CN102329789B - Positive control composition for reverse transcription polymerase chain reaction kit and preparation method thereof - Google Patents
Positive control composition for reverse transcription polymerase chain reaction kit and preparation method thereof Download PDFInfo
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Abstract
The invention provides a positive control composition for a reverse transcription polymerase chain reaction kit and a preparation method thereof, and the positive control composition is characterized in that a non-target virus is taken as positive control, a pair of primers are simultaneously designed for amplification, and the length of an amplified fragment is the same with that of a target gene of the reverse transcription polymerase chain reaction kit; and the non-target virus is inactivated by guanidine isothiocyanate. The positive control prepared by the method is safe, thereby avoiding virus expansion caused by direct use of a virus infection material in the prior art, playing a control role during an RNA (ribonucleic acid) extraction process and an RNA reverse transcription process when serving as the reverse transcription positive control of the RNA virus, proving that an RNA extraction reagent and substances used in a reaction system are normal in activity, facilitating the positive control when serving as the commercial kit and having very high practical value.
Description
Technical field
The invention belongs to field of virus detection, relate to a kind of positive control for inverse transcription polymerase chain reaction test kit, more specifically relate to a kind of positive control composition for inverse transcription polymerase chain reaction test kit and preparation method thereof.
Background technology
The effect of the control sample of inverse transcription polymerase chain reaction test kit in experiment is: when each RT-PCR detects, negative control sample and the positive control sample set up can not lack, when negative control sample detection is negative, show that the reagent of testing all processes is not polluted; During positive control sample test positive, show that RNA extraction, RNA reverse transcription, DNA cloning and electrophoresis evaluation work system are normal.Under the prerequisite of all setting up in negative control sample and positive control sample detected result, could judge detecting sample.Therefore, each test all should set up RNA extraction, RNA reverse transcription, DNA cloning and electrophoresis to identify contrast, only sets up the omnidistance contrast of test, could prove test-results establishment.Therefore, inverse transcription polymerase chain reaction test kit will be set up positive control, and in test kit, positive control need to meet following 3 points: 1) good stability, 2) reliably effective, 3) meet Biosafety.
Biosafety refers to that biological Vector of infection causes the true or potential danger to the mankind, animal or plant by direct infection or indirect welding.Therefore, meeting Biosafety refers to and has avoided above-mentioned danger.Described danger is for example highly pathogenic pathogenic micro-organism, the first kind and Equations of The Second Kind pathogenic micro-organism are referred to as highly pathogenic pathogenic micro-organism, first kind pathogenic micro-organism, refer to and can cause the very microorganism of serious disease of the mankind or animal, and China not yet finds or announced the microorganism of elimination.Equations of The Second Kind pathogenic micro-organism, refers to and can cause the mankind or animal serious disease, than being easier to direct or being connected on the microorganism of propagating between person to person, animals and human beings, animal and animal.Positive control for the inverse transcription polymerase chain reaction test kit of some highly pathogenic pathogenic micro-organisms, this test kit way of most is that object fragment is cloned on carrier, its shortcoming be can not extract as RNA, RNA transcriptive process,reversed contrast, and easily pollute.
Therefore, need badly a kind of good stability, reliable effectively, meet the preparation method of the inverse transcription polymerase chain reaction test kit positive control of Biosafety.
Summary of the invention
Technical problem to be solved by this invention is for the deficiencies in the prior art, a kind of positive composition contrast of inverse transcription polymerase chain reaction test kit is provided, positive control composition and preparation method thereof particularly, the positive control good stability that the method makes, safe and reliable, can greatly reduce the possibility that test is polluted.
Technical problem to be solved by this invention is achieved through the following technical solutions.The preparation method who the invention provides a kind of positive control composition of inverse transcription polymerase chain reaction test kit, is characterized in that, chooses a kind of non-target virus as positive control template.
One aspect of the present invention, the non-target virus of choosing in the preparation method of the positive control composition of inverse transcription polymerase chain reaction test kit is RNA viruses, it can be the RNA viruses that meets arbitrarily Biosafety, include but not limited to newcastle disease virus and porcine reproductive and respiratory syndrome virus, and the virus strain that is selected from safer, inheritance stability, easily cultivates.
In the specific embodiment of the present invention, selected non-target virus strain newcastle disease virus lasota strain and porcine reproductive and respiratory syndrome virus MLV strain.
An also aspect of the present invention, also comprises the nutrient solution that uses non-target virus described in deactivation agent treated in the preparation method of the positive control composition of inverse transcription polymerase chain reaction test kit.Described deactivation reagent is high density strong denaturant, and it destroys rapidly cellularstructure, and RNA is discharged from cell, also makes intracellular RNA enzyme deactivation, and the RNA discharging is not degraded.Wherein, high density strong denaturant includes but not limited to the salt that guanidinium isothiocyanate, guanidine thiocyanate, Guanidinium hydrochloride etc. contain guanidine radicals.Deactivation reagent can be used for the deactivation of RT-PCR, real-time fluorescence RT-PCR positive control.That is to say, in method of the present invention, non-target virus-culturing fluid is diluted with denaturing agent, the multiple of dilution can easily be determined by those skilled in the art, generally should dilute and be not less than 7 times, preferably dilutes 50-100 doubly, more preferably dilutes 50 times.Extension rate is relevant with viral level, and wherein the requirement of positive control is that viral level can not be too high, can not be too low; Too high, easily contamination reagent box, too low, makes the preservation period of test kit shorter.According to the requirement of test kit separately, dilute.
An also aspect of the present invention, in the preparation method of the positive control composition of inverse transcription polymerase chain reaction test kit, also comprise a pair of non-target virus primer of design, the described non-target virus of choosing is increased, the fragment length of amplification and inverse transcription polymerase chain reaction test kit identical for the length of goal gene
The present invention also provides a kind of positive control composition for inverse transcription polymerase chain reaction test kit, it is characterized in that comprising that a kind of non-target virus is as positive control template.
In the specific embodiment of the present invention, the non-target virus of choosing in positive control composition is RNA viruses, it can be the RNA viruses that meets arbitrarily Biosafety, include but not limited to newcastle disease virus and porcine reproductive and respiratory syndrome virus, and be selected from safety, inheritance stability, easy virus strain of cultivating, preferred described virus strain comprises newcastle disease virus lasota strain and porcine reproductive and respiratory syndrome virus MLV strain.
In one aspect of the invention, positive control composition also comprises deactivation reagent.Deactivation reagent is high density strong denaturant.Wherein, high density strong denaturant includes but not limited to the salt that contains guanidine radicals, preferably guanidinium isothiocyanate, guanidine thiocyanate, Guanidinium hydrochloride.Deactivation reagent can be used for the deactivation of RT-PCR, real-time fluorescence RT-PCR positive control.The nutrient solution of described non-target virus is used according to deactivation reagent described in viral level and is diluted, and generally should dilute and be not less than 7 times, preferably dilutes 50-100 doubly, more preferably dilutes 50 times.And extension rate can easily be determined according to the requirement of test kit separately by those skilled in the art.
In one aspect of the invention, positive control composition also comprises a pair of non-target virus primer, so that described non-target virus is increased, the fragment length of amplification and inverse transcription polymerase chain reaction test kit for the length of goal gene identical.
The present invention also provides the purposes of positive control composition mentioned above, and it is as the part of inverse transcription polymerase chain reaction test kit.Positive control composition of the present invention can be placed in inverse transcription polymerase chain reaction test kit.
In the specific embodiment of the present invention, applicable inverse transcription polymerase chain reaction test kit includes but not limited to avian influenza virus RT-PCR test kit and foot and mouth disease virus RT-PCR test kit, wherein avian influenza virus RT-PCR test kit can be used for bird, for example chicken, duck, goose, sparrow, pigeon etc.; Foot and mouth disease virus RT-PCR test kit can be used for detecting artiodactyl, for example ox.
Compared with prior art, positive control safety prepared by the inventive method, easily preservation, can play the effect that RNA extracts, RNA transcriptive process,reversed contrasts, the ThermoScript II isoreactivity that proof RNA extracts in reagent, reaction system is normal, has avoided directly using virus infection material easily to cause the shortcoming of virus diffusion in prior art.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example is only for the present invention is described, and should not be considered as limiting scope of the present invention.
Utilizing and detecting target virus is a kind of desirable method as positive control.Because bird flu generation meeting causes serious financial loss to country, and bird flu belongs to zoonosis.Therefore, avian influenza virus detection kit Biosafety is very important.Particularly avian influenza virus may produce some unsafe impacts to laboratory environment as positive control.
Newcastle disease virus lasota strain is a strain attenuated vaccine strain, and widespread use China newcastle disease immunization is being brought into play very important effect aspect prevention newcastle disease.This virus stain hemagglutinative titer in chicken embryo culture is very high simultaneously, can reach 2
-13, be safer, inheritance stability, the virus strain of easily cultivating.Therefore newcastle disease virus lasota strain is to set up the ideal of reverse transcription positive control virus.We adopt newcastle disease virus lasota strain as the reverse transcription positive control of RNA viruses, have played the effect that RNA extraction, RNA transcriptive process,reversed contrast, and have proved that in RNA extraction reagent, reaction system, ThermoScript II isoreactivity is normal.Design a pair of primer for newcastle disease virus, this primer requires the clip size of amplification just consistent with detected avian influenza virus expanding fragment length simultaneously.Add the deactivation reagent containing guanidinium isothiocyanate, by newcastle disease virus deactivation, positive control as avian influenza virus inverse transcription polymerase chain reaction test kit, owing to having adopted newcastle disease virus lasota strain as extracting contrast in RNA leaching process, greatly reduce the possibility that test is polluted, be relatively applicable to carrying out for a long time laboratory or the large-scale monitoringof bird flu of bird flu detection.
Embodiment
Unreceipted concrete technology or condition person in embodiment, according to the described technology of the document in this area or condition (such as with reference to works such as J. Pehanorm Brookers, the < < molecular cloning experiment guide > > that Huang Peitang etc. translate, the third edition, Science Press) or carry out according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
The preparation of embodiment 1. avian influenza virus RT-PCR test kit positive controls
1, the preparation of deactivation reagent: 4mol/L guanidinium isothiocyanate, 25m mol/L Sodium Citrate, usp, Dihydrate Powder, 0.5% (m/v) sodium lauryl sulphate, 0.1mol/L beta-mercaptoethanol.
2, non-target virus newcastle disease virus lasota strain (deriving from China Veterinery Drug Inspection Office, original seed code name AV100 (L)): blood clotting valency is merged, mixed higher than 2048 newcastle disease virus lasota strain chick embryo allantoic liquid.
3, the preparation of positive control: non-target virus newcastle disease virus lasota strain, with after 100 times of dilutions of sex change liquid, be can be used as to avian influenza virus RT-PCR test kit positive control and uses.
The application of embodiment 2. avian influenza virus RT-PCR test kit positive controls
1, the composition of test kit
0.2mL thin-walled PCR pipe, RNA extract test kit (Rneasy Mini Kit， qiagen company), 50 times of TAE electrophoretic buffers, staining fluid (Goldview), sample-loading buffer (tetrabromophenol sulfonphthalein sucrose solution), negative control (being sterilizing deionized water), positive control (being positive control prepared in embodiment 1), RT-PCR reaction solution (primer, dNTPs, TaqDNA polysaccharase with reaction buffer, Mg
2+), enzyme mixation (AMV ThermoScript II, RNA inhibitor, TaqDNA polysaccharase), X liquid (as described below).
2, sample preparation
2.1 sample collectings: larynx tracheae and the lung tissue of the chicken that dies of illness.
2.2 sample preparation: every duplicate samples is processed respectively.
2.2.1 tissue sample is processed: organize respectively for every part and take the about 1g of sample from three different positions, with operating scissors, shred and get 0.05g after mixing and grind in mill, add 1.5mL physiological saline to continue to grind, after homogenate, go in 1.5mL sterilizing centrifuge tube, the centrifugal 2min of 8000rpm, gets supernatant liquor 100 μ L in 1.5mL sterilizing centrifuge tube.
2.2.2 positive control is processed: get positive control 100 μ L, put in 1.5mL sterilizing centrifuge tube.
2.2.3 negative control is processed: get negative control 100 μ L, put in 1.5mL sterilizing centrifuge tube.
3, the extraction of viral RNA (operating according to test kit specification sheets)
4, RT-PCR schedule of operation
Every part of cumulative volume 20 μ L, containing 16.8 μ L RT-PCR reaction solutions (with front mixing), 1.2 μ L enzyme mixations, 2 μ L template ribonucleic acids.For example: n duplicate samples (n < 10), preparation n+1 part, 16.8 * (n+1) RT-PCR reaction solution, add again 1.2 * (n+1) enzyme mixation mixes, get 18 μ L and be distributed into n part, add respectively 2 μ L template ribonucleic acids (positive control pipe adds 1 μ L positive control RNA and 1 μ L X liquid, and negative control pipe adds 1 μ L negative control RNA and 1 μ LX liquid), perform mark.
5, X liquid: be the aqueous solution of the primer sequence in non-target virus newcastle disease virus lasota pnca gene group, concentration 10pmoL/ μ L.
6, RT-PCR amplification program
On pcr amplification instrument, carry out following program: 42 ℃ of 45min, 95 ℃ of 3min; Amplification condition is 95 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s, 35 circulations; 72 ℃ are extended 10min.
7, positive control result
Pcr amplification product is carried out to agarose gel electrophoresis, there is the 330bp amplified band of expection in positive control, and result is as Fig. 1.The fragment length of positive control amplification and inverse transcription polymerase chain reaction test kit for the length of goal gene identical.
Preparation and the application of embodiment 3. foot and mouth disease virus RT-PCR test kit positive controls
1, non-target virus porcine reproductive and respiratory syndrome virus MLV strain (being obtained commercially): every milliliter of viral level should be not less than 10
7.0tCID
50virus-culturing fluid.
2, the preparation of positive control: non-target virus porcine reproductive and respiratory syndrome virus MLV strain cell culture fluid, with after 50 times of dilutions of deactivation reagent, be can be used as to foot and mouth disease virus RT-PCR test kit positive control and uses.
3, non-target virus primer sequence: be mixed with X liquid with concentration 10pmoL/ μ L
4, sample preparation
4.1 sample collectings: blister skin or the blister liquid of getting ox are placed in 50% glycerine physiological saline.
4.2 sample preparation: every duplicate samples is processed respectively.
4.2.1 blister liquid is processed: get 100 μ L, put in 1.5mL sterilizing centrifuge tube.
4.2.2 positive control is processed: get positive control 100 μ L, put in 1.5mL sterilizing centrifuge tube.
4.2.3 negative control is processed: get negative control 100 μ L, put in 1.5mL sterilizing centrifuge tube.
The method of 5, the extraction of viral RNA, RT-PCR schedule of operation is with embodiment 2.
6, RT-PCR amplification program
On pcr amplification instrument, carry out following program: 42 ℃ of 45min, 95 ℃ of 3min; Amplification condition is 95 ℃ of 15s, 60 ℃ of 30s, 35 circulations; 60 ℃ are extended 3min.
7, positive control result
Pcr amplification product is carried out to agarose gel electrophoresis, there is the 131bp amplified band of expection in positive control, and result is as Fig. 2.The fragment length of positive control amplification and inverse transcription polymerase chain reaction test kit for the length of goal gene identical.
Accompanying drawing explanation
Fig. 1. the amplification of positive control in embodiment 2.Wherein each swimming lane is respectively 1.Marker 2. positive control 3. sample 4. negative controls.
Fig. 2. the amplification of positive control in embodiment 3.Wherein each swimming lane is respectively 1.Marker 2. positive control 3. samples 4.Negative control.
Claims (23)
1. the preparation method for the positive control composition of inverse transcription polymerase chain reaction test kit, it is characterized in that, choose a kind of non-target virus as positive control template, and comprise a pair of non-target virus primer, so that described non-target virus is increased, the fragment length of amplification and inverse transcription polymerase chain reaction test kit for the length of goal gene identical.
2. the preparation method of claim 1, is characterized in that the non-target virus of choosing is RNA viruses.
3. claim 1 or 2 preparation method, is characterized in that described virus comprises newcastle disease virus and porcine reproductive and respiratory syndrome virus.
4. claim 1 or 2 preparation method, is characterized in that described virus comprises newcastle disease virus lasota strain and porcine reproductive and respiratory syndrome virus MLV strain.
5. claim 1 or 2 preparation method, characterized by further comprising the nutrient solution that uses non-target virus described in deactivation agent treated.
6. preparation method according to claim 5, wherein said deactivation reagent is high density strong denaturant.
7. preparation method according to claim 6, wherein said high density strong denaturant comprises the salt that contains guanidine radicals.
8. preparation method according to claim 6, wherein said high density strong denaturant is guanidine thiocyanate or Guanidinium hydrochloride.
9. preparation method claimed in claim 5, the nutrient solution of wherein said non-target virus dilutes with described deactivation reagent according to viral level, and dilution is not less than 7 times.
10. preparation method claimed in claim 5, the nutrient solution of wherein said non-target virus dilutes with described deactivation reagent according to viral level, and dilution 50-100 is doubly.
11. preparation methods claimed in claim 5, the nutrient solution of wherein said non-target virus dilutes with described deactivation reagent according to viral level, dilutes 50 times.
12. 1 kinds of positive control compositions for inverse transcription polymerase chain reaction test kit, it is characterized in that comprising that a kind of non-target virus is as positive control template, and comprise a pair of non-target virus primer, so that described non-target virus is increased, the fragment length of amplification and inverse transcription polymerase chain reaction test kit for the length of goal gene identical.
Positive control composition described in 13. claims 12, is characterized in that the non-target virus of choosing is RNA viruses.
14. claims 12 or 13 positive control composition, is characterized in that described virus comprises newcastle disease virus and porcine reproductive and respiratory syndrome virus.
15. claims 12 or 13 positive control composition, is characterized in that described virus comprises newcastle disease virus lasota strain and porcine reproductive and respiratory syndrome virus MLV strain.
Positive control composition described in 16. claims 12 or 13, characterized by further comprising deactivation reagent.
Positive control composition described in 17. claims 16, wherein deactivation reagent is high density strong denaturant.
Positive control composition described in 18. claims 17, wherein, high density strong denaturant comprises the salt that contains guanidine radicals.
Positive control composition described in 19. claims 17, wherein, high density strong denaturant is guanidine thiocyanate or Guanidinium hydrochloride.
Positive control composition described in 20. claims 16, the nutrient solution of wherein said non-target virus dilutes with described deactivation reagent according to viral level, and dilution is not less than 7 times.
Positive control composition described in 21. claims 16, the nutrient solution of wherein said non-target virus dilutes with described deactivation reagent according to viral level, and dilution 50-100 is doubly.
Positive control composition described in 22. claims 16, the nutrient solution of wherein said non-target virus dilutes with described deactivation reagent according to viral level, dilutes 50 times.
The purposes of the positive control composition described in 23. claims 12 or 13, it is as the part of inverse transcription polymerase chain reaction test kit.
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Citations (2)
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| US20030224017A1 (en) * | 2002-03-06 | 2003-12-04 | Samal Siba K. | Recombinant Newcastle disease viruses useful as vaccines or vaccine vectors |
| CN101798602A (en) * | 2010-03-18 | 2010-08-11 | 山东出入境检验检疫局检验检疫技术中心 | Composite kit for detecting avian influenzas and Newcastle disease viruses and detection method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030224017A1 (en) * | 2002-03-06 | 2003-12-04 | Samal Siba K. | Recombinant Newcastle disease viruses useful as vaccines or vaccine vectors |
| CN101798602A (en) * | 2010-03-18 | 2010-08-11 | 山东出入境检验检疫局检验检疫技术中心 | Composite kit for detecting avian influenzas and Newcastle disease viruses and detection method |
Non-Patent Citations (4)
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| S.Rashid et al..Multiplex polymerase chain reaction for the detection and differentiation of avian influenza viruse and other poultry respiratory pathogens.《Poultry Science》.2009,第88卷2526-2531. * |
| 范忠军等.美洲型猪繁殖与呼吸综合征病毒荧光RT-PCR检测方法的建立.《中国预防兽医学报》.2007,第29卷(第2期),134-137. * |
| 赵虎等.新城疫单抗酶联免疫试剂盒的完善及其应用.《中国兽医学报》.2002,第22卷(第1期),25-28. * |
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