is S-type, and all negative sample occur without amplification curve, and the CoV detected result of sample to be measured is effective, otherwise, result is invalid, it is necessary to again detect, and carrying out positive sample detection by quantitative according to standard substance, result is such as Fig. 1/2.
Embodiment 3: coronavirus single stage method fluorescence real-time quantitative RT-PCR detection kit clinical detection uses
Being detected by other doubtful coronavirus infection patient's sputum sample 18 parts with aforesaid method, wherein positive 4 examples of CoV detected result, FLuorescent quantitative PCR amplification curve is shown in Fig. 3, according to the C of this 4 example positive findingstValue, in conjunction with amplification curve, is automatically analyzed the virus concentration obtaining this 4 example CoV positive sample by RocheLightCycler480 analysis software, and concrete outcome is in table 1.
Table 14 example CoV positive sample virus concentration
| Sample number into spectrum | Ct value | CoV virus concentration (copy number/�� l) |
| Sample 1 | 21.34 | 2.35��102 |
| Sample 2 | 20.98 | 2.92��102 |
| Sample 3 | 22.46 | 2.48��103 |
| Sample 4 | 21.68 | 3.48��102 |
Claims (4)
1. a RT-PCR detection kit for coronavirus, this test kit comprises: RNA extracts reagent, RT-PCR amplification reaction solution, negative quality control product, positive quality control product, coronavirus positive criteria product; Wherein, described RT-PCR amplification reaction solution include can with the forward primer CoV-F of the Article 1 chain specific binding of double-strand target polynucleotide, can with the reverse primer CoV-R of the Article 2 chain specific binding of double-strand target polynucleotide, can with target polynucleotide specific binding and two ends are combined with the oligonucleotide probe CoV-P of fluorescent reporter group and fluorescent quenching group respectively, wherein:
The sequence of described forward primer CoV-F is: 5'-GGGAAGGCCAGGACTTAAGC-3';
The sequence of described reverse primer CoV-R is: 5'-CGGTTAAGAGCTTCCGTACT-3';
The sequence of described oligonucleotide probe CoV-P is: 5'-GGCTAAAGGCTTGGATTAGCCATT-3';
RT-PCR amplification reaction solution described in test kit also comprises archaeal dna polymerase, reversed transcriptive enzyme, RNase inhibitor, dNTPs and PCR damping fluid; Wherein, archaeal dna polymerase is Taq DNA polymerase, and its optimum concn is 5U/ reaction; The optimum concn of reversed transcriptive enzyme is 100U/ reaction; The optimum concn of RNase inhibitor is 20U/ reaction; DNTPs optimum concn is 0.2mmol/L/ reaction;
The optimum concn of forward primer and reverse primer is 1umol/L, and the optimum concn of oligonucleotide probe is 0.5umol/L;
The reaction conditions of described reverse transcription is: 37 DEG C of 60min, 94 DEG C of 5min; PCR reaction conditions is 94 DEG C of 5min; 94 DEG C of 10s, 60 DEG C of 30s, 72 DEG C of 1s, totally 30 circulations.
2., for detecting an oligonucleotide composition for coronavirus, described composition comprises:
1) forward primer CoV-F, its sequence is: 5'-GGGAAGGCCAGGACTTAAGC-3';
2) reverse primer CoV-R, its sequence is: 5'-CGGTTAAGAGCTTCCGTACT-3';
3) oligonucleotide probe CoV-P, its sequence is: 5'-GGCTAAAGGCTTGGATTAGCCATT-3'.
3. oligonucleotide composition according to claim 2 detects the application in the reagent of coronavirus in preparation.
4. application according to claim 3, wherein said reagent is the reagent for RT-PCR.
Priority Applications (1)
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| CN111363849A (en) * | 2020-02-13 | 2020-07-03 | 深圳市梓健生物科技有限公司 | Novel coronavirus nucleic acid detection kit and detection method |
| WO2021214467A1 (en) * | 2020-04-23 | 2021-10-28 | Cellbio | A diagnostic tool improvement comprising a pathogen binding molecule |
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| CN1618982A (en) * | 2003-11-20 | 2005-05-25 | 中国人民解放军军事医学科学院生物工程研究所 | Fluorescene quantitative PCR reagant box for detecting SARS-Cov virus |
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| CN1188530C (en) * | 2003-05-16 | 2005-02-09 | 浙江大学医学院附属第一医院 | Gene detection reagent kit for SARS virus and its detection method |
| CN1548550A (en) * | 2003-05-20 | 2004-11-24 | 吴秉铨 | Target sequence RT-PCR primer, short-handle ring probe, reagent kit and detection method for SARS related coronavirus distinction and their application |
| WO2006022459A1 (en) * | 2004-08-23 | 2006-03-02 | Mogam Biotechnology Institute | Primer and probe for detection of sars coronavirus, kit comprising the primer and/or the probe, and detection method thereof |
| CN102181583B (en) * | 2011-04-25 | 2013-10-16 | 河南科技学院 | Primers, probe and kit for detecting duck-origin coronavirus through fluorescence quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) |
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| CN1618982A (en) * | 2003-11-20 | 2005-05-25 | 中国人民解放军军事医学科学院生物工程研究所 | Fluorescene quantitative PCR reagant box for detecting SARS-Cov virus |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111363849A (en) * | 2020-02-13 | 2020-07-03 | 深圳市梓健生物科技有限公司 | Novel coronavirus nucleic acid detection kit and detection method |
| WO2021214467A1 (en) * | 2020-04-23 | 2021-10-28 | Cellbio | A diagnostic tool improvement comprising a pathogen binding molecule |
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