CN1289692C - Magnetic capture of SARS coronary virus and PCR detection therewith - Google Patents
Magnetic capture of SARS coronary virus and PCR detection therewith Download PDFInfo
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- CN1289692C CN1289692C CNB2004100479597A CN200410047959A CN1289692C CN 1289692 C CN1289692 C CN 1289692C CN B2004100479597 A CNB2004100479597 A CN B2004100479597A CN 200410047959 A CN200410047959 A CN 200410047959A CN 1289692 C CN1289692 C CN 1289692C
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Abstract
The present invention provides a method for capturing SARS coronavirus RNA. A capture probe labeled by biotin is combined with a magnetic bead coated with avidine; target RNA in specimens is captured by the capture probe, then is collected by a magnetic frame and is washed. The capture method can remove protein, non-target RNA and substances inhibiting PCR in specimens to purify the target RNA. The method can simultaneously concentrate and enrich the target RNA so as to achieve the purpose of enhancing the concentration of the target RNA. In addition, the present invention also provides a double-probe capture method. Before the target RNA is captured by the capture probe, the target RNA is prehybridized with target RNA in specimens by a prehybridization probe so as to open the secondary structure of the target RNA and further enhance capture efficiency. The present invention also provides an RT-PCR detection method of SARS coronavirus RNA, which uses the capture method to capture the target RNA. The RT-PCR detection method is capable of greatly enhancing detection sensitivity and is good for early diagnosing patients.
Description
Technical field
The present invention relates to a kind of method of catching sars coronavirus (Coronavirus) RNA, particularly a kind of method that adopts the magnetic laws on arrests to catch sars coronavirus RNA.The invention still further relates to the PCR detection method of the patient being carried out early diagnosis, particularly adopt the magnetic laws on arrests to catch sars coronavirus RNA, and it is carried out the method that RT+PCR detects.
Background technology
SARS is the abbreviation of severe acute respiratory syndrome (Severe Acute Respiratory Syndrome).For being different from the typicalness pneumonia, be called severe acute respiratory syndrome again.SARS has the infectivity of height and certain mortality ratio, therefore the SARS suspected patient is made diagnosis early and is very important, and the timely isolation and the treatment that can be the patient gain time.Disease controlling is worsened and prevents that the epidemic situation diffusion from having great importance.
Detect about SARS etiology laboratory at present and mainly contain three class methods: viral separation, antibody test and polymerase chain reaction (PCR) detection method.Preceding two kinds of methods are not suitable for the early diagnosis of SARS.RT-PCR, promptly the reverse transcription polymerase chain reaction method is the main method that can be used for early diagnosis at present.This method can be used for the detection of sars coronavirus in the various samples of patient (as: body fluid, ight soil, respiratory secretions, tissue slice etc.), can detect SARS patient's sars coronavirus gene in the collect specimen in 10 days of falling ill, and can utilize the order-checking of PCR product, do further checking.
10 pairs of Auele Specific Primers that detect SARS virus have been announced in the WHO website, but simultaneously WHO think the higher but susceptibility deficiency of the specificity of the existing PCR method of inspection (Burkardt HJ.Standardization and quality control of PCR analyses.Clin Chem Lab Med.2000,38:87-91).Clinical detection result also shows, utilize common RT-PCR method, for the sample of morbidity in 3 days, recall rate only is 22% (Poon LL, Chan KH, Wong OK, et al.Earlydiagnosis of SARS coronavirus infection by real time RT-PCR.J Clin Virol.2003,28:233-238).So the key of SARS early diagnosis at present is to improve the susceptibility of PCR detection method.
CN 1468965A discloses a kind of PCR fluorescence detection method, designs the primer and the probe of the high specific that is fit to SARS virus.This method adopts traditional RNA catching method, and it is low and false-negative problem occurs to fail to solve in the early stage patient sample the low detection sensitivity of bringing of viral level.CN 1442488A discloses a kind of method of fluorescence quantitative PCR detection, this method adopts different fluorescent marks to come the different probe of mark, in a reaction system, comprise many group primers and fluorescent probe, can detect the different sections of SARS virus or different virus variants simultaneously.Though this method has improved the susceptibility that detects to a certain extent, its PCR product is not easy to order-checking, can not do last conclusive evidence to amplified production.
Burkard thinks in PCR detects, causing the major cause that susceptibility is low, false negative rate is high is to exist some interfering substances can suppress the PCR reaction in people's sample, low with the template amount in addition relevant (the Bidawid S of technical problem, Farber JM, Sattar SA.Rapid concentration anddetection of hepatitis A virus from lettuce and strawberries.J Virol Methods.2000,88:175-185).So improving the committed step of PCR detection sensitivity is exactly as much as possible template to be concentrated, as much as possible template is carried out purifying to eliminate or to reduce PCR and suppress simultaneously.This just requires carrying out in the RNA leaching process detecting sample (especially low viral sample), need RNA is carried out enrichment and purifying, and the at present existing RNA extracting method commonly used of relatively generally acknowledging comprises that TrizolLS, QIAGEN RNA extract test kit, and the ability of RNA enrichment and purifying especially still is subject to certain restrictions at lower concentration RNA sample.
The magnetic laws on arrests is based on the specific combination of vitamin H and avidin and the principle of base complementrity, biotin labeled special oligonucleotide probe is bonded to bag by on the magnetic bead of avidin, utilization is combined with the magnetic bead and the hybridization of the target nucleic acid in the liquid phase of specific probe, collect with the magnetic force frame then, wash through low salt buffer, remove impurity such as protein, non-target nucleic acid, finally obtain the specific nucleic acid molecule of purifying.The magnetic laws on arrests is applied to immunologic assay the earliest, develops into afterwards a kind ofly new directly from sample target nucleic acid separated, concentrate and the effective ways of purifying.At present this method be used to multiple virus especially the nucleic acid PCR of viruses such as RNA viruses such as HCV, HAV, enterovirus, poliovirus detect (Jothikumar N, Cliver DO, Mariam TW.Immunomagnetic capture PCR forrapid concentration and detection of hepatitis A Virus from environmentalsamples.Appl Environ Microbiol.1998,64:504-508; Van Doorn LJ, Kleter B, Voermans J, et al.Rapid detection of hepatitis C virus RNA by direct capturefrom blood.J Med Virol.1994,42:22-8; Muir P, Nicholson F, Jhetam M, et al.Rapid diagnosis of enterovirus infection by magnetic bead extraction andpolymerase chain reaction detection of enterovirus RNA in clinical specimens.JClin Microbiol, 1993,31:31-38; Regan PM, Margolin AB.Development of anucleic acid capture probe with reverse transcriptase-polymerase chain reactionto detect poliovirus in groundwater.J Virol Methods, 1997,64:65-72), this method has powerful enrichment and purifying function to target nucleic acid.But have not yet to see based on this method sars coronavirus RNA is caught pertinent literature and the report that the performing PCR of going forward side by side detects.
Summary of the invention
The purpose of this invention is to provide and a kind ofly sars coronavirus RNA is had specific, and can effectively remove non-target RNA in the sample and suppress the catching method of the material of PCR.The present invention simultaneously also provides a kind of RT-PCR detection method with high specific, high sensitive sars coronavirus RNA.By this method, can infect in early days at SARS suspected patient is made diagnosis accurately, so that take the corresponding treatment measure as early as possible.
For achieving the above object, the invention provides the catching method of a kind of SARS virus RNA.This method comprises following step:
1) clean surface is coated with the magnetic bead of avidin, to remove the RNA enzyme, prevents the degraded of RNA;
2) capture probe P1 is carried out mark at 5 ' end with vitamin H;
3) magnetic bead that will clean with mix through the capture probe P1 of mark in right amount, by the specific combination of vitamin H and avidin, make capture probe P1 be coated on magnetic bead surfaces, 4 ℃ of preservations;
4) sample is used TRIzol LS cracking;
5) in step 4) gained solution, add the magnetic bead that is coated with capture probe P1, make capture probe P1 catch sars coronavirus RNA; With
6) with the magnetic bead of magnetic force frame collection in the solution of step 5), after the washing, it is dissolved in an amount of DEPC water-80 ℃ of preservations;
Wherein the sequence of capture probe P1 is:
5′-GTAGCCTGGTTAATGTGC-3’；
Described sample is selected from a kind of in patient's serum, blood plasma, saliva, urine, cerebrospinal fluid or the respiratory secretions.
Further, the present invention also provides a kind of catching method that adopts two probes, further comprises the steps:
Solution with the step 4) gained before carrying out step 5) mixes with prehybridization probe PR1, carries out prehybridization; Proceed step 5) and 6 with gained solution again); Wherein the sequence of prehybridization probe PR1 is:
5′-CAAACAATTGGCTTATGCAGT-3′。
Another object of the present invention provides a kind of high specific and high sensitive SARS virus RT-PCR detection method.This method comprises the steps:
A) the catching of sars coronavirus RNA in the sample;
B) will change cDNA into by the target RNA that a) catches by reverse transcription;
C) at b) in the cDNA that obtains carry out PCR; With
D) to c) PCR result detect;
It is characterized by with the catching method of above-mentioned SARS virus RNA and catch sars coronavirus RNA.Wherein reverse transcription, PCR and detection method adopt method of the prior art, and these methods are as well known to those skilled in the art.
Further, RT-PCR method provided by the invention, wherein PCR adopts nest-type PRC, and used primer sequence is:
Outside primer 1:5 '-GAAAGTCAACAACCCACCT-3 ',
Outside primer 2: 5 '-TTCCACATAAGCAGCCAT-3 ';
Inboard primer 1:5 '-ACTACCGAAGTTGTAGGCAATG-3 ',
Inboard primer 2: 5 '-CCACATAAGCAGCCATAAGA-3 '.
Wherein in the reverse transcription step of RNA, adopt the M-MLV reversed transcriptive enzyme of 200U, and reaction conditions is preferably 40 ℃～50 ℃, 30 minutes～10 minutes; Most preferably be 50 ℃, 10 minutes.
Adopt the magnetic laws on arrests to catch the material that SARA coronavirus RNA can remove the protein in the sample, non-target RNA and suppress PCR, make target RNA obtain purifying; This method can be carried out enrichment method to target RNA simultaneously, plays the purpose that improves target RNA concentration.In addition, adopt two probe catching methods,, open the secondary structure of target RNA, can further improve capture rate by prehybridization.Therefore adopt the RT-PCR detection method of above-mentioned RNA catching method, for the method that adopts the nest-type PRC amplification, can improve the susceptibility of detection greatly especially, help the patient is carried out early diagnosis.
Description of drawings
Fig. 1 is the synoptic diagram of catching of magnetic laws on arrests SARS virus RNA.
Fig. 2 is without the RT-PCR detected result (2A) of catching of the SARS virus standard rna of prehybridization and contrast through the RT-PCR detected result (2B) of catching of the SARS virus standard rna of prehybridization.
Fig. 3 is that the magnetic laws on arrests is caught the electrophorogram that back RT-PCR detects to the SARS virus RNA that cultivates when adopting different magnetic bead amount.
Fig. 4 adopts the magnetic laws on arrests that the SARS virus RNA that cultivates is caught the electrophorogram that back RT-PCR detects under different extent of dilution.
Embodiment
Below in conjunction with the drawings and specific embodiments content of the present invention is described in detail.
An object of the present invention is to provide the catching method of a kind of SARS virus RNA.
As shown in Figure 1, this catching method is to design capture probe P1 at the conservative region in the sars coronavirus, and this probe is carried out mark at 5 ' end with vitamin H.The capture probe P1 of mark and the magnetic bead that is coated with avidin are combined by the interaction between vitamin H and the avidin.Because the gene sequencing work of sars coronavirus is finished, this lays a good foundation for the design of capture probe.Utilize BLAST that the genome sequence of sars coronavirus genome sequence and all biological species system is compared, homology between the observation sequence, to determine the conservative region in the sars coronavirus gene order, on this conservative region, catch the capture probe P1 of sars coronavirus RNA with software set such as Oligo6.0, Primer5.0.Wherein the sequence of this capture probe P1 is:
5′-GTAGCCTGGTTAATGTGC-3’。
The magnetic bead that is coated with avidin can be that any being suitable for carried out isolating magnetic bead to target nucleic acid.In one embodiment, described magnetic bead is the M-280 type magnetic bead of Dynal company.This magnetic bead need at first clean when being used to catch RNA, to remove RNA enzyme (RNase) wherein, prevents the degraded of RNA.
With bag by the magnetic bead of capture probe P1 hybridize with RNA in TRIzol LS cracking sample, collect and wash the target RNA that is caught with the magnetic force frame.The target RNA that catches is transferred in an amount of DEPC water,, promptly can be used for the amplification of reverse transcription and nest-type PRC in-80 ℃ of preservations.
Wherein the RNA sample is a kind of in patient's serum, blood plasma, saliva, urine, cerebrospinal fluid or the respiratory secretions.
Owing to adopted specific capture probe to combine, make the sars coronavirus RNA that captures by capture probe P1 be attached to the surface of magnetic bead, and under the action of a magnetic field, assemble with magnetic bead with magnetic bead.Not only can remove a large amount of protein and non-target nucleic acids that exist in the sample by washing, also can will have inhibiting material to remove to PCR.And, make target RNA in the solution of smaller size smaller, have higher relatively concentration, thereby obtain enrichment by adding an amount of solution.This is greatly improved the specificity and the susceptibility of the PCR detection of carrying out subsequently.Thereby improved the recall rate of the early diagnosis of SARS suspected patient.
The step (as shown in Figure 1) that also comprises a prehybridization in a preferred embodiment of the invention.In the acquisition procedure of RNA,,, finally cause susceptibility slightly to descend so when single use capture probe, can cause losing of target RNA because its secondary structure relatively is difficult to open; If before RNA catches, RNA at first with prehybridization probe that capture probe adjoins is mutually carried out prehybridization, can increase capture rate, finally improve detection sensitivity.For opening the secondary structure of RNA better, improve the capture rate of capture probe to target RNA, in same conservative region, catch the prehybridization probe PR1 of sars coronavirus RNA with software set such as Oligo6.0, Primer5.0.The PR1 probe sequence is:
5′-CAAACAATTGGCTTATGCAGT-3′。
This prehybridization probe and sars coronavirus RNA bonded position and capture probe P1 institute bonded position are adjoined mutually, have effectively improved capture rate.
In an embodiment of the present invention, contrasted the RT-PCR detection effect that adopts prehybridization probe RP1 that the SARS virus RNA that catches is carried out prehybridization and do not adopt prehybridization.As shown in Figure 2,1～6 correspond respectively to 1 * 10
1Copy/reaction, 2.5 * 10
1Copy/reaction, 5.0 * 10
1Copy/reaction, 1 * 10
2Copy/reaction, 1 * 10
3Copy/reaction, 1 * 10
4Copy/reaction; M:100bp dna ladder scale standard; N: with water as blank.The detection sensitivity that makes two probes has improved 4 times.Proved and adopted the catching method of the magnetic laws on arrests SARS virus RNA of two probes will further improve the susceptibility that RT-PCR detects.
In addition in the embodiments of the invention also the consumption to magnetic bead study.As shown in Figure 3, adopt bigger magnetic bead amount (100 μ g) obviously to help the susceptibility that PCT detects.Wherein 1～5 be respectively 10
-1, 10
-2, 10
-3, 10
-4With 10
-5The RNA diluted sample; M:100bp dna ladder scale standard; N: with water as blank; P: positive control.But considering from the angle of cost, is not that the magnetic bead amount is big more just good more.Because the target rna content in the sample is very low, so the amount of magnetic bead will be tending towards saturated in certain value, even if that is: increase the magnetic bead consumption again, the contribution of susceptibility also no longer significantly be increased.As seen from Figure 3, adopt the magnetic bead of 50 μ g also can detect 10
-5Dilute sample, but susceptibility is poor slightly.Therefore it is appropriate adopting 100 μ g.
The present invention also provides a kind of high specific and high sensitive sars coronavirus RT-PCR detection method.This method comprises the steps:
A) the catching of sars coronavirus RNA in the sample;
B) will change cDNA into by the target RNA that a) catches by reverse transcription;
C) at b) in the cDNA that obtains carry out PCR; With
D) to c) PCR result detect;
It is characterized by on the basis of the catching method of above-mentioned sars coronavirus RNA, the RNA that catches is carried out reverse transcription, make it to change into cDNA.Carry out pcr amplification and detection then.
Carry out pcr amplification and can adopt common amplification method, also can adopt the nido TRAP.Adopt the nido TRAP on the basis of magnetic laws on arrests, detection sensitivity further to be improved.For the very low situation of template amount in the sample, can obtain more accurate believable result.
In a preferred embodiment of the invention, adopt the nido TRAP to increase.Wherein adopt outside primer 1 and outside primer 2 to carry out first round pcr amplification, get first round pcr amplification product again and carry out second with inboard primer 1 and inboard primer 2 and take turns pcr amplification.Wherein the primer sequence that is adopted is:
Outside primer 1:5 '-GAAAGTCAACAACCCACCT-3 ',
Outside primer 2: 5 '-TTCCACATAAGCAGCCAT-3 ';
Inboard primer 1:5 '-ACTACCGAAGTTGTAGGCAATG-3 ',
Inboard primer 2: 5 '-CCACATAAGCAGCCATAAGA-3 '.
Wherein reverse transcription can adopt any suitable reverse transcription method of the prior art.In a preferred embodiment, adopt the M-MLV reversed transcriptive enzyme of 200U; Reaction conditions is preferably 40 ℃～50 ℃, and 30 minutes～10 minutes, more preferably 50 ℃, 10 minutes.Under preferred temperature, can reach same effect fully, but shortened detection time greatly.
Because the hyperinfection of SARS, so the use of SARS virus and clinical samples is strictly controlled in the laboratory study work.Therefore do not adopt the sample of clinical patients in following examples, but the method disclosed in the present can be applied to the detection to the clinical patients sample fully.
Embodiment
Preparation is coated with the magnetic bead of special capture probe P1:
Used M-280 type magnetic bead in the experiment, available from Dynal company, granular size is 2.8 ± 0.2 μ m, and pan coating has avidin, and the probe of biotin-binding mark or antibody are used for nucleic acid or antigenic catching.
Magnetic bead at first cleans when being used to catch RNA, to remove the RNase in the magnetic bead.At first use with the solution A of volume and wash 2 times, use then with the solution B of volume and wash 2 times, with the B﹠amp of volume; W damping fluid washing 2 times is dissolved in isopyknic B﹠amp at last; In the W damping fluid.Wherein solution A, solution B and B﹠amp; The W damping fluid is the washings that Dynal company provides prescription.Compound method is as follows:
1) preparation of DEPC water
Get about 980ml deionized water, add 1ml tetra-sodium diethyl ester (DEPC), replenish deionized water to 1L, after processing is spent the night, high-pressure decomposing DEPC, packing, 4 ℃ of preservations are standby.
2) solution A:
DEPC-treated?0.1M?NaOH
DEPC-treated?0.05M?NaCl
Take by weighing 0.8g NaOH, 0.5844g NaCl is dissolved in about 160ml deionized water, until dissolving fully, add DEPC 0.2ml with magnetic stirrer, replenishing deionized water to overall solution volume is 200ml, stir at a slow speed and spend the night, behind the autoclaving, 4 ℃ of preservations are standby.
3) solution B:
DEPC-treated?0.1M?NaCl
Take by weighing 1.1688g NaCl, be dissolved in about 160ml deionized water, until dissolving fully, add DEPC 0.2ml with magnetic stirrer, replenishing deionized water to overall solution volume is 200ml, stirs at a slow speed and spends the night, and behind the autoclaving, 4 ℃ of preservations are standby.
4) B﹠amp; The W damping fluid:
10mM?Tris-HCl(pH7.5)
1mM?EDTA
2.0M?NaCl
The preparation of 1M Tris solution: take by weighing 12.1g Tris, be dissolved in the DEPC water that about 90ml handled, stirring and evenly mixing is to dissolving fully, and replenishing DEPC water to cumulative volume is 100ml, room temperature preservation behind the autoclaving.
0.5M the preparation of DEPC-treated EDTA solution: take by weighing 18.61g EDTA, be dissolved in about 90ml deionized water, stirring and evenly mixing adds DEPC 0.1ml after dissolving extremely fully, stirs at a slow speed and spends the night, and behind the autoclaving, 4 ℃ of preservations are standby.
5.0M the preparation of DEPC-treated NaCl solution: take by weighing 146.1g NaCl, be dissolved in about 460ml deionized water, magnetic stirrer is until dissolving fully, add DEPC 0.5ml, replenishing deionized water to overall solution volume is 500ml, stir at a slow speed and spend the night, behind the autoclaving, 4 ℃ of preservations are standby.
100ml B﹠amp; The preparation of W damping fluid: measure the 1M Tris solution 1ml for preparing, 0.5MDEPC-treated EDTA solution 0.2ml, 5.0M DEPC-treated NaCl solution 40ml replenishes DEPC water to 100ml.
Then, get the magnetic bead 10 μ l (magnetic bead concentration is 10 μ g/ μ l) after the cleaning, add the biotin labeled capture probe P1 that 300pmol waits to wrap quilt, gently behind the mixing, incubated at room 45min.Use 6 * SSPE washing 3 times then, be dissolved at last among 1ml 6 * SSPE, 4 ℃ of preservations are standby.
Wherein the collocation method of 12 * SSPE is as follows:
Take by weighing 21.036g NaCl, 3.312g NaH
2PO
4H
2O, 0.888g EDTA, NaOH regulator solution pH value to 7.4 adds DEPC 0.2ml, replenishes deionized water to 200ml, stirs at a slow speed and spends the night, and behind the autoclaving, 4 ℃ of preservations are standby.
The preparation of SARS virus RNA reference material:
1) extraction of plasmid construction and plasmid DNA
At first utilize the RT-PCR technology with purpose cDNA fragment amplification, utilize TOPO clone's test kit (Invitrogen company) to carry out plasmid construction, extract having the segmental plasmid DNA of purpose then.The plasmid DNA of having extracted is measured its OD260 and OD280 value, estimate its purity and concentration, verify with 1% agarose gel electrophoresis then.
2) plasmid DNA linearization for enzyme restriction
Utilize restriction enzyme Hind III, will contain the segmental plasmid DNA linearization for enzyme restriction of purpose cDNA, reaction system is:
10 * M damping fluid, 20 μ l
Hind?III 75U
Aqua sterilisa adds to 200 μ l
Hatch 1h for 37 ℃, get 5 μ l enzymes and cut liquid, carry out 1% agarose electrophoresis detection enzyme and cut effect.
3) linear DNA is refining
After enzyme is cut into merit, gets the 400ul enzyme and cut liquid, add 400 μ l phenol: chloroform: primary isoamyl alcohol is 25: 24: 1 a mixture, the centrifugal 5min of 12000rpm gets supernatant liquor, and add 400 μ l chloroforms: primary isoamyl alcohol is 24: 1 a mixture, the centrifugal 5min of 12000rpm gets supernatant liquor, adds 40 μ l 3M sodium-acetate and 1ml dehydrated alcohols, the centrifugal 15min of 13500rpm, abandoning supernatant precipitates 2 times with 70% washing with alcohol, after the drying, be dissolved in the 20 μ l DEPC water, get 1 μ l and carry out electrophoresis detection.
4) in-vitro transcription reaction
Utilize the linear DNA after making with extra care to be template, carry out responsive transcription, reaction system is:
10 * T7 damping fluid, 10 μ l
1M?DTT 1μl
100mM?rNTP 0.5μl(each)
T7 rna polymerase 20U
RNase inhibitor 20U
DEPC water adds to 100 μ l
37 ℃ of overnight incubation are got the checking of 2 μ l electrophoresis.
5) DNA enzyme (DNase) I of transcription product handles
The reactant of transcribing success is handled with DNase I, and the ratio with 10U/200ng in reactant adds DNase I, with the digestion residual DNA, gets 2～5 μ l reaction solution electrophoresis detection digestion effect.
6) transcription product RNA's is refining
The transcription product that has been digested through the electrophoresis validating DNA is carried out RNA to be made with extra care.Add isopyknic phenol in reaction product: chloroform: primary isoamyl alcohol is 25: 24: 1 a mixture, the centrifugal 5min of 12000rpm gets supernatant liquor, adds isopyknic chloroform: primary isoamyl alcohol is 24: 1 a mixture, the centrifugal 5min of 12000rpm, get supernatant liquor, add the 10 μ M ammonium acetates and the 1ml dehydrated alcohol of 1/4 volume, the centrifugal 15min of 13500rpm, abandoning supernatant, precipitate 2 times with 70% washing with alcohol, after the drying, be dissolved in 20 μ l DEPC water or RNA and preserve in the liquid.Measure the OD260 and the OD280 of transcribe rna ,-80 ℃ of preservations are standby.Calculate the copy number concentration for preparing RNA according to the OD260 that measures, be used for the evaluation of detection architecture as reference material with this.
Embodiment 1: without catching and the RT-PCR detection of prehybridization standard rna sample
Get the standard SARS virus RNA that obtains by aforesaid method of 50 μ l, add magnetic bead 100 μ l, incubated at room 90min in conjunction with capture probe P1.Utilize the magnetic force frame to collect and washing RNA B﹠amp; W damping fluid washing 2 times, 1 * PCR damping fluid is dissolved in RNA in the 10 μ l DEPC water after washing 2 times, is used for the amplification of reverse transcription and nest-type PRC.
The reverse transcription of RNA:
Utilize the M-MLV reversed transcriptive enzyme, get 10 μ l RNA samples and carry out reverse transcription, reaction system is:
5 * the first chain cDNA damping fluids, 4.0 μ l
0.1M?DTT 2.0μl
10mM?dNTP 1.0μl
M-MLV 200U
RNA template 10 μ l
DEPC water adds to 20 μ l
Reaction conditions is respectively: 42 ℃, and 30min.
The nest-type PRC amplification:
Utilize outside primer 1 and outside primer 2 to carry out the amplification of first round PCR, reaction system is:
10 * Taq damping fluid, 5.0 μ l
dNTP(2.5mM) 4.0μl
Outside primer 1 (25pmol/ μ l) 1.0 μ l
Outside primer 2 (25pmol/ μ l) 1.0 μ l
Reverse transcription product 10 μ l
Taq enzyme 1.25U
Aqua sterilisa adds to 50l
Reaction conditions is: 95 ℃, and 2min; 30 circulations: 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 30s; 72 ℃ of 10min.
Get first round PCR product 2 μ l and utilize inboard primer 1 and inboard primer 2 to carry out second to take turns the amplification of PCR, reaction system is:
10 * Taq damping fluid, 5.0 μ l
dNTP(2.5mM) 4.0μl
Inboard primer (1) (25pmol/ μ l) 1.0 μ l
Inboard primer (2) (25pmol/ μ l) 1.0 μ l
First round PCR product 2.0 μ l
Taq enzyme 1.25U
Aqua sterilisa adds to 50 μ l
Reaction conditions is: 95 ℃, and 5min; 30 circulations: 94 ℃ of 15s, 56 ℃ of 15s, 72 ℃ of 30s; 72 ℃ of 7min.Getting second takes turns PCR product 10 μ l and carries out 2.5% agarose gel electrophoresis.Nest-type PRC amplified production length is 100bp.Electrophoresis result is shown in Fig. 2 A.
Embodiment 2: through catching and the RT-PCR detection of prehybridization standard rna sample
At first 50 μ l standard rnas and 300pmol prehybridization probe PR1 are carried out prehybridization, 54 ℃ of incubation 15min in 6 * SSPE.Add magnetic bead 100 μ l, incubated at room 90min in conjunction with capture probe P1.Utilize the magnetic force frame to collect and washing RNA B﹠amp; W damping fluid washing 2 times, 1 * PCR damping fluid is dissolved in RNA in the 10 μ l DEPC water after washing 2 times, is used for the amplification of reverse transcription and nest-type PRC.Adopt identical reverse transcription and nest-type PRC amplification method among the embodiment 1.Electrophoresis result is shown in Fig. 2 B.
Embodiment 3: hit catching of RNA and RT-PCR of sars coronavirus nutrient solution detected
In the P3 laboratory, get and cultivate successfully and through the sars coronavirus 100 μ l of TCID50 definite value, add 300 μ l TRIzol LS lysates, room temperature adds 80 μ l chloroforms after placing 5min, thermal agitation, after room temperature was placed 5min, 4 ℃ of following centrifugal 10min of 12000 * g got the superiors' supernatant liquor 100 μ l, add among the 6 * SSPE that contains 300pmol prehybridization probe PR1 54 ℃ of incubation 15min.Add among 10,50, the 100 μ g embodiment 1 magnetic bead respectively in conjunction with specific probe P1.The reaction solution incubated at room 90min that will add magnetic bead then.Utilize the magnetic force frame to collect and wash the RNA that is combined on the magnetic bead, B﹠amp; W damping fluid washing 2 times, after 1 * PCR damping fluid washed 2 times, the RNA that will have magnetic bead was dissolved in 10 μ lDEPC water, and-80 ℃ of preservations are standby.
Adopt identical reverse transcription and nest-type PRC amplification method among the embodiment 1, difference is that the reverse transcription condition is: 50 ℃, and 10min.Electrophoresis result as shown in Figure 3.
Embodiment 4: magnetic laws on arrests sars coronavirus RNA catches the extraction effect of system to the sars coronavirus RNA of different concns
Will be through 6.3 * 10
4The sars coronavirus nutrient solution of TCID50/100 μ l definite value is with substratum 10 times of dilutions successively.Get the diluent 100 μ l of different concns respectively, add 300 μ l TRIzol LS lysates respectively, after room temperature is placed 5min, add 80 μ l chloroforms, thermal agitation is after room temperature is placed 5min, 4 ℃ of following centrifugal 10min of 12000 * g, get upper strata supernatant 100 μ l respectively, add among the 6 * SSPE that contains 300pmol prehybridization probe PR1 54 ℃ of incubation 15min.Add respectively among the 100 μ g embodiment 1 obtain in conjunction with the magnetic bead of specific probe P1, carry out RNA and catch.Catch and RT-PCR adopts the method identical with last embodiment 3.Nest-type PRC amplified production length is 100bp.Electrophoresis result as shown in Figure 4, wherein 1～6 is 10
-1, 10
-2, 10
-3, 10
-4, 10
-5With 10
-6Viral dilution liquid, M are 100bp dna ladder scale standard; N is as blank with water; The positive contrast of P promptly contains the sample of standard rna.
The result adopts this method can detect 6.3 * 10 as shown in Figure 4
-1The virus-culturing fluid of TCID50/100 μ l, the higher neutralization test result of this result and the susceptibility that carries out simultaneously is consistent.
The foregoing description is an explanation of the invention, rather than limitation of the present invention.One skilled in the art will understand that any specific form or the change that do not deviate from the spirit and scope of the present invention, all should think to be included within the scope of claims of the present invention.
Claims (7)
1, the catching method of a kind of sars coronavirus RNA, this method comprises following step:
1) clean surface is coated with the magnetic bead of avidin, to remove the RNA enzyme, prevents the degraded of RNA;
2) capture probe P1 is carried out mark at 5 ' end with vitamin H;
3) magnetic bead that will clean with mix through the capture probe P1 of mark in right amount, by the specific combination of vitamin H and avidin, make capture probe P1 be coated on magnetic bead surfaces, 4 ℃ of preservations;
4) sample is used TRIzol LS cracking;
5) in step 4) gained solution, add the magnetic bead that is coated with capture probe P1, make capture probe P1 catch sars coronavirus RNA; With
6) with the magnetic bead of magnetic force frame collection in the solution of step 5), after the washing, it is dissolved in an amount of DEPC water-80 ℃ of preservations;
Wherein the sequence of capture probe P1 is:
5′-GTAGCCTGGTTAATGTGC-3’；
Described sample is selected from a kind of in patient's serum, blood plasma, saliva, urine, cerebrospinal fluid or the respiratory secretions.
2, the method for claim 1 further comprises following step:
Solution with the step 4) gained before carrying out step 5) mixes with prehybridization probe PR1, carries out prehybridization; Proceed step 5) and 6 with gained solution again); Wherein the sequence of prehybridization probe PR1 is:
5′-CAAACAATTGGCTTATGCAGT-3′。
3, a kind of RT-PCR method that detects sars coronavirus RNA in the sample, this method comprises the steps:
A) the catching of sars coronavirus RNA in the sample;
B) will change cDNA into by the target RNA that a) catches by reverse transcription;
C) at b) in the cDNA that obtains carry out PCR; With
D) to c) PCR result detect;
It is characterized by step a) comprises:
1) clean surface is coated with the magnetic bead of avidin, to remove the RNA enzyme, prevents the degraded of RNA;
2) capture probe P1 is carried out mark at 5 ' end with vitamin H;
3) magnetic bead that will clean with mix through the capture probe P1 of mark in right amount, by the specific combination of vitamin H and avidin, make capture probe P1 be coated on magnetic bead surfaces, 4 ℃ of preservations;
4) sample is used TRIzol LS cracking;
5) in step 4) gained solution, add the magnetic bead that is coated with capture probe P1, make capture probe P1 catch sars coronavirus RNA; With
6) with the magnetic bead of magnetic force frame collection in the solution of step 5), after the washing, it is dissolved in an amount of DEPC water-80 ℃ of preservations;
Wherein the sequence of capture probe P1 is:
5′-GTAGCCTGGTTAATGTGC-3’；
Described sample is selected from a kind of in patient's serum, blood plasma, saliva, urine, cerebrospinal fluid or the respiratory secretions.
4, method as claimed in claim 3, wherein catching of sars coronavirus RNA further comprises following step:
Solution with the step 4) gained before carrying out step 5) mixes with prehybridization probe PR1, carries out prehybridization; Proceed step 5) and 6 with gained solution again); Wherein the sequence of prehybridization probe PR1 is:
5′-CAAACAATTGGCTTATGCAGT-3′。
5, method as claimed in claim 4, wherein PCR is a nest-type PRC, wherein the primer sequence that is adopted is:
Outside primer 1:5 '-GAAAGTCAACAACCCACCT-3 ',
Outside primer 2: 5 '-TTCCACATAAGCAGCCAT-3 ';
Inboard primer 1:5 '-ACTACCGAAGTTGTAGGCAATG-3 ',
Inboard primer 2: 5 '-CCACATAAGCAGCCATAAGA-3 '.
6, method as claimed in claim 5, wherein the reverse transcription system adopts the M-MLV reversed transcriptive enzyme of 200U, and condition is 40 ℃～50 ℃ and hatched 30 minutes～10 minutes.
7, method as claimed in claim 5, wherein the reverse transcription system adopts the M-MLV reversed transcriptive enzyme of 200U, and condition is 50 ℃ and hatched 10 minutes.
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| CN103388032B (en) * | 2012-05-07 | 2016-08-03 | 上海仁度生物科技有限公司 | A kind of coxsackie virus A 16-type (CA16) real-time fluorescence nucleic acid isothermal amplification detection kit |
| CN102703603B (en) * | 2012-05-07 | 2014-01-08 | 上海仁度生物科技有限公司 | Real-time fluorescence nucleic acid constant temperature amplification detection kit of general influenza a virus (IAV) |
| CN103388033B (en) * | 2012-05-07 | 2016-08-10 | 上海仁度生物科技有限公司 | A kind of enterovirns type 71 (EV71) real-time fluorescence nucleic acid isothermal amplification detection kit |
| CN103525946B (en) * | 2012-07-02 | 2018-12-18 | 上海仁度生物科技有限公司 | The real-time fluorescence nucleic acid isothermal amplification detection kit of people seasonal current Influenza Virus H1N1 (HuH1N1) |
| CN103525945B (en) * | 2012-07-02 | 2018-08-31 | 上海仁度生物科技有限公司 | H1N1virus (2009) real-time fluorescence nucleic acid isothermal amplification detection kit |
| CN104278022B (en) * | 2014-09-12 | 2017-03-15 | 中国人民解放军第四军医大学 | A kind of preparation method and application of DNA capture probes |
| CN104946774A (en) * | 2015-07-08 | 2015-09-30 | 北京科技大学 | Method for gathering and detecting DON toxin production genes in corn |
| CN109321637A (en) * | 2018-10-31 | 2019-02-12 | 武汉利恩达医疗科技有限公司 | A kind of pathogen nucleic acid capture amplification detection method |
| CN112159835B (en) * | 2020-09-30 | 2022-09-23 | 北京吉检医疗科技有限公司 | Method for capturing and enriching nucleic acid through probe |
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