CN115074424A - PCR reaction solution and aerosol pollution prevention PCR amplification method - Google Patents
PCR reaction solution and aerosol pollution prevention PCR amplification method Download PDFInfo
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Abstract
The invention provides a PCR reaction solution and a PCR amplification method for preventing aerosol pollution, which are applied to the technical field of enzyme or microorganism detection, wherein an upstream primer and a downstream primer containing a single RNA base are respectively designed, and in a PCR reaction system of alpha-type DNA polymerase or mixed enzyme containing the alpha-type DNA polymerase, RNase H2 can effectively degrade RNA-DNA hybrid chains under the condition of reacting for 10 minutes at 37 ℃, so that RNase H2 can effectively prevent false positive caused by aerosol pollution of the RNA-DNA hybrid chains; and has no influence on the sensitivity and precision of the PCR kit.
Description
Technical Field
The invention is applied to the technical field of enzyme or microorganism detection, and particularly relates to a PCR reaction solution and a PCR amplification method for preventing aerosol pollution.
Background
The biggest characteristic of Polymerase Chain Reaction (PCR) relying on thermostable DNA polymerase is that it has a large amplification capacity and a very high sensitivity, and there are three types of thermostable DNA polymerase as a key raw material for PCR reaction: pol type I, alpha type and mixed type. Type α: has 3'→ 5' exonuclease activity, i.e. a calibration function, which is the ability to remove erroneously bound bases and re-bind the correct bases in the PCR reaction. 3'→ 5' since the exonuclease takes some time to function, the elongation rate is slower than pol type I, but the heat resistance is higher than pol type I. pol I type: this enzyme is characterized by high DNA extension activity and addition of A at the 3' end of the PCR product. However, since it has no 3'→ 5' exonuclease activity, it lacks accuracy, and the bases may be mistakenly taken in the PCR extension, so that the DNA cannot be repaired. Mixing: the PCR kit is a mixture of pol I type and alpha type, has the advantages of rapidness and accuracy, can obviously improve the PCR efficiency, and becomes a choice of a plurality of PCR amplification kits.
Aerosol contamination in the operating environment is a common factor contributing to false positives in PCR results. A commonly used measure to prevent aerosol contamination is the addition of UNG enzyme (Uracil-N-Glycosylase ) and dUTP (deoxyuridine triphosphate) to the hot-start PCR kit. UNG enzyme can effectively hydrolyze uracil in single-stranded or double-stranded DNA and is inactive to RNA, and is mainly used for preventing or eliminating contamination of PCR amplification products. The action mechanism of the enzyme is as follows: dUTP is used for replacing dTTP in the PCR reaction, T in the amplified product fragment is completely replaced by U, and a PCR amplified product containing dU base is formed. UNG enzyme can selectively break glycosidic bonds of U basic groups in single-stranded and double-stranded DNA, degrade DNA containing U in a reaction system, effectively eliminate residual pollution of PCR products, and greatly reduce false positive caused by pollution of amplification products, thereby ensuring the specificity and accuracy of amplification.
However, the dUTP-UNG anti-contamination system is suitable for being combined with Pol type I DNA enzyme to carry out PCR reaction, and the alpha type enzyme with the correction activity can generate PCR inhibition after being combined with a template containing uracil, so that the amplification performance of the PCR reaction can be greatly reduced if the dUTP-UNG anti-contamination system is combined with the alpha type PCR enzyme and a mixed enzyme containing the alpha type enzyme. The addition of an anti-contamination system to a system to reduce the probability of false positive has become a factor that must be considered in the development of a PCR kit, and therefore how to add an effective anti-contamination system to a PCR kit using an alpha-type PCR enzyme or a mixed enzyme containing an alpha-type enzyme has become a problem to be solved urgently.
Thus, referring to FIG. 1, the present application first theorizes that RNase H, Ribonuclase H, known in Chinese as Ribonuclease H, is an endoribonuclease that specifically hydrolyzes RNA in DNA-RNA hybrid strands, and that RNase H is not capable of hydrolyzing the phosphodiester bonds in single-or double-stranded DNA or RNA, i.e., is not capable of digesting single-or double-stranded DNA or RNA. Use of RNase H: removing RNA from the DNA-RNA hybrid strand; realizing fixed-point cutting of RNA through complementary DNA sequences; removing poly (A) from the mRNA hybridized to poly (dT); in vitro polyadenylation reaction (polyadenylation reaction) product studies.
RNase H is roughly classified into two subtypes, RNase H1 and RNase H2. The RNase H1 enzyme requires at least four ribonucleotides containing base pairs in the substrate and cannot remove a single ribonucleotide from the strand consisting of deoxyribonucleotides. Therefore, the RNase H1 enzyme does not participate in the treatment of the Okazaki fragment RNA primer during DNA replication. Whereas both prokaryotic and eukaryotic RNase H2 enzymes can cleave a single ribonucleotide in one strand.
Therefore, theoretically, the site-directed cleavage of the position containing RNA base can be achieved by adding 1 RNA base to the appropriate position in the designed upstream primer and downstream primer, respectively, forming a DNA double strand containing a single ribonucleotide RNA under the action of PCR reaction, and then recognizing and processing the DNA double strand by RNase HII (FIG. 1), and the cleaved DNA double strand is not completely matched with the primer, so that the PCR amplification cannot be continued. And when the PCR reaction is carried out for 3-10 minutes at 95 ℃, the RNase HII can be identified without influencing the subsequent PCR amplification.
Based on the above, the invention provides a PCR reaction solution and a PCR amplification method for preventing aerosol pollution.
Disclosure of Invention
The invention aims to solve the problem that a dUTP-UNG anti-pollution system is the most common anti-pollution means of a PCR kit, but is influenced by different types of DNA polymerases used by different PCR kits, the dUTP-UNG anti-pollution system is only suitable for the PCR kit only containing pol I type DNA polymerase, the dUTP-UNG anti-pollution system is not suitable for the alpha type PCR kit containing corrective activity, if the PCR kit does not have reasonable anti-pollution measures, the risk of false positive of an amplification result caused by aerosol pollution is greatly increased, and a PCR reaction solution and an aerosol pollution prevention PCR amplification method are provided.
The invention adopts the following technical means for solving the technical problems:
the present invention provides a PCR reaction solution comprising:
upstream primer containing RNA base 0.1-1 μ M;
downstream primer containing RNA base 0.1-1 μ M;
0.05-0.5 mu M of Taqman probe;
1-3U/test of alpha-type DNA polymerase or mixed enzyme containing alpha-type DNA polymerase;
1 part of PCR buffer solution containing Mg2+ and dNTP;
mixing the upstream primer containing RNA base, the downstream primer containing RNA base, a Taqman probe, alpha-type DNA polymerase or a mixed enzyme containing alpha-type DNA polymerase, and PCR buffer containing Mg2+ and dNTP to 25-30 mu L by using water.
Further, the PCR reaction solution includes:
upstream primer containing RNA base 0.1-1 μ M;
downstream primer containing RNA base 0.1-1 μ M;
0.05-0.5 mu M of Taqman probe;
1-3U/test of mixed enzyme containing alpha type DNA polymerase;
1 part of PCR buffer solution containing Mg2+ and dNTP;
5 mu M of reaction template;
1 part of the upstream primer containing RNA bases, the downstream primer containing RNA bases, a Taqman probe, a mixed enzyme containing alpha-type DNA polymerase, a reaction template, and a PCR buffer containing Mg2+ and dNTP was mixed with water to 25. mu.L.
Further, the PCR reaction solution includes:
upstream primer containing RNA base 0.1-1 μ M;
downstream primer containing RNA base 0.1-1 μ M;
0.05-0.5 mu M of Taqman probe;
1-3U/test of mixed enzyme containing alpha type DNA polymerase;
1 part of PCR buffer solution containing Mg2+ and dNTP;
RNase H2 enzyme 1-3U/test
RNA-DNA hybrid strand template 10. mu.M;
1 part of the upstream primer containing RNA bases, the downstream primer containing RNA bases, a Taqman probe, a mixed enzyme containing alpha-type DNA polymerase, RNase H2 enzyme, a reaction template, and a PCR buffer containing Mg2+ and dNTP was mixed with water to 30. mu.L.
The invention also provides a PCR amplification method for preventing aerosol pollution, which is carried out by adopting the PCR reaction solution material and comprises the following steps:
respectively designing an upstream primer containing RNA base and a downstream primer containing RNA base aiming at a target fragment of DNA;
designing a Taqman probe for fluorescent PCR detection aiming at the target fragment;
preparing a first PCR reaction solution to obtain a first experimental group and a first control group;
amplifying the first experimental group and the first control group according to a preset environmental condition, obtaining a product RNA-DNA hybrid chain from the first experimental group, and obtaining a product DNA double chain without RNA base from the first control group;
preparing a second PCR reaction solution through the obtained RNA-DNA hybrid chain and the DNA double chain without RNA base to obtain a second experimental group and a second control group;
and amplifying the second experimental group and the second control group again according to the preset environmental conditions to obtain an anti-pollution test result and an amplification result.
Further, the step of designing an upstream primer containing RNA base and a downstream primer containing RNA base for the target fragment of DNA respectively comprises:
designing an upstream primer containing an RNA base which can be located near the middle and 3 'end of the primer, but cannot be located 4 bases before the 3' end;
designing a downstream primer containing one RNA base which can be located near the middle and 3 'end of the primer, but can not be located at the first 4 bases of the 3' end.
Further, designing a Taqman probe for fluorescence PCR detection aiming at the target fragment, wherein the Taqman probe comprises a fluorescent group and a quenching group;
the fluorescent group marks the 5' end of the target fragment;
the quenching group marks the 3' end of the target fragment;
the fluorescent group is one of FAM, HEX, TET, JOE, VIC, ROX, Cy3 and Cy5, and the quenching group is one of TAMRA, BHQ1, BHQ2 and DABCYL
Further, the step of preparing the first PCR reaction solution to obtain the first experimental group and the first control group includes:
the first experimental group and the first control group both comprise
Upstream primer containing RNA base 0.1-1 μ M;
downstream primer containing RNA base 0.1-1 μ M;
0.05-0.5 mu M of Taqman probe;
1-3U/test of mixed enzyme containing alpha type DNA polymerase;
1 part of PCR buffer solution containing Mg2+ and dNTP;
5 mu M of reaction template;
1 part of the upstream primer containing RNA bases, the downstream primer containing RNA bases, a Taqman probe, a mixed enzyme containing alpha-type DNA polymerase, a reaction template, and a PCR buffer containing Mg2+ and dNTP was mixed with water to 25. mu.L.
Further, in the step of amplifying the first experimental group and the first control group according to a preset environmental condition, obtaining the product RNA-DNA hybrid strand from the first experimental group, and obtaining the product DNA double strand without RNA base from the first control group, the preset environmental condition includes:
pre-denaturation: the temperature is 95 ℃, the time is 3 minutes, no fluorescence signal is collected, and the cycle is 1 time;
denaturation: the temperature is 95 ℃, the time is 15 seconds, no fluorescence signal is collected, and the cycle is 10-12 times;
annealing/extending: collecting fluorescence signals at the temperature of 60 ℃ for 40 seconds, and circulating for 10-12 times.
Further, the step of preparing a second PCR reaction solution by using the obtained RNA-DNA hybrid strand and the DNA double strand not containing the RNA base to obtain a second experimental group and a second control group includes:
the second experimental component:
upstream primer containing RNA base 0.1-1 μ M;
downstream primer containing RNA base 0.1-1 μ M;
0.05-0.5 mu M of Taqman probe;
1-3U/test of mixed enzyme containing alpha type DNA polymerase;
1 part of PCR buffer solution containing Mg2+ and dNTP;
RNase H2 enzyme 1-3U/test
RNA-DNA hybrid strand template 10. mu.M;
1 part of water is mixed with the upstream primer containing RNA base, the downstream primer containing RNA base, a Taqman probe, a mixed enzyme containing alpha-type DNA polymerase, RNase H2 enzyme, a reaction template and PCR buffer solution containing Mg2+ and dNTP to 30 mu L;
the second control group component:
upstream primer containing RNA base 0.1-1 μ M;
downstream primer containing RNA base 0.1-1 μ M;
0.05-0.5 mu M of Taqman probe;
1-3U/test of mixed enzyme containing alpha type DNA polymerase;
1 part of PCR buffer solution containing Mg2+ and dNTP;
RNase H2 enzyme 1-3U/test
DNA double-stranded template 10. mu.M;
1 part of the upstream primer containing RNA bases, the downstream primer containing RNA bases, a Taqman probe, a mixed enzyme containing alpha-type DNA polymerase, RNase H2 enzyme, a reaction template, and a PCR buffer containing Mg2+ and dNTP was mixed with water to 30. mu.L.
Further, the step of amplifying the second experimental group and the second control group again according to the preset environmental conditions to obtain the anti-contamination test result and the amplification result, wherein the second experimental group and the second control group again according to the preset environmental conditions include:
anti-pollution treatment: the temperature is 37 ℃, the time is 10 minutes, no fluorescence signal is collected, and the cycle is 1 time;
pre-denaturation: the temperature is 95 ℃, the time is 3 minutes, no fluorescence signal is collected, and the cycle is 1 time;
denaturation: the temperature is 95 ℃, the time is 15 seconds, no fluorescence signal is collected, and the cycle is 45 times;
annealing/extending: the temperature is 60 ℃, the time is 40 seconds, and the fluorescence signal is collected and circulated for 45 times.
The invention provides a PCR reaction solution and a PCR amplification method for preventing aerosol pollution, and the PCR amplification method has the following beneficial effects:
respectively designing an upstream primer and a downstream primer containing a single RNA base, and effectively degrading an RNA-DNA hybrid chain under the condition that RNase H2 reacts for 10 minutes at 37 ℃ in a PCR reaction system of alpha-type DNA polymerase or mixed enzyme containing the alpha-type DNA polymerase, thereby showing that RNase H2 can effectively prevent false positive caused by aerosol pollution of the RNA-DNA hybrid chain; and has no influence on the sensitivity and precision of the PCR kit.
Drawings
FIG. 1 is a diagram illustrating the site-directed cleavage of a single RNA-containing DNA duplex by RNase H2 in the background of the invention;
FIG. 2 is a schematic flow chart of an embodiment of the PCR amplification method for preventing aerosol contamination according to the present invention;
FIG. 3 shows a DNA double strand (1X 10) of the second control reagent of the present invention 6 Copies/mL) as a template;
FIG. 4 is a graph showing the amplification results of a second control reagent of the present invention using a DNA double strand (100 Copies/mL) as a template;
FIG. 5 shows a second experimental group of reagents according to the present invention using RNA-DNA hybrid strand template (1X 10) 6 Copies/mL and 100 Copies/mL) as template.
The implementation, functional features and advantages of the present invention will be further described with reference to the accompanying drawings.
Detailed Description
It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The alpha-type DNA polymerase containing the corrective activity has high accuracy, and the mixed enzyme containing the alpha-type DNA polymerase not only has high accuracy and good amplification efficiency, but also becomes a choice of more and more PCR kits, but once aerosol pollution exists in a laboratory, the risk that the kit cannot avoid the aerosol pollution to cause false positive is increased because a dUTP-UNG anti-pollution system cannot be added. The invention aims to add an anti-pollution system which is high in efficiency and does not influence the performance of a kit and mainly takes RNase HII into a PCR kit containing alpha-type DNA polymerase so as to avoid aerosol pollution.
The specific scheme of the PCR amplification method for preventing aerosol pollution is as follows:
respectively designing an upstream primer and a downstream primer containing an RNA base aiming at a DNA target fragment, wherein the RNA base is close to the middle and the 3 'end of the primer, but cannot be positioned at the first four bases of the 3' end; and aiming at the target DNA fragment, a Taqman probe for fluorescence PCR detection is designed, wherein the 5 'end of the probe is marked with a fluorescent group, and the 3' end of the probe is marked with a quenching group. The fluorescent group is one of FAM, HEX, TET, JOE, VIC, ROX, Cy3 and Cy5, and the quenching group is one of TAMRA, BHQ1, BHQ2 and DABCYL.
Preparing a PCR reaction solution; preparing the designed upstream primer, downstream primer and Taqman probe into PCR reaction solution, wherein the enzyme is alpha-type DNA polymerase or mixed enzyme containing alpha-type DNA polymerase, so as to obtain an experimental group and a control group; with specific reference to table 1:
PCR amplification
PCR amplification was performed according to the following reaction conditions, see Table 2:
finally, PCR amplification results were obtained, see table 3:
in view of the above, referring to fig. 2, a schematic flow chart of the PCR amplification method for preventing aerosol contamination includes the steps of:
p1, designing an upstream primer containing RNA base and a downstream primer containing RNA base respectively aiming at the target fragment of DNA;
p2, designing a Taqman probe for fluorescent PCR detection aiming at the target fragment;
p3, preparing a first PCR reaction solution to obtain a first experiment group and a first control group;
p4, amplifying the first experimental group and the first control group according to the preset environmental conditions, obtaining the product RNA-DNA hybrid chain from the first experimental group, and obtaining the product DNA double chain without RNA base from the first control group;
p5, preparing a second PCR reaction solution by the obtained RNA-DNA hybrid chain and the DNA double chain without RNA base, and obtaining a second experimental group and a second control group;
and P6, amplifying the second experimental group and the second control group again according to the preset environmental conditions to obtain the anti-pollution test result and the amplification result.
Taking Hepatitis B Virus (HBV) as an example, the mixed enzyme containing alpha-type DNA polymerase can meet the high-sensitivity amplification requirement of an HBV PCR amplification kit, so an anti-pollution system based on the principle of RNase HII site-directed RNA base shearing is designed, and the specific steps are as follows:
designing an upstream primer containing an RNA base which can be located at the position close to the middle and 3 'end of the primer but can not be located at the position of the first 4 bases of the 3' end, and can be any one of seq1-seq 8:
Seq1：5’CTAGGACCC/rC/TGCTCGTGTTA 3’
Seq2：5’CTAGGACCCC/rT/GCTCGTGTTA 3’
Seq3：5’CTAGGACCCCT/rG/CTCGTGTTA 3’
Seq4：5’CTAGGACCCCTG/rC/TCGTGTTA 3’
Seq5：5’CTAGGACCCCTGC/rT/CGTGTTA 3’
Seq6：5’CTAGGACCCCTGCT/rC/GTGTTA 3’
Seq7：5’CTAGGACCCCTGCTC/rG/TGTTA 3’
Seq8：5’CTAGGACCCCTGCTCG/rT/GTTA 3’
designing a downstream primer containing one RNA base, wherein the RNA base can be positioned at the position close to the middle and the 3 'end of the primer, but can not be positioned at the position of the first 4 bases of the 3' end, and the RNA base can be any one of seq 9-seq 15:
Seq9：5’AATTGAGAGAA/rG/TCCACCACGA 3’
seq10：5’AATTGAGAGAAG/rT/CCACCACGA 3’
seq11：5’AATTGAGAGAAGT/rC/CACCACGA 3’
seq12：5’AATTGAGAGAAGTC/rC/ACCACGA 3’
seq13：5’AATTGAGAGAAGTCC/rA/CCACGA 3’
seq14：5’AATTGAGAGAAGTCCA/rC/CACGA 3’
seq15：5’AATTGAGAGAAGTCCAC/rC/ACGA 3’
designing a taqman hydrolysis probe without RNA base, wherein the sequence is as follows:
seq16：5FAM-TTGACAAAAATCCTCACAATACCACAGAGTCTAGA-BHQ1 3’；
seq17 and seq18 are the forward primer and the reverse primer, respectively, without RNA bases for comparative validation, and the sequences are as follows:
Seq17：5’CTAGGACCCCTGCTCGTGTTA 3’
Seq18：5’AATTGAGAGAAGTCCACCACGA 3’
the above steps Seq1-Seq18 are cycled sequentially so that the experimental group and the control group are not two separate groups, but are experimental control processes in the same reagent.
Preparing a first PCR reaction solution, i.e. a first experimental group and a first control group, using seq1 as an upstream primer, seq9 as a downstream primer, and seq16 as a probe, to prepare the first PCR reaction solution of the first experimental group and the first control group, which is shown in table 4:
the PCR amplification was performed according to the following reaction conditions, the products of the first experimental group were RNA-DNA hybrid strands, and the products of the first control group were DNA duplexes containing no RNA base, see Table 5:
to obtain an RNA-DNA hybrid strand and a DNA double strand not containing an RNA base;
then, the obtained RNA-DNA hybrid strand and DNA double strand are respectively used as templates to prepare a second PCR reaction solution, 1-3U/test RNase HII is respectively added to a second experimental group and a second control group, the second experimental group uses the RNA-DNA hybrid strand containing a single RNA base as a template, the second control group uses the DNA double strand as a template, and the reference table 6:
finally, the experimental and control reaction solutions were subjected to PCR amplification by the following reaction conditions, see table 7:
results of anti-contamination test
As shown in FIGS. 3 and 4, the control group using the DNA double-stranded template as the template has a distinct S-shaped amplification curve, while the experimental group using the RNA-DNA hybrid strand as the template has no amplification curve in FIG. 5. Therefore, the RNase HII can effectively remove RNA-DNA hybrid chains under proper treatment conditions, thereby achieving the anti-pollution effect.
Similarly, any one upstream primer in seq1-seq 5, any one downstream primer in seq 9-seq 15 and the fluorescent probe seq15 are combined to prepare a PCR reaction solution containing RNase HII, and an RNA-DNA hybrid chain (which is a target fragment at the same time) is used as a template, so that the test result can be achieved.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A PCR reaction solution, comprising:
upstream primer containing RNA base 0.1-1 μ M;
downstream primer containing RNA base 0.1-1 μ M;
0.05-0.5 mu M of Taqman probe;
1-3U/test of mixed enzyme containing alpha type DNA polymerase;
1 part of PCR buffer solution containing Mg2+ and dNTP;
10 mu L of DNA double-stranded template;
mixing the upstream primer containing RNA base, the downstream primer containing RNA base, a Taqman probe, alpha-type DNA polymerase or a mixed enzyme containing alpha-type DNA polymerase, and PCR buffer containing Mg2+ and dNTP to 25-30 mu L by using water.
2. The PCR reaction solution according to claim 1, comprising:
upstream primer containing RNA base 0.1-1 μ M;
downstream primer containing RNA base 0.1-1 μ M;
0.05-0.5 mu M of Taqman probe;
1-3U/test of mixed enzyme containing alpha type DNA polymerase;
1 part of PCR buffer solution containing Mg2+ and dNTP;
5 mu M of reaction template;
1 part of the upstream primer containing RNA bases, the downstream primer containing RNA bases, a Taqman probe, a mixed enzyme containing alpha-type DNA polymerase, a reaction template, and a PCR buffer containing Mg2+ and dNTP was mixed with water to 25. mu.L.
3. The PCR reaction solution according to claim 1, comprising:
upstream primer containing RNA base 0.1-1 μ M;
downstream primer containing RNA base 0.1-1 μ M;
0.05-0.5 mu M of Taqman probe;
1-3U/test of mixed enzyme containing alpha type DNA polymerase;
1 part of PCR buffer solution containing Mg2+ and dNTP;
RNase H2 enzyme 1-3U/test
RNA-DNA hybrid strand template 10. mu.M;
1 part of the upstream primer containing RNA bases, the downstream primer containing RNA bases, a Taqman probe, a mixed enzyme containing alpha-type DNA polymerase, RNase H2 enzyme, a reaction template, and a PCR buffer containing Mg2+ and dNTP was mixed with water to 30. mu.L.
4. A PCR amplification method for preventing aerosol contamination, which is carried out by using the PCR reaction solution material according to any one of claims 1 to 3, and which comprises:
respectively designing an upstream primer containing RNA base and a downstream primer containing RNA base aiming at a target fragment of DNA;
designing a Taqman probe for fluorescent PCR detection aiming at the target fragment;
preparing a first PCR reaction solution to obtain a first experimental group and a first control group;
amplifying the first experimental group and the first control group according to a preset environmental condition, obtaining a product RNA-DNA hybrid chain from the first experimental group, and obtaining a product DNA double chain without RNA base from the first control group;
preparing a second PCR reaction solution through the obtained RNA-DNA hybrid chain and the DNA double chain without RNA base to obtain a second experimental group and a second control group;
and amplifying the second experimental group and the second control group again according to the preset environmental conditions to obtain an anti-pollution test result and an amplification result.
5. The PCR amplification method for preventing aerosol contamination according to claim 4, wherein the step of designing an upstream primer containing RNA base and a downstream primer containing RNA base for the target fragment of DNA respectively comprises:
designing an upstream primer containing an RNA base which can be located near the middle and 3 'end of the primer, but cannot be located 4 bases before the 3' end;
the downstream primer is designed to contain an RNA base that can be located near the middle and 3 'end of the primer, but not the first 4 bases of the 3' end.
6. The aerosol contamination prevention PCR amplification method of claim 5, wherein the step of designing a Taqman probe for fluorescence PCR detection for the target fragment comprises a fluorescent group and a quencher group;
the fluorescent group marks the 5' end of the target fragment;
the quenching group marks the 3' end of the target fragment;
the fluorescent group is one of FAM, HEX, TET, JOE, VIC, ROX, Cy3 and Cy5, and the quenching group is one of TAMRA, BHQ1, BHQ2 and DABCYL.
7. The method for PCR amplification according to claim 4, wherein the step of preparing the first PCR reaction solution to obtain the first experimental group and the first control group comprises:
the first experimental group and the first control group both comprise
Upstream primer containing RNA base 0.1-1 μ M;
downstream primer containing RNA base 0.1-1 μ M;
0.05-0.5 mu M of Taqman probe;
1-3U/test of mixed enzyme containing alpha type DNA polymerase;
1 part of PCR buffer solution containing Mg2+ and dNTP;
5 mu M of reaction template;
1 part of the upstream primer containing RNA bases, the downstream primer containing RNA bases, a Taqman probe, a mixed enzyme containing alpha-type DNA polymerase, a reaction template, and PCR buffer containing Mg2+ and dNTP is mixed to 25 mu L by using water.
8. The method for PCR amplification according to claim 7, wherein the amplification of the first experimental group and the first control group is performed under predetermined environmental conditions, the steps of obtaining the RNA-DNA hybrid strand from the first experimental group and obtaining the DNA double strand without RNA base from the first control group comprise:
pre-denaturation: the temperature is 95 ℃, the time is 3 minutes, no fluorescence signal is collected, and the cycle is 1 time;
denaturation: the temperature is 95 ℃, the time is 15 seconds, no fluorescence signal is collected, and the cycle is 10-12 times;
annealing/extending: collecting fluorescence signals at the temperature of 60 ℃ for 40 seconds, and circulating for 10-12 times.
9. The method for PCR amplification against aerosol contamination according to claim 4, wherein the step of preparing a second PCR reaction solution by using the obtained RNA-DNA hybrid strand and the DNA double strand without RNA base to obtain a second experimental group and a second control group comprises:
the second experimental component:
upstream primer containing RNA base 0.1-1 μ M;
downstream primer containing RNA base 0.1-1 μ M;
0.05-0.5 mu M of Taqman probe;
1-3U/test of mixed enzyme containing alpha type DNA polymerase;
1 part of PCR buffer solution containing Mg2+ and dNTP;
RNase H2 enzyme 1-3U/test
RNA-DNA hybrid strand template 10. mu.M;
1 part of water is mixed with the upstream primer containing RNA base, the downstream primer containing RNA base, a Taqman probe, a mixed enzyme containing alpha-type DNA polymerase, RNase H2 enzyme, a reaction template and PCR buffer solution containing Mg2+ and dNTP to 30 mu L;
the second control component:
upstream primer containing RNA base 0.1-1 μ M;
downstream primer containing RNA base 0.1-1 μ M;
0.05-0.5 mu M of Taqman probe;
1-3U/test of mixed enzyme containing alpha type DNA polymerase;
1 part of PCR buffer solution containing Mg2+ and dNTP;
RNase H2 enzyme 1-3U/test
DNA double-stranded template 10. mu.M;
1 part of the upstream primer containing RNA bases, the downstream primer containing RNA bases, a Taqman probe, a mixed enzyme containing alpha-type DNA polymerase, RNase H2 enzyme, a reaction template, and a PCR buffer containing Mg2+ and dNTP was mixed with water to 30. mu.L.
10. The aerosol contamination prevention PCR amplification method according to claim 9, wherein the second experimental group and the second control group are amplified again according to preset environmental conditions to obtain the contamination prevention test result and the amplification result, and the further according to the preset environmental conditions include:
anti-pollution treatment: the temperature is 37 ℃, the time is 10 minutes, the fluorescence signal is not collected, and the cycle is 1 time;
pre-denaturation: the temperature is 95 ℃, the time is 3 minutes, no fluorescence signal is collected, and the cycle is 1 time;
denaturation: the temperature is 95 ℃, the time is 15 seconds, no fluorescence signal is collected, and the cycle is 45 times;
annealing/extending: the temperature is 60 ℃, the time is 40 seconds, and the fluorescence signal is collected and circulated for 45 times.
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