CN105525040A - Real-time fluorescent RPA kit and test strip RPA kit for rapid detection of porcine type 2 circovirus and application of real-time fluorescent RPA kit and test strip RPA kit - Google Patents
Real-time fluorescent RPA kit and test strip RPA kit for rapid detection of porcine type 2 circovirus and application of real-time fluorescent RPA kit and test strip RPA kit Download PDFInfo
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Abstract
The invention discloses a real-time fluorescent RPA kit for rapid detection of porcine type 2 circovirus and application of test strip RPA kit, and also discloses a test strip RPA kit for rapid detection of the porcine type 2 circovirus and application of the test strip RPA kit. The two kits respectively include primers, as shown in SEQ ID NO.1 and SEQ ID NO.2, as well as corresponding probes, and the primers and the probes, although for a same target sequence, are different in modified base and testing platform. Experiments prove that the sensitivities of both kits are 102 copies/reaction and the kits are capable of achieving the specific detection of the porcine type 2 circovirus. Results of detecting suspected samples of the porcine type 2 circovirus with the two kits show that the detection results of the two kits are completely consistent with that of qPCR. Therefore, the two kits of the invention can be used for rapidly, effectively and sensitively detecting the porcine type 2 circovirus; and an effective technical means is provided for the differentiation and the diagnosis of the porcine type 2 circovirus.
Description
Technical field
The present invention relates to and a kind ofly detect test kit of pig 2 type PCV-II and uses thereof, in particular to a kind of real-time fluorescence RPA test kit for rapid detection pig 2 type PCV-II based on RPA reaction and test strip RPA test kit, purposes both also relating in rapid detection pig 2 type PCV-II, the invention belongs to Preventive Veterinary Medicine inspection field.
Background technology
30 years have been had since round pcr is born.From classical PCR, real-time quantitative PCR digital pcr till now again, this technology is in our visual field of but never fading out of constantly changing in quality.But this technology needs the thermal cycler instrument of special expensive and skilled operator, wastes time and energy, be difficult to the place being used in field diagnostic and experiment condition difference.In developing country laboratory, infrastructure device required for PCR implements and technology limit, most developing countries still concentrates on and uses traditional test method, such as serological method, microscopy, or cultivates and identify infectivity and Non Communicable Diseases (NCD).In order to fill up the vacancy between traditional method and PCR, new isothermal molecular diagnostic techniques is developed, and the method is particularly useful for Infrastructure, experimental installation and experimental technique and is difficult to support to use PCR to carry out the place diagnosed.
Compared with ordinary method, isothermal duplication experiment has very high Sensitivity and Specificity, the isothermal amplification technique that these reasons impel different company tradeization different, such as, and the amplification (LAMP of ring mediation; Eiken, Japan), RPA (RPA; Alere, USAandTwistDx, UK), strand displacement amplification (stranddisplacementamplification, BectonDickson, USA).In these techniques, LAMP uses the most extensively, and the most ripe test method.Compared with RPA method, LAMP technology has test design cumbersome (3 pairs of primers), specificity relatively weak (can increase a lot of band), and the time is still relatively long, and is easy to the place of deficiencies such as polluting.
The recombinase polymeric enzymatic amplification (RecombinasePolymeraseAmplification, RPA) of TwistDxInc company of Britain exploitation is known as and can be substituted the nucleic acid detection technique of PCR.Based on this
nucleic acid amplification products in 15 minutes, can carry out detection of nucleic acids between 37 DEG C-42 DEG C.Present use maximum, the most ripe system for carrying out monitoring in real time (as
exo test kit, real-timeRPA) and based on test strip end point determination (
nfo test kit, RecombinasePolymeraseAmplificationAssaycombinedwithlater alflowtest, LFSRPA).
Real-time fluorescence RPA (real-timeRPA) test needs to excite and detects the thermostatical instrument of fluorophor, the thermal cycler of such as microplate reader or detection in real time.Since exploitation, this technology has been widely used in human diseases, veterinary medicine, foodstuffs industry and agricultural, the shiga toxin such as produced for Francisella tularensis, leptospira, HIV-1DNA, plague bacillus, anthrax bacillus, variola virus, Type B suis, intestinal bacteria, Middle East respiration syndrome coronavirus, Rift Valley fever virus, foot and mouth disease virus, bovine coronavirus, Ebola virus, Sudan virus, Marburg virus, executes the detection of the cause of diseases such as Maron shellfish lattice virus, bovine viral diarrhea virus, yellow fever virus and Gordon be viral.
For test strip RPA (LFSRPA) test, as long as temperature can control between 37-42 DEG C, such as can react in water-bath etc. is arranged, or the body temperature of directly employment heats, Sidestream chromatography test strip LFS (Hybridetect2T can be passed through subsequently, MileniaBiotecGmbH, Germany) read test-results.Since exploitation, this technology has been widely used in human diseases, veterinary medicine, foodstuffs industry and agricultural, such as and haematopoietic necrosis virus (IHHNV) subcutaneous for infectivity, plasmodium falciparum, plum pox virus, Qiang worm, makes every effort to overcome the detection of time body, Cryptosporidium and yellow fever virus etc.Compared with ordinary method, this experiment has very high Sensitivity and Specificity.
Pig circular ring virus (Porcinecircovirus, PCV) belongs to PCV-II section, the member of Circovirus, PCV be little, without the DNA virus of cyst membrane, diameter is approximately 17 nanometers.According to DNA sequence dna, antigenicity and pathogenicly can be divided into two different genotype-PCV1 and PCV2.PCV1 is considered to not have pathogenic, and persistent infection in porcine kidney cell line (PK-15).; the main pathogen of PCV2 postweaning multisystemic wasting syndrome (PMWS); show as immunity system loss; the pathology such as lymphopenia, lymphadenectasis; cause the secondary infection of other cause of diseases simultaneously, become serious at present and hindered one of main pathogen of pig industry development.Furthermore, PCV2 is a kind of immunosuppressant virus, infects the pig of this virus more easily by other virus or bacteriological infection.Investigation display, PCV2 extensively exists in pig body.Therefore, set up a kind of quick, simple, special, responsive detection method, to detect PCV2, key is played a part for the prevention and control of this disease.
(the Zhou such as Zhou, S., etal., Loop-mediatedisothermalamplificationfordetectionofporcin ecircovirustype2.VirolJ, 2011.8:p.497.) disclose a kind of ring mediated isothermal amplification method detecting pig 2 type PCV-II.Shown by the amplification of LAMP, can be there is a lot of amplified band in the method, therefore the method exists non-specific amplification, and the method needs design three pairs of primers to increase, because this increasing the difficulty of experimental design, and add its difficulty combined with other detection platform.And detected temperatures higher (63 DEG C), detection time longer (60min).And need nucleic acid electrophoresis to detect amplified production, add the possibility of pollution.
(the Yang such as Yang, Z.Z., etal., DetectionofPCV2DNAbySYBRGreenI-basedquantitativePCR.JZhe jiangUnivSciB, 2007.8 (3): p.162-9.) disclose a kind of quantitative real-time PCR for detecting PCV2DNA based on SYBRgreenI.The method needs complicated testing installation and skilled operator, and the method needs longer test period (about 1.5h).Due to the double-stranded DNA that SYBRGreenI non-specific binding is all, therefore, the specificity of the method is relatively poor.
The present invention is directed to the ORF2 gene of pig 2 type PCV-II, set up and have evaluated RPA (real-timeRPA) detection kit based on fluorescent probe and the LFSRPA detection kit based on test strip to realize the object of rapid detection pig 2 type PCV-II, as far as we know, there is no both at home and abroad and set up such test kit for detecting pig 2 type PCV-II.
Summary of the invention
Technical problem to be solved by this invention is to provide detection method and the test kit of quick, simple, the special qualification pig 2 type PCV-II of a kind of energy.
In order to achieve the above object, present invention employs following technique means:
Inventor is for the ORF2 gene design primer of pig 2 type PCV-II and probe.Simultaneously by comparing to the ORF2 DNA homolog sequence of KR559725.1, KR559695.1, KM455975.1, KP768481.1, JN181905.1, JN181902.1, KF850461.1, KC620515.1, KF035059.1, KF951567.1, KF951570.1, KM624031.1 and JQ002672.1 of coming from GenBank, further determined that the conservative region of pig 2 type PCV-II ORF2 gene, for this zone design primer and probe, to pig 2 type PCV-II as much as possible can be detected.All primers and probe are all by raw work biology (Shanghai, China) synthesis.The present invention distinguishes design and synthesis three pairs of upstream primers and three pairs of downstream primers, and by carrying out expanding effect evaluation to primer and probe combinations, primer pair and the probe combinations that wherein can produce the strongest amplified signal for a pair are applied in the present invention.For real-time fluorescence RPA (real-timeRPA) and test strip RPA (LFSRPA, RecombinasePolymeraseAmplificationAssaycombinedwithlater alflowtest) difference detect feature, the present invention devises primer and the probe sequence of real-time fluorescence RPA and the test strip RPA detection detected for pig 2 type PCV-II respectively, these two kinds of test kits are for same target sequence, but primer is different with detection platform with the modified base of probe.
A kind of real-time fluorescence RPA (real-timeRPA) detection kit for rapid detection pig 2 type PCV-II of the present invention, includes pair of primers and a probe,
Wherein, the sequence of described pair of primers and a probe is as follows:
Upstream primer: 5 '-AAACCTGTCCTAGATTCCACTATTGATTACTTCCA-3 ' (shown in SEQIDNO.1)
Downstream primer: 5 '-TTGTATTCCTGGTCGTATATACTGTTTTCGAACGC-3 ' (shown in SEQIDNO.2)
Probe: 5 '-ATTACTTCCAACCAAACAACAAAAGAAATCAGCTG
-FAM-dT-G-THF-C-BHQ1-dT-GAGACTACAAACTGC-P-3 ' (shown in SEQIDNO.3)
Wherein, FAM-dT represents the thymidylic acid carrying fluorescein base group, and THF represents tetrahydrofuran (THF) connexon, and BHQ1-dT represents the thymidylic acid carrying fluorescent quenching group B HQ1, and P represents phosphoric acid, for stoping the extension of chain.
In real-time fluorescence RPA detection kit of the present invention, preferably, in described test kit, also comprise lysis buffer, magnesium acetate and ddH
2o.
Real-time fluorescence RPA detection kit of the present invention is used to detect pig 2 type PCV-II, preferably, reaction system is as follows: the lysis buffer of 14.75 μ L, 10 μMs of upstream primers of 1.05 μ L, 10 μMs of downstream primers of 1.05 μ L, 10 μMs of probes of 0.075 μ L, the viral DNA template of 2 μ L, the ddH of 4.825 μ L
2the 280mM magnesium acetate of O and 1.25 μ L;
Amplified reaction is set as carrying out in the real-time fluorescence quantitative PCR instrument of 37-39 DEG C in temperature, and the reaction times is 20min-1h, is analyzed after terminating by Mx3005P software to result.
Preferred, amplified reaction is set as carrying out in the real-time fluorescence quantitative PCR instrument of 37 DEG C in temperature, and the reaction times is 20min, is analyzed after terminating by Mx3005P software to result.
Test the detection sensitivity of real-time fluorescence RPA (real-timeRPA) test kit of the present invention and specificity, sensitivity test result shows that the susceptibility using this test kit to detect pig 2 type PCV-II is 10
2copy/reaction, and there is wider sensing range, at least 10
6-10
2sample in copy/reaction range all can be detected.Specific detection result shows, this test kit is used to detect pig 2 type PCV-II, porcine reproductive and respiratory syndrome virus, Pestivirus suis, PRV (Pseudorabies virus), pig parvoviral and foot and mouth disease virus respectively, result only has pig 2 type PCV-II to can be good at increasing, other viruses all can not increase, therefore, illustrate that this test kit has good specificity.We detect the sample (n=8) of the clinical sample (n=45), the clinical sample (n=17) in Gansu Province, the serum sample (n=12) of health pig and the PCV1 positive that gather from Shandong Province by the real-timeRPA method set up, and by detected result compared with the detected result of qPCR, result show the detected result of real-timeRPA and qPCR completely the same, namely there is the degree of conformity of 100%.
Therefore, further, the invention allows for the purposes of above-described real-time fluorescence RPA detection kit in preparation detection pig 2 type PCV-II reagent.
The invention allows for a kind of test strip RPA (LFSRPA) detection kit for rapid detection pig 2 type PCV-II, containing Sidestream chromatography test strip (Hybridetect2T, MileniaBiotecGmbH, Germany), and have pair of primers and a probe
Wherein, the sequence of described pair of primers and a probe is as follows:
Upstream primer: 5 '-AAACCTGTCCTAGATTCCACTATTGATTACTTCCA-3 ' (shown in SEQIDNO.1)
Downstream primer: 5 '-biotin-TTGTATTCCTGGTCGTATATACTGTTTTCGAACGC-3 ' (shown in SEQIDNO.2)
Probe: 5 '-FAM-TACTTCCAACCAAACAACAAAAGAAATCAGCTGTG
-THF-CTGAGACTACAAACT-P-3 ' (shown in SEQIDNO.4).
Wherein, biotin represents vitamin H, and FAM represents Fluoresceincarboxylic acid, and THF represents tetrahydrofuran (THF) connexon, and P represents phosphoric acid, for stoping the extension of chain.
In test strip RPA detection kit of the present invention, preferably, in described test kit, also comprise lysis buffer, magnesium acetate and ddH
2o.
Test strip RPA detection kit of the present invention is used to detect pig 2 type PCV-II, preferably, reaction system is as follows: the lysis buffer of 14.75 μ L, 10 μMs of upstream primers of 1.05 μ L, 10 μMs of downstream primers of 1.05 μ L, 10 μMs of probes of 0.075 μ L, the viral DNA template of 2 μ L, the ddH of 4.825 μ L
2the 280mM magnesium acetate of O and 1.25 μ L;
Amplified reaction is set as carrying out in the water-bath of 35-45 DEG C in temperature, and the reaction times is 15min-30min, terminates rear use Sidestream chromatography test strip (Hybridetect2T, MileniaBiotecGmbH, Germany) and detects result.
Preferred, amplified reaction is set as carrying out in the water-bath of 37 DEG C in temperature, and the reaction times is 20min, terminates rear use Sidestream chromatography test strip and detects result.
Test the detection sensitivity of test strip RPA of the present invention (LFSRPA) test kit and specificity, sensitivity test result shows that the susceptibility using this test kit to detect pig 2 type PCV-II is 10
2copy/reaction, and there is wider sensing range, at least 10
6-10
2sample in copy/reaction range all can be detected.Specific detection result shows, this test kit is used to detect pig 2 type PCV-II, porcine reproductive and respiratory syndrome virus, Pestivirus suis, PRV (Pseudorabies virus), pig parvoviral and foot and mouth disease virus respectively, result only has pig 2 type PCV-II to can be good at increasing, other viruses all can not increase, therefore, illustrate that this test kit has good specificity.We detect the sample (n=8) of the clinical sample (n=45), the clinical sample (n=17) in Gansu Province, the serum sample (n=12) of health pig and the PCV1 positive that gather from Shandong Province by the LFSRPA method set up, and by detected result compared with the detected result of qPCR, result show the detected result of real-timeRPA and qPCR completely the same, namely there is the degree of conformity of 100%.
Therefore, further, the invention allows for the purposes of described test strip RPA detection kit in preparation detection pig 2 type PCV-II reagent.
Compared to prior art, method of the present invention has the following advantages:
(1) can save test period: the whole process of the test of RPA only needs 25min, this time is well below the 60min of 1.5 hours of qPCR method and LAMP.Add sample preparation and prepare the time of test, the whole testing process of RPA can complete within one hour.
(2) can reduce temperature of reaction: RPA only needs constant temperature 37 DEG C to complete test, this temperature is well below 63 DEG C of 60 DEG C-95 DEG C of qPCR method and LAMP.
(3) method more simple, be easy to carry: the enzyme needed for amplification and some other requirement freeze-drying are preserved, can place for a long time at normal temperatures, only need during amplification to add lysis buffer, primer, probe and template, and add magnesium ion initial action, LFSRPA method only needs a water-bath to complete test, does not need skilled testing crew.
(4) high specificity: with the addition of probe in test kit of the present invention, adds the specificity of detection, and based on the LAMP method of fluorescent reagent because do not have probe, specificity is relatively poor.
(5) be more not easy to pollute: with the addition of exonuclease III in test kit of the present invention, cut amplified production, because this reducing the possibility of product pollution, qPCR method does not add the enzyme of cleaved products, LAMP needs to run nucleic acid electrophoresis glue, therefore, all has the possibility of pollution.
(6) detected result is true and reliable: be 100% with the degree of conformity of existing qPCR method.
Accompanying drawing explanation
The standard substance plasmid for PCV2 primer and probe of synthesis is carried out ten times of doubling dilutions by Figure 1A, and dilution range is 10
6-10
1copy/reaction, carries out sensitivity Detection with Real-timeRPA method subsequently, as shown is the result of the real-time fluorescence quantitative PCR instrument after 20min of increasing.This figure can find out that primer designed by the present invention and probe under 37 DEG C of conditions, well can detect 10
6-10
2the template of copy/reaction.Wherein NC represents negative control.
Figure 1B verifies for utilizing the repeatability of PRISM5.0 software (GraphPadSoftware, USA) to Real-timeRPA method.Threshold value is mean value ± standard variance (SD).4 replica tests were carried out in this test.
Fig. 1 C carries out degeneration analysis to 4 repeated results of Real-timeRPA method.We mark the detection restriction of 95% possibility with triangle.
Fig. 2 (A) is the susceptibility detecting LFSRPA method, and the standard substance plasmid of synthesis carries out quantitatively by we, and dilutes concentration for 10 in the mode of ten times of doubling dilutions
6-10
1the plasmid DNA of copy/reaction is tested as template, and test conditions is above-mentioned optimum test condition (37 DEG C, 20min), and as shown in the figure, NC represents negative control to test-results; (B) for detecting the specificity of LFSRPA method, we test as template with the DNA/RNA that porcine reproductive and respiratory syndrome virus, Pestivirus suis, PRV (Pseudorabies virus), pig parvoviral, foot and mouth disease virus are extracted respectively, as shown in the figure, NC represents negative control to test-results.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
Real-time fluorescence RPA (Real-timeRPA) detection kit of embodiment 1 rapid detection pig 2 type PCV-II and the foundation of detection method
1, the design of primer and probe sequence and preparation
Inventor is by comparing to the ORF2 DNA homolog sequence of KR559725.1, KR559695.1, KM455975.1, KP768481.1, JN181905.1, JN181902.1, KF850461.1, KC620515.1, KF035059.1, KF951567.1, KF951570.1, KM624031.1 and JQ002672.1 of coming from GenBank, determine the conservative region of pig 2 type PCV-II ORF2 gene, primer and probe for this zone design, to can detect pig 2 type PCV-II as much as possible.All primers and probe are all by raw work biology (Shanghai, China) synthesis.The present invention distinguishes design and synthesis three pairs of upstream primers, three pairs of downstream primers and a probe sequence, as shown in table 1 below.
The pig 2 type PCV-II Real-timeRPA primer of table 1, the present invention's design and probe
2. strain, cell and clinical sample
The all strains used in this research are preserved by this laboratory: PCV2NX strain, HP-PRRSV (SD0907) strain, C-PRRSV (CH-1R) strain, CSF (C-strain) strain, PRV (Fa) strain, PPV (AV30) strain; (FMDV)/O/CHA strain.The serum that we collect respectively from 45 parts of clinical samples of Shandong Province, the 17 parts of clinical samples coming from Gansu Province, 8 parts of PCV1 positive and 12 parts of health pig is tested.PK-15 cell is preserved by this laboratory, with the MEM containing 10% serum at 37 DEG C, and 5%CO
2condition under cultivate.
3. viral genome is extracted
Use highly purified viral nucleic acid extracting method (Roche) to extract viral DNA/RNA to specifications, and finally with 50 μ L without RNase water elution.The DNA/RNA extracted is stored in-80 DEG C of refrigerators to treat use subsequently.
4. produce plasmid DNA standard substance
Jin Weizhi synthesizes pig 2 type PCV-II ORF2 gene fragment (320bp), and is cloned into pUC57 carrier, called after pPCV2/RPA.PPCV2/RPA plasmid is extracted by plasmid extraction method (Promega, USA).Carry out the DNA of quantitative purifying with Nanovue (GElifescience), and carry out ten times of doubling dilutions subsequently, and be stored in subsequently-80 DEG C for subsequent use.
5, real-timeRPA tests amplified conditions optimization
Be that template increases with the viral DNA/RNA of extraction purification or standard DNA plasmid, experimental system is as follows: 14.75 μ L lysis buffer (rehydrationbuffer, TwistDxexoKit, Cambridge, UnitedKingdom), 1.05 μ L upstream primers (10 μMs), 1.05 μ L downstream primers (10 μMs), the RPAexo probe (10 μMs) of 0.075 μ L, the RNA template of 2 μ L, the ddH of 4.825 μ L
2the magnesium acetate (magnesiumacetate, 280mM) of O and 1.25 μ L.
Wherein, the sequence of described pair of primers and a probe is as follows:
Upstream primer: 5 '-AAACCTGTCCTAGATTCCACTATTGATTACTTCCA-3 ' (shown in SEQIDNO.1)
Downstream primer: 5 '-TTGTATTCCTGGTCGTATATACTGTTTTCGAACGC-3 ' (shown in SEQIDNO.2)
Probe: 5 '-ATTACTTCCAACCAAACAACAAAAGAAATCAGCTG
-FAM-dT-G-THF-C-BHQ1-dT-GAGACTACAAACTGC-P-3 ' (shown in SEQIDNO.3).
Carrying out the temperature of reaction timing really of RPA primer, add reactant according to above-mentioned system.We test 37 DEG C, 38 DEG C, 39 DEG C different temperature of reaction respectively, and the reaction times is 20min, is analyzed after terminating by Mx3005P software to result.By showing interpretation of result, 37 DEG C of amplification the bests.So the pig 2 type PCV-II real-timeRPA designed by the present invention increases, temperature is set as 37 DEG C.Carrying out the reaction times timing really of real-timeRPA primer, add reactant according to above-mentioned system.And when temperature being set as 37 DEG C, test reaction different time amplification difference.We will be set as 20min, 30min and 1h the time respectively, result shows, the feminine gender after amplification 20min is still negative after one hour, and the positive after 20min is still positive after one hour, therefore, we select 20min to be amplification setting-up time of the present invention.
The present invention simultaneously evaluates three pairs of upstream primers of synthesis and the combination of three pairs of downstream primers and probe by this system respectively, evaluation result shows that wherein pair of primers (Fe3/Re1) can produce the strongest amplified signal with the combination of probe, therefore, this primer pair and probe carry out Combination application in the present invention.
Real-timeRPA testing inspection result judges: the revision test that a sample is all, what within the specific time, (20min) amplification was all greater than background value 3.5 standard variances (3.5SD) is the positive, otherwise this sample is negative.
6, real-timeRPA assay sensitivity and specific detection
When carrying out real-timeRPA method reaction sensitivity and detecting, add reactant according to above-mentioned system.Use template is the standard plasmid of above-mentioned synthesis, and measure densitometer by nucleic acid determination instrument and calculate copy number, diluting concentration gradient is respectively 10
6-10
16 gradients altogether of individual copy/reaction are as template.Amplified reaction is set as carrying out in the qPCR instrument of 37 DEG C in temperature, and the time is set as 20min, monitors amplification in real time by real-time fluorescence.From this result, result as shown in Figure 1A, can find out that the susceptibility of the method is 10
2copy/reaction, and there is wider sensing range, at least 10
6-10
2sample in copy/reaction range all can be detected.
In real-timeRPA sensitivity test, we have carried out 4 times for same test and have repeated, and result shows that the detected result tested for 4 times is consistent, all can be good at detecting minimum 10
2the standard DNA of individual copy/reaction, the present invention utilizes the repeatability of PRISM5.0 software (GraphPadSoftware, USA) to test to carry out statistical study, and result as shown in Figure 1B, shows that this invention has good repeatability.Threshold value is mean value ± standard variance (SD).Meanwhile, the present invention utilizes Excel software to carry out degeneration analysis to four repeated results that real-timeRPA tests, and result as shown in Figure 1 C, illustrates that the method can be good at detecting pig 2 type PCV-II.
When the detection of atopic carrying out real-timeRPA method, add reactant according to above-mentioned system.Wherein use template be respectively pig 2 type PCV-II (PCV2), porcine reproductive and respiratory syndrome virus (HP-PRRSV), Pestivirus suis (CSFV), PRV (Pseudorabies virus) (PRV), pig parvoviral (PPV),
Foot and mouth disease virus (FMDV).Amplified reaction is set as carrying out in the qPCR instrument of 37 DEG C in temperature, and the reaction times is set as 20min.Result shows, only have PCV2 to can be good at increasing, other viruses all can not increase, and therefore, the method has good specificity, and result is as shown in table 2.
QPCR method reference literature (Yang, Z.Z., etal., DetectionofPCV2DNAbySYBRGreenI-basedquantitativePCR.JZhe jiangUnivSciB, 2007.8 (3): p.162-9.) method is carried out.
The specificity of PCV2real-timePRA method assessed by table 2
Neg: represent feminine gender
7, real-timeRPA testing inspection clinical sample
We detect the serum sample (n=12) gathered from the clinical sample (n=45) of Shandong Province, the clinical sample (n=17) in Gansu Province and health pig by the real-timeRPA method set up, and by detected result compared with the detected result of qPCR.Result shows that the serum sample real-timeRPA detected result of 12 parts of health pig is consistent with qPCR detected result, is all negative.In 62 parts of clinical samples, real-timeRPA detected result is that (time is 31 parts of positive: 6.6min-8.6min), this and qPCR detected result completely the same (CT value is 18-30).We have detected the sample (n=8) of the PCV1 positive equally with real-timeRPA, result shows, PCV2real-timeRPA detected result is feminine gender, and therefore, the method has good specificity.Based on the 82 increment product detected, compared with qPCR detected result, the Sensitivity and Specificity of real-timeRPA is 100%, and result is as shown in table 3.
QPCR method reference literature (Yang, Z.Z., etal., DetectionofPCV2DNAbySYBRGreenI-basedquantitativePCR.JZhe jiangUnivSciB, 2007.8 (3): p.162-9.) method is carried out.
Table 3 compares the clinical sample detected result of real-timePRA method and qPCR method
As fully visible, the pair of primers of design and synthesis of the present invention and a probe gene order and its real-time fluorescence PRA (real-timeRPA) method formed can quick diagnosis pig 2 type PCV-II, and this detection method is simple, fast, detected result is true and reliable.
Test strip RPA (LFSRPA) detection kit of embodiment 2 rapid detection pig 2 type PCV-II and the foundation of detection method
1, the design of primer and probe sequence and preparation
Inventor is by comparing to the ORF2 DNA homolog sequence of KR559725.1, KR559695.1, KM455975.1, KP768481.1, JN181905.1, JN181902.1, KF850461.1, KC620515.1, KF035059.1, KF951567.1, KF951570.1, KM624031.1 and JQ002672.1 of coming from GenBank, determine the conservative region of pig 2 type PCV-II ORF2 gene, primer and probe for this zone design, to can detect pig 2 type PCV-II as much as possible.All primers and probe are all by raw work biology (Shanghai, China) synthesis.The present invention distinguishes design and synthesis three pairs of upstream primers, three pairs of downstream primers and a probe sequence, as shown in table 4 below.
The LFSRPA primer of table 4, the present invention's design and probe
2. strain, cell and clinical sample
The all strains used in this research are preserved by this laboratory: PCV2NX strain, HP-PRRSV (SD0907) strain, C-PRRSV (CH-1R) strain, CSF (C-strain) strain, PRV (Fa) strain, PPV (AV30) strain; (FMDV)/O/CHA strain.The serum that we collect respectively from 45 parts of clinical samples of Shandong Province, the 17 parts of clinical samples coming from Gansu Province, 8 parts of PCV1 positive and 12 parts of health pig is tested.PK-15 cell is preserved by this laboratory, with the MEM containing 10% serum at 37 DEG C, and 5%CO
2condition under cultivate.
3. viral genome is extracted
Use highly purified viral nucleic acid extracting method (Roche) to extract viral DNA/RNA to specifications, and finally with 50 μ L without RNase water elution.The DNA/RNA extracted is stored in-80 DEG C of refrigerators to treat use subsequently.
4. produce plasmid DNA standard substance
Jin Weizhi synthesizes pig 2 type PCV-II ORF2 gene fragment (320bp), and is cloned into pUC57 carrier, called after pPCV2/RPA.PPCV2/RPA plasmid is extracted by plasmid extraction method (Promega, USA).Carry out the DNA of quantitative purifying with Nanovue (GElifescience), and carry out ten times of doubling dilutions subsequently, and be stored in subsequently-80 DEG C for subsequent use.
5.LFSRPA method amplified conditions optimization
We are that template increases with the viral DNA/RNA of extraction purification or standard DNA plasmid, experimental system is as follows: 14.75 μ L lysis buffer (rehydrationbuffer, TwistDxexoKit, Cambridge, UnitedKingdom), 1.05 μ L upstream primers (10 μMs), 1.05 μ L downstream primers (10 μMs), the RPAexo probe (10 μMs) of 0.075 μ L, the DNA/RNA template of 2 μ L, the ddH of 4.825 μ L
2the magnesium acetate (magnesiumacetate, 280mM) of O and 1.25 μ L.
Wherein, the sequence of described pair of primers and a probe is as follows:
Upstream primer: 5 '-AAACCTGTCCTAGATTCCACTATTGATTACTTCCA-3 ' (shown in SEQIDNO.1)
Downstream primer: 5 '-biotin-TTGTATTCCTGGTCGTATATACTGTTTTCGAACGC-3 ' (shown in SEQIDNO.2)
Probe: 5 '-FAM-TACTTCCAACCAAACAACAAAAGAAATCAGCTGTG
-THF-CTGAGACTACAAACT-P-3 ' (shown in SEQIDNO.4).
First, we determine the optimal reaction temperature of LFSRPA method, and we test 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 37 DEG C, 40 DEG C, 45 DEG C, 50 DEG C differential responses temperature respectively, and incubation time is set as 30min.When result shows temperature of reaction lower than 30 DEG C, test strip does not occur test band, more weak test band is there is 30 DEG C time, between 35 DEG C-45 DEG C, test band there is no observable difference, do not occur when temperature is greater than 50 DEG C testing band, result shows that the optimal reaction temperature of this test is 35 DEG C-45 DEG C, and we select 37 DEG C as the temperature of reaction of the method.
Subsequently, under we test 37 DEG C of conditions, different incubation time is for the impact of amplification, we tested the test-results of 0min, 1min, 5min, 10min, 15min, 20min, 25min, 30min, result shows, when amplification 10min, faint band to appear in test band, tests band and do not occur observable difference time when reacted between 15min-30min, therefore, we select 20min as the incubation time of this test.
We evaluate three of design and synthesis pairs of upstream primers, three pairs of downstream primers and probe combinations by this system, evaluation result shows that wherein a pair (Fn3/Rn1) primer and probe combinations can produce the strongest amplified signal, therefore, this primer pair and probe carry out Combination application in the present invention.
Detected result judges: sample under given conditions (37 DEG C, 20min) test strip upper control line be positive, and the sample that test wire is the positive is positive; Test strip upper control line is positive, and the sample that test wire is feminine gender is negative sample.
6, reaction sensitivity detects
When carrying out the sensitivity technique of LFSRPA method, add reactant according to above-mentioned system.Use template is the standard plasmid of above-mentioned synthesis, and measure densitometer by nucleic acid determination instrument and calculate copy number, diluting concentration gradient is respectively 10
6-10
16 gradients altogether of copy/reaction are as template.Amplified reaction is set as carrying out in the water-bath of 37 DEG C in temperature, and the time is set as 20min, by test strip end point determination amplification.As shown in Figure 2 A, as can be seen from this result, the susceptibility of the method is 10
2copy/reaction.And there is wider sensing range, at least 10
6-10
2sample in copy/reaction range all can be detected.
When the specific detection carrying out LFSRPA method, add reactant according to above-mentioned system.Template is wherein used to be respectively pig 2 type PCV-II (PCV2), porcine reproductive and respiratory syndrome virus (HP-PRRSV), Pestivirus suis (CSFV), PRV (Pseudorabies virus) (PRV), pig parvoviral (PPV), foot and mouth disease virus (FMDV).Temperature of reaction is set in the water-bath of 37 DEG C carries out, and the reaction times is set as 20min, terminates rear use Sidestream chromatography test strip and detects result.Result shows, only have PCV2 to can be good at increasing, other viruses all can not increase, and therefore, the method has good specificity, and result is as shown in table 5.
QPCR method reference literature (Yang, Z.Z., etal., DetectionofPCV2DNAbySYBRGreenI-basedquantitativePCR.JZhe jiangUnivSciB, 2007.8 (3): p.162-9.) method is carried out.
The specificity of LFSRPA method assessed by table 5
Neg: represent feminine gender; Pos: represent the positive.
7, LFSRPA method detects clinical sample
First, we test 7 parts of positive and 7 parts of negative samples of qPCR confirmation by LFSRPA method, and LFSRPA test-results shows: 7 parts of positive that qPCR confirms are the positive, and 7 parts of negative samples that qPCR confirms are feminine gender.We detect the serum sample (n=12) gathered from the clinical sample (n=45) of Shandong Province, the clinical sample (n=17) in Gansu Province and health pig by the LFSRPA method set up, and by detected result compared with the detected result of qPCR.Result shows that the serum sample LFSRPA detected result of 12 parts of health pig is consistent with qPCR detected result, is all negative.In 62 parts of clinical samples, LFSRPA detected result is 31 parts of positive, this and qPCR detected result completely the same (CT value is 18-30).We have detected the sample (n=8) of the PCV1 positive equally by LFSRPA method, result shows, PCV2LFSRPA detected result is feminine gender, and therefore, the method has good specificity.Based on the 82 increment product detected, compared with qPCR detected result, the Sensitivity and Specificity of LFSRPA is 100%, and result is as shown in table 6.
QPCR method reference literature (Yang, Z.Z., etal., DetectionofPCV2DNAbySYBRGreenI-basedquantitativePCR.JZhe jiangUnivSciB, 2007.8 (3): p.162-9.) method is carried out.
Table 6 compares the clinical sample detected result of LFSRPA method and qPCR method
As fully visible, the pair of primers of design and synthesis of the present invention and a probe gene order and its test strip RPA (LFSRPA) method formed can quick diagnosis pig 2 type PCV-II, and this detection method is simple, fast, detected result is true and reliable.
Claims (10)
1., for a real-time fluorescence RPA detection kit for rapid detection pig 2 type PCV-II, it is characterized in that including pair of primers and a probe,
Wherein, the sequence of described pair of primers and a probe is as follows:
Upstream primer: 5 '-AAACCTGTCCTAGATTCCACTATTGATTACTTCCA-3 '
Downstream primer: 5 '-TTGTATTCCTGGTCGTATATACTGTTTTCGAACGC-3 '
Probe: 5 '-ATTACTTCCAACCAAACAACAAAAGAAATCAGCTG
-FAM-dT-G-THF-C-BHQ1-dT-GAGACTACAAACTGC-P-3’。
2. real-time fluorescence RPA detection kit as claimed in claim 1, is characterized in that also comprising lysis buffer in described test kit, magnesium acetate and ddH
2o.
3. real-time fluorescence RPA detection kit as claimed in claim 1 or 2, when it is characterized in that for detecting pig 2 type PCV-II, reaction system is as follows: the lysis buffer of 14.75 μ L, 10 μMs of upstream primers of 1.05 μ L, 10 μMs of downstream primers of 1.05 μ L, 10 μMs of probes of 0.075 μ L, the viral DNA template of 2 μ L, the ddH of 4.825 μ L
2the 280mM magnesium acetate of O and 1.25 μ L;
Amplified reaction is set as carrying out in the real-time fluorescence quantitative PCR instrument of 37-39 DEG C in temperature, and the reaction times is 20min-1h, is analyzed after terminating by Mx3005P software to result.
4. real-time fluorescence RPA detection kit as claimed in claim 3, it is characterized in that amplified reaction is set as carrying out in the real-time fluorescence quantitative PCR instrument of 37 DEG C in temperature, the reaction times is 20min, is analyzed after terminating by Mx3005P software to result.
5. the real-time fluorescence RPA detection kit described in any one of claim 1-4 detects the purposes in pig 2 type PCV-II reagent in preparation.
6., for a test strip RPA detection kit for rapid detection pig 2 type PCV-II, containing Sidestream chromatography test strip, characterized by further comprising pair of primers and a probe,
Wherein, the sequence of described pair of primers and a probe is as follows:
Upstream primer: 5 '-AAACCTGTCCTAGATTCCACTATTGATTACTTCCA-3 '
Downstream primer: 5 '-biotin-TTGTATTCCTGGTCGTATATACTGTTTTCGAACGC-3 '
Probe: 5 '-FAM-TACTTCCAACCAAACAACAAAAGAAATCAGCTGTG-THF-CTGAGACTACAA ACT-P-3 '.
7. test strip RPA detection kit as claimed in claim 6, is characterized in that also comprising lysis buffer in described test kit, magnesium acetate and ddH
2o.
8. test strip RPA detection kit as claimed in claims 6 or 7, when it is characterized in that for detecting pig 2 type PCV-II, reaction system is as follows: the lysis buffer of 14.75 μ L, 10 μMs of upstream primers of 1.05 μ L, 10 μMs of downstream primers of 1.05 μ L, 10 μMs of probes of 0.075 μ L, the viral DNA template of 2 μ L, the ddH of 4.825 μ L
2the 280mM magnesium acetate of O and 1.25 μ L;
Amplified reaction is set as carrying out in the water-bath of 35-45 DEG C in temperature, and the reaction times is 15min-30min, terminates rear use Sidestream chromatography test strip and detects result.
9. test strip RPA detection kit as claimed in claim 8, it is characterized in that amplified reaction is set as carrying out in the water-bath of 37 DEG C in temperature, the reaction times is 20min, terminates rear use Sidestream chromatography test strip and detects result.
10. the test strip RPA detection kit described in any one of claim 6-9 detects the purposes in pig 2 type PCV-II reagent in preparation.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106521027A (en) * | 2016-11-03 | 2017-03-22 | 河北出入境检验检疫局检验检疫技术中心 | A real-time isothermal recombinase-polymerase amplification detection kit for African swine fever viruses |
| CN107385112A (en) * | 2017-08-22 | 2017-11-24 | 河北省检验检疫科学技术研究院 | Detect nucleic acid, real-time fluorescence RPA kits and the method for the type of pig circular ring virus 3 |
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| CN109517929A (en) * | 2018-12-21 | 2019-03-26 | 武汉科前生物股份有限公司 | Primer sets and kit for pig circular ring virus detection and 2 type partings |
| CN110004240A (en) * | 2019-04-17 | 2019-07-12 | 中国农业科学院兰州兽医研究所 | The real-time fluorescence detection kit of chicken virus mycoplasma based on RPA, test strips detection kit and application thereof |
| CN113322350A (en) * | 2021-05-06 | 2021-08-31 | 深圳海关动植物检验检疫技术中心 | Kit for simultaneously detecting porcine circovirus type 2 and porcine circovirus type 3 viruses and application |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102382900A (en) * | 2010-08-31 | 2012-03-21 | 上海市农业科学院 | Real-time fluorescence quantitative PCR (polymerase chain reaction) detection method for porcine circovirus type 2 |
-
2016
- 2016-01-28 CN CN201610060957.4A patent/CN105525040B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102382900A (en) * | 2010-08-31 | 2012-03-21 | 上海市农业科学院 | Real-time fluorescence quantitative PCR (polymerase chain reaction) detection method for porcine circovirus type 2 |
Non-Patent Citations (2)
| Title |
|---|
| 孙魁等: "重组酶聚合酶扩增技术的研究进展", 《军事医学》 * |
| 陈蓉等: "猪圆环病毒2型TaqMan荧光定量PCR检测方法的建立", 《中国生物制品学杂志》 * |
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