Multiple anti-pollution PCR (Polymerase Chain Reaction) premixed solution and application thereof in sex identification of pandas
By developing multiple prevention PCR premissions, combined with multiple PCR auxiliary agents and polluting components, the problem of DNA inhibitors and air -soluble glue pollution in the giant panda gender identification significantly improves the accuracy of the results.
Patent Information
- Application Number
- CN202411657045.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-19
- Publication Date
- 2025-01-28
AI Technical Summary
During the PCR process of gender identification of giant pandas, various inhibitors and environmental aerosol pollution in DNA lead to insufficient accuracy of the results, and the existing PCR reaction system is difficult to solve these problems at the same time.
Develop a multi -anti -pollution PCR premix, containing a variety of PCR auxiliary agents and anti -polluting components, such as DNA polymerase, beef serum albumin, surfactant, antioxidant, buffer, one -price cationic component, metal ion The chelating agent, DNA glycase and amplification promoter can effectively reduce the impact of inhibitors and eliminate gas -soluble pollution.
It significantly improves the accuracy of the results of the giant panda gender, enhances the tolerance of the PCR reaction system to inhibitors, and effectively prevent the interference of subsequent expansion of gas -soluble glue pollution.
Smart Images
Figure CN119372297A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of molecular biological detection, and in particular to a multiple anti-pollution PCR premix and application thereof in sex identification of giant pandas. Background Art
[0002] The giant panda (Ailuropoda melanoleuca) is an endangered and rare protected animal unique to my country, and is also a flagship species for biodiversity conservation in the world. However, accurate identification of the sex of giant pandas has always been a difficult problem in the sex ratio survey of wild giant panda populations and the targeted feeding and management of captive giant pandas. On the one hand, it is difficult for researchers to determine the sex of wild giant pandas by their behavior because wild giant pandas are not easy to observe closely. On the other hand, giant pandas have dense fur and no obvious secondary sexual characteristics, so researchers cannot distinguish between male and female individuals by appearance characteristics. Young giant pandas have the physiological characteristics that the appearance of male and female genitals is not obviously different, so it is more difficult to accurately determine the sex of young individuals at an early stage. The method of identifying sex by combining animal behavior, appearance and genital characteristics is not only less accurate, but also heavily dependent on the subjective experience of the operator. With the development of molecular biology technology, PCR detection has provided a reliable method for identifying the sex of giant pandas. Zhan et al. developed two pairs of combined primers included in the "Technical Regulations for the Establishment of Genetic Archives of Giant Panda Populations" (People's Republic of China Forestry Industry Standard LY / T 2896-2017), which are specifically used for giant panda sex identification. One pair is the ZX1 primers (ZX1-F: 5'-CTACATAGCCCCAACCCCTTTTC-3'; ZX1-R: 5'-GGTTCTGGCCGCTGTCTCTACTAT-3') for amplifying the SRY gene on the Y chromosome, with a target fragment size of 210 bp, which is used to identify male individuals. The other pair is the ZFX primers (ZFX-F: 5'-ATAATCACATGGAGAGCCACAAGCT-3'; ZFX-R: 5'-CTCCTTTTTCCTTATGCACC-3') for amplifying the ZFX gene on the X chromosome, with a target fragment size of 130 bp, which is used to distinguish cases where PCR amplification fails or is identified as female individuals.
[0003] The source of DNA for sex identification of giant pandas is usually non-damage samples such as excrement, or damaged samples such as tissues and blood, among which non-damage fecal samples are the most common choice. The effect of PCR amplification is easily affected by various inhibitors in DNA. Although the "Technical Regulations for the Establishment of Genetic Archives of Giant Panda Populations" (People's Republic of China Forestry Industry Standard LY / T 2896-2017) recommends adding bovine serum albumin (BSA) to the PCR system to bind certain inhibitors in DNA, relying on a single PCR adjuvant is still not enough to avoid the increase of nonspecific amplification or the inhibition of Taq enzyme activity. Fecal DNA not only contains a large amount of background DNA that is easy to cause nonspecific amplification (DNA from microorganisms, food and other substances other than host DNA), but also easily retains various amplification inhibitors that destroy the stability of the PCR system, which will increase nonspecific amplification, weaken the brightness of the target band, and even cause amplification failure. Therefore, if you want to obtain stable and reliable sex identification results, you must increase the types of PCR adjuvants to adjust the tolerance of the reaction system to various inhibitors. Ultimately, the PCR reaction system can achieve a balance between the template concentration sufficient to amplify the target band and the inhibitor concentration that destroys the stability of the system. In addition to the adverse effects of various PCR inhibitors in DNA, the PCR process for giant panda sex identification is also susceptible to aerosol contamination in the environment. As the number of PCR rounds increases, the amplified product is prone to overflowing the centrifuge tube due to repeated opening of the lid, shaking, liquid evaporation, repeated blowing and suction of liquids, etc., causing the next round of PCR reaction system to be contaminated by the PCR products of the previous rounds. These environmental aerosol pollutions are not easy to eliminate and can easily interfere with the accuracy of the giant panda sex identification results. However, the PCR reaction system currently commonly used in giant panda sex identification cannot solve the above two problems at the same time. Therefore, it is very necessary to develop a multiplex PCR premix that can tolerate various inhibitors in DNA and prevent aerosol contamination to increase the accuracy of giant panda sex identification results. Summary of the invention
[0004] The purpose of the present invention is to solve the technical problems of insufficient tolerance of PCR premix to inhibitors and insufficient ability to prevent environmental aerosol pollution in giant panda sex identification, and to provide a multiple anti-pollution PCR premix and its application in giant panda sex identification. The PCR premix contains multiple PCR adjuvants and anti-pollution components, which can effectively reduce the destructive effect of complex inhibitors in template DNA on the balance of PCR reaction system, eliminate the interference of aerosol pollution of amplification products on subsequent amplification, and greatly improve the accuracy of sex identification results of giant pandas and even other animals.
[0005] In order to achieve the above technical objectives, the technical solutions adopted in this application are as follows: In a first aspect, the present invention provides a multiple anti-contamination PCR premix, wherein the premix comprises, according to the final concentration, 1-5 U of DNA polymerase; 100-700 nM of substrate; 1-5 mM of MgCl2; 0.02-1 mg / mL of bovine serum albumin (BSA); 0.1%-1% V / V of surfactant; 0.1-3 mM of antioxidant; 10-30 mM of buffer; 10-60 mM of monovalent cation component; 0.1-4 mM of metal ion chelator; 0.5-3 U of DNA glycosylase; 0.01-3 M of stabilizer; 4%-20% V / V of amplification accelerator; and the solvent is water; wherein the substrate is at least one of dNTP, dATP, dTTP, dGTP, dCTP and dUTP; and the monovalent cation component is a compound that provides potassium ions and ammonium ions, respectively.
[0006] Preferably, the premix comprises, according to the final concentration, the following components: DNA polymerase, 1-2.5 U; substrate, 500-700 nM; MgCl2, 1-2.5 mM; bovine serum albumin (BSA), 0.025-0.05 mg / mL; surfactant, 0.3%-0.7% V / V; antioxidant, 0.5-2 mM; buffer, 10-20 mM; monovalent cation component, 20-50 mM; metal ion chelator, 0.5-2 mM; DNA glycosylase, 1-2 U; stabilizer, 1.2-2.0 M; amplification accelerator, 4%-10% V / V; and the solvent is water.
[0007] Preferably, the DNA polymerase is at least one of Taq polymerase, Pfu polymerase, Taq Plus polymerase, HotStart Taq polymerase, PrimerStar HS DNA polymerase and AmpliTaq Gold DNA polymerase.
[0008] Preferably, the surfactant is at least one of Tween 20, Tween 21, Tween 40, Tween 60, Tween 61, Tween 80, Span 20, Span 40, Span 60, Span 80, Span 65, Span 85, TritonX100 and NP40, more preferably at least one of Tween 20, TritonX100 and NP40.
[0009] Preferably, the antioxidant is at least one of dithiothreitol (DTT) and dithioerythritol (DTE).
[0010] Preferably, the buffer is at least one of tris-hydroxymethylaminomethane hydrochloride (Tris-HCL), phosphate buffered saline (PBS), 3-morpholinepropanesulfonic acid (MOPS) and 4-hydroxyethylpiperazineethanesulfonic acid (HEPES).
[0011] Preferably, in the monovalent cation component, the compound providing potassium ions is selected from one of potassium chloride, potassium sulfate, potassium nitrate, and potassium acetate, and the compound providing ammonium ions is selected from one of ammonium sulfate, ammonium chloride, ammonium nitrate, and ammonium acetate.
[0012] Preferably, the metal ion chelating agent is at least one of ethylene glycol ditetraacetic acid (EGTA) and ethylenediaminetetraacetic acid (EDTA).
[0013] Preferably, the DNA glycosylase is uracil DNA glycosylase or heat-sensitive uracil DNA glycosylase. Uracil DNA glycosylase is abbreviated as UDG or UNG, and heat-sensitive uracil DNA glycosylase is abbreviated as Heat-labile UDG or Heat-labile UNG.
[0014] Preferably, the stabilizer is at least one of trehalose and betaine.
[0015] Preferably, the amplification accelerator is at least one of PEG200-8000 and DMSO.
[0016] Preferably, the PCR premix solution further comprises a pH adjuster to adjust the pH of the PCR premix solution to 7.5-8.5; more preferably, the pH adjuster is a sodium hydroxide solution.
[0017] In a second aspect, the present invention provides a method for preparing the PCR premix solution described in the first aspect, comprising the following steps: preparing ingredients according to the components of the PCR premix solution, and mixing them into a solution.
[0018] In a third aspect, the present invention provides use of the PCR premix described in the first aspect in preparing a PCR kit.
[0019] Preferably, the PCR kit is used to identify the sex of giant pandas; more preferably, the source of the DNA sample used for PCR is the excrement, blood, hair, skin, cells and tissues of giant pandas.
[0020] In a fourth aspect, the present invention provides a PCR kit for identifying the sex of a giant panda, comprising amplification primers and the PCR premix according to any one of claims 1-13.
[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. Various adjuvants that increase the stability of the PCR system are added to the PCR premix of the present invention, which can enhance the tolerance of the reaction system to various inhibitors, reduce the inhibitory effect of the inhibitors on the activity of DNA polymerase, and improve the success rate of PCR amplification.
[0022] Second, the present invention adds anti-pollution components to the multiplex PCR premix. During continuous amplification, the PCR reaction system can eliminate the influence of the previous round of PCR amplification products on the current round of reaction system, prevent the interference of amplification product aerosols in the environment on the sex identification results, improve the accuracy of the sex identification results of giant pandas, and is expected to be further expanded to multiple anti-pollution PCR amplification of other animal DNA. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 This is the appearance map of fecal DNA in Application Example 1 of the present invention.
[0024] Figure 2 This is the electrophoresis detection pattern of fecal DNA in Application Example 1 of the present invention.
[0025] Figure 3 This is a comparative graph of electrophoresis detection of template detection performance in Application Example 2 of the present invention.
[0026] Figure 4 This is a comparative graph of electrophoresis detection of inhibitor tolerance in Application Example 3 of the present invention.
[0027] Figure 5 This is a comparative graph of electrophoresis detection when amplifying DNA from other sources in Application Example 4 of the present invention.
[0028] Figure 6 This is a comparative graph of electrophoresis detection of anti-pollution performance in Application Example 5 of the present invention.
[0029] Figure 7 This is the electrophoresis detection pattern of fecal DNA in Application Example 6 of the present invention.
[0030] Figure 8 This is the electrophoresis detection spectrum of the anti-pollution performance in Application Example 7 of the present invention.
[0031] Fig. 9 This is the electrophoresis detection pattern of fecal DNA in Application Example 8 of the present invention. DETAILED DESCRIPTION
[0032] In the embodiment of the present invention, the control PCR premix is prepared according to the PCR system components recommended in the "Technical Regulations for Establishing Genetic Archives of Giant Panda Populations". In a 25 μL PCR reaction system, the specific composition of the control PCR premix is: TaqPCR Premix, 12.5 μL; MgCl2 (25mM), 1 μL; bovine serum albumin (BSA), 1 mg / mL.
[0033] In the description of the present invention, it should be noted that, if the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.
[0034] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, which are intended to explain the present invention rather than to limit it. Example 1
[0035] This embodiment provides a multiple anti-contamination PCR premix solution, which includes, according to the final concentration, DNA polymerase, 2.5U; substrate, 700 nM; MgCl2, 1.25 mM; bovine serum albumin (BSA), 0.05 mg / mL; surfactant, 0.5% V / V; antioxidant, 1 mM; buffer, 10 mM; monovalent cation component, 20 mM; metal ion chelator, 1 mM; Heat-labile UDG enzyme, 2 U; system stabilizer, 1.7 M; amplification accelerator, 6% V / V. The pH of the PCR premix solution is 8.0.
[0036] Among them, the DNA polymerase was 2 U of HotStart Taq polymerase and 0.5 U of Pfu polymerase, the substrate was 300 nM dNTP and 400 nM dUTP, the surfactant was NP40, the antioxidant was DTT, the buffer was Tris-HCL, the monovalent cation components were 10 mM potassium chloride and 10 mM ammonium sulfate, the metal ion chelators were 0.5 mM EGTA and 0.5 mM EDTA, the system stabilizers were 0.2 M trehalose and 1.5 M betaine, the amplification promoters were 1% DMSO and 5% PEG8000, and the pH adjuster was 0.1 M sodium hydroxide solution. Example 2
[0037] This embodiment provides a multiple anti-contamination PCR premix, which is different from Example 1 in that: the DNA polymerase is 2U PrimerStar HS DNA polymerase, the surfactant is Tween 20, the antioxidant is DTE, the monovalent cation component is potassium chloride, the system stabilizer is 2 M betaine, and the amplification promoter is 4% PEG6000. Example 3
[0038] This embodiment provides a multiplex anti-contamination PCR premix, which is different from Example 1 in that: the DNA polymerase is 2 U of AmpliTaq Gold DNA polymerase and 0.5 U of Pfu polymerase, the metal ion chelator is 1 mM EGTA, the system stabilizer is 0.3 M trehalose and 1 M betaine, the DNA glycosylase is UDG enzyme, and the amplification accelerator is 8% PEG400. Example 4
[0039] This embodiment provides a multiplex anti-contamination PCR premix solution, which is different from Embodiment 1 in that the DNA polymerase is 2 U of Taq polymerase and 0.5 U of Pfu polymerase. Example 5
[0040] This embodiment provides a multiple anti-contamination PCR premix solution, which is different from Embodiment 1 in that the surfactant is TritonX100. Example 6
[0041] This embodiment provides a multiple anti-contamination PCR premix solution, which is different from Embodiment 1 in that the buffer solution is PBS. Example 7
[0042] This embodiment provides a multiple anti-contamination PCR premix solution, which is different from Embodiment 1 in that the DNA glycosylase is UDG enzyme.
[0043] Comparative Example 1: This comparative example provides a multiple anti-contamination PCR premix solution, which is different from Example 1 in that it does not contain Heat-labileUDG enzyme.
[0044] Comparative Example 2: This comparative example provides a multiple anti-contamination PCR premix solution, which is different from Example 1 in that bovine serum albumin (BSA) is replaced by bovine submandibular gland mucin (BSM).
[0045] Application Example 1: Fresh feces of a male giant panda was extracted using a commercial fecal DNA extraction kit (QIAamp Fast DNA Stool Mini Kit, Cat. No. / ID: 51604). The absorbance of the extracted four fecal DNAs was analyzed, and the DNA concentration was tested by Qubit and the DNA purity was tested by Nanodrop. A 1.5% agarose gel was prepared to perform electrophoresis analysis on 2 μL of DNA to detect the integrity of the DNA structure.
[0046] The absorbance test results are shown in Table 1. The Qubit concentration of fecal DNA is 7.6-30.8 ng / μL, and the purity is OD260 / 280=1.69-2.09, OD260 / 230=0.86-1.54. Figure 1 As shown in Figure 1, 2, and 4 DNA are light yellow to brown in color, and 3 DNA is transparent. Figure 2 As shown, the main DNA band is obvious, but the electrophoresis lane is diffuse. The above results show that the structural integrity of fecal DNA is average, the purity OD260 / 230 is low, and some DNA samples have pigment residues, indicating that 1#, 2# and 4# DNA have more impurities, and 3# DNA is of average quality.
[0047] Table 1 Absorbance test results of fecal DNA extraction
[0048] Application Example 2: The 3# DNA obtained in Example 1 was used for PCR analysis of the sex identification of giant pandas. The number of target bands of the PCR product electrophoresis should be two, and the sex determination result should be male. The PCR premixes used in the PCR analysis were the control PCR premix, Example 1, Example 2, and Example 3; the amount of template DNA added was 20 ng; the primer combination was ZF1 and ZFX, and the primers were derived from: "Technical Regulations for the Establishment of Genetic Archives of Giant Panda Populations" (People's Republic of China Forestry Industry Standard LY / T 2896-2017), and the target fragment sizes were 210bp and 130bp respectively. The PCR reaction program was 37℃ 5min, 95℃ 10min; 95℃ 10s, 60℃ 10s, 72℃ 30s, 35 cycles; 72℃ 10min. Other experimental conditions were carried out in accordance with the "Technical Regulations for the Establishment of Genetic Archives of Giant Panda Populations".
[0049] The results are as follows Figure 3 As shown, the electrophoresis results show that the PCR product of the control PCR premix group has only one band, located around 130bp. In the electrophoresis results of Examples 1 to 3, the PCR product has two target fragments, with sizes around 210bp and 130bp, respectively. According to the above electrophoresis results, the judgment result of the control PCR premix group is female, which is inconsistent with theoretical expectations; the judgment results of Examples 1 to 3 are male, which is consistent with theoretical expectations.
[0050] In this application example, the amount of template DNA added is 20 ng / 20 μL reaction system, which is lower than the amount of template DNA added (250 ng / 20 μL reaction system) recommended in the "Technical Regulations for Establishing Genetic Archives of Giant Panda Populations". Therefore, when using the control PCR premix, the gender determination results of male individuals may be misjudged due to the small amount of template added. However, the determination results of Examples 1 to 3 are in line with theoretical expectations, indicating that the multiple anti-contamination PCR premixes of Examples 1 to 3 have a lower detection limit for templates and have better template detection performance than the control PCR premix.
[0051] Application Example 3: The 1# DNA obtained in Example 1 was used for PCR analysis of the sex identification of giant pandas to evaluate the tolerance of the multiple anti-contamination PCR premix to inhibitors in low-quality DNA (with pigment residue and OD260 / 230<1). The number of target bands in the electrophoresis of the PCR product should be two, and the sex determination result should be male. The PCR premixes used in the PCR analysis were the control PCR premix and Example 1; the template DNA addition amount was 20 ng; the primer combination was ZF1 and ZFX, and the target fragment sizes were 210bp and 130bp, respectively. The PCR reaction program was 37℃ 5min, 95℃ 10min; 95℃ 10s, 60℃ 10s, 72℃ 30s, 35 cycles; 72℃ 10min. Other experimental conditions were carried out in accordance with the "Technical Regulations for the Establishment of Genetic Archives of Giant Panda Populations".
[0052] The results are as follows Figure 4 As shown, in the electrophoresis results of Example 1, the PCR product has two target fragments, with sizes of about 210bp and 130bp respectively, but the brightness of the electrophoresis band of one of the PCR repeats is weak; no target band amplification is found in the control PCR premix group. The judgment result of Example 1 group is male, which is in line with theoretical expectations; the control PCR premix group fails to amplify. The above results show that the multiple anti-contamination PCR premix of Example 1 can tolerate various PCR inhibitors in low-quality fecal DNA to a certain extent, and has better inhibitor tolerance than the control PCR premix.
[0053] Application example 4: PCR analysis of the sex identification of giant pandas was performed on DNA from individual tissues and blood of male giant pandas (Qubit concentration 10.1-76.6 ng / μL, purity OD260 / 280=1.98-2.09, OD260 / 230=1.72-1.93). The number of target bands of PCR product electrophoresis should be two, and the sex determination result should be male. The PCR premixes used in the PCR analysis were respectively those in Example 1, Example 2, and Example 3; the amount of template DNA added was 4 ng; the primer combination was ZF1 and ZFX, and the target fragment sizes were 210 bp and 130 bp, respectively. The PCR reaction program was 37°C 5min, 95°C 10min; 95°C 10s, 60°C 10s, 72°C 30s, 35 cycles; 72°C 10min. Other experimental conditions were carried out in accordance with the "Technical Regulations for the Establishment of Genetic Archives of Giant Panda Populations".
[0054] The results are as follows Figure 5 As shown, in the electrophoresis results of Examples 1 to 3, all PCR products have two target fragments, with sizes of about 210 bp and 130 bp, respectively. According to the above electrophoresis results, the results of Examples 1 to 3 are male, which is consistent with theoretical expectations. This shows that the multiple anti-contamination PCR premix solutions of Examples 1 to 3 still have stable detection performance for tissue and blood-derived DNA.
[0055] Application Example 5: The PCR amplification product of Application Example 4 was diluted to 10 -6 concentration. The dilution was used to simulate aerosol pollutants to test the tolerance of the PCR premix to aerosol pollutants. The PCR premix was the control PCR premix and Example 1; no DNA was added to the PCR reaction system, only 2 μL of pollutants were added; the primer combination was ZF1 and ZFX, and the target fragment sizes were 210 bp and 130 bp, respectively. The PCR reaction procedure was 37°C 5 min, 95°C 10 min; 95°C 10s, 60°C 10s, 72°C 30s, 35 cycles; 72°C 10 min. Other experimental conditions were carried out in accordance with the "Technical Regulations for the Establishment of Genetic Archives of Giant Panda Populations".
[0056] The results are as follows Figure 6 As shown, the PCR product of the control PCR premix group has multiple bands, and non-specific amplification is obvious, while the PCR product of the Example 1 group has only a primer dimer band. The above results show that the multiple anti-contamination PCR premix of Example 1 has a certain tolerance to aerosol contaminants of PCR amplification products, and has better anti-contamination performance than the control PCR premix.
[0057] Application Example 6: The 1# DNA obtained in Example 1 was used for PCR analysis of the sex identification of giant pandas. The number of target bands in the electrophoresis of the PCR product should be two, and the sex determination result should be male. The PCR premixes used in the PCR analysis were respectively those in Example 1, Example 4, Example 5, Example 6 and Example 7; the amount of template DNA added was 20 ng; the primer combinations were ZF1 and ZFX, and the target fragment sizes were 210 bp and 130 bp, respectively. The PCR reaction procedure was 37°C 5 min, 95°C 10 min; 95°C 10 s, 60°C 10 s, 72°C 30 s, 35 cycles; 72°C 10 min. Other experimental conditions were carried out in accordance with the "Technical Regulations for the Establishment of Genetic Archives of Giant Panda Populations".
[0058] The results are as follows Figure 7 As shown, the electrophoresis results show that in the electrophoresis results of Example 1, Example 4, Example 5, Example 6 and Example 7, the PCR product has two target fragments, the sizes of which are around 210bp and 130bp respectively. According to the above electrophoresis results, the judgment results of Example 1, Example 4, Example 5, Example 6 and Example 7 are all male, which is in line with theoretical expectations.
[0059] In this application example, the amount of template DNA added is 20 ng / 20 μL reaction system, which is lower than the amount of template DNA added (250 ng / 20 μL reaction system) recommended in the Technical Regulations for Establishing Genetic Archives of Giant Panda Populations. The determination results of all groups are consistent with theoretical expectations, indicating that the multiple anti-contamination PCR premixes of Examples 1, 4, 5, 6 and 7 have a low detection limit for templates and stable template detection performance.
[0060] Application Example 7: The PCR amplification product of Application Example 4 was diluted to 10 -6 concentration. The dilution was used to simulate aerosol pollutants to test the tolerance of the PCR premix to aerosol pollutants. The PCR premixes were from Example 1 and Comparative Example 1 respectively; no DNA was added to the PCR reaction system, only 2 μL of pollutants were added; the primer combinations were ZF1 and ZFX, and the target fragment sizes were 210bp and 130bp, respectively. The PCR reaction procedure was 37°C 5min, 95°C 10min; 95°C 10s, 60°C 10s, 72°C 30s, 35 cycles; 72°C 10min. Other experimental conditions were carried out in accordance with the "Technical Regulations for the Establishment of Genetic Archives of Giant Panda Populations".
[0061] The results are as follows Figure 8As shown, the PCR product of Comparative Example 1 has obvious target bands, and the electrophoresis lane has serious diffusion phenomenon, and non-specific amplification is obvious; the PCR product of Example 1 group has only primer dimer bands. The above results show that the multiple anti-pollution PCR premix of Example 1 has a certain tolerance to aerosol pollutants of PCR amplification products, and has better anti-pollution performance than Comparative Example 1.
[0062] Application Example 8: The 1# DNA obtained in Example 1 was used for PCR analysis of the sex identification of giant pandas to evaluate the tolerance of the multiple anti-contamination PCR premix to inhibitors in low-quality DNA (with pigment residue and OD260 / 230<1). The number of target bands in the electrophoresis of the PCR product should be two, and the sex determination result should be male. The PCR premixes used in the PCR analysis were respectively Example 1 and Comparative Example 2; the amount of template DNA added was 20ng; the primer combinations were ZF1 and ZFX, and the target fragment sizes were 210bp and 130bp, respectively. The PCR reaction program was 37℃ 5min, 95℃ 10min; 95℃ 10s, 60℃ 10s, 72℃ 30s, 35 cycles; 72℃ 10min. Other experimental conditions were carried out in accordance with the "Technical Regulations for the Establishment of Genetic Archives of Giant Panda Populations".
[0063] The results are as follows Fig. 9 As shown, in the electrophoresis results of Example 1, the PCR product has two target fragments, with sizes of about 210 bp and 130 bp respectively; no target band amplification was found in Comparative Example 2. The judgment result of Example 1 was male, which was in line with theoretical expectations; amplification failed in Comparative Example 2. The above results show that the multiple anti-contamination PCR premix of Example 1 can tolerate various PCR inhibitors in low-quality fecal DNA to a certain extent, and has better inhibitor tolerance than Comparative Example 2.
[0064] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.
Claims
1. A multiplex anti-contamination PCR premix, characterized in that: The premix comprises, according to the final concentration, 1-5 U of DNA polymerase; 100-700 nM of substrate; 1-5 mM of MgCl2; 0.02-1 mg / mL of bovine serum albumin (BSA); 0.1%-1% V / V of surfactant; 0.1-3 mM of antioxidant; 10-30 mM of buffer; 10-60 mM of monovalent cation component; 0.1-4 mM of metal ion chelator; 0.5-3 U of DNA glycosylase; 0.01-3 M of stabilizer; 4%-20% V / V of amplification accelerator; and the solvent is water; wherein the substrate is at least one of dNTP, dATP, dTTP, dGTP, dCTP and dUTP; and the monovalent cation component is a compound that provides potassium ions and ammonium ions, respectively.
2. The PCR premix according to claim 1, characterized in that: The premix comprises, according to the final concentration, the following components: DNA polymerase, 1-2.5 U; substrate, 500-700 nM; MgCl2, 1-2.5 mM; bovine serum albumin (BSA), 0.025-0.05 mg / mL; surfactant, 0.3%-0.7% V / V; antioxidant, 0.5-2 mM; buffer, 10-20 mM; monovalent cation component, 20-50 mM; metal ion chelator, 0.5-2 mM; DNA glycosylase, 1-2 U; stabilizer, 1.2-2.0 M; amplification promoter, 4%-10% V / V; and the solvent is water.
3. The PCR premix according to claim 1 or 2, characterized in that: The DNA polymerase is at least one of Taq polymerase, Pfu polymerase, Taq Plus polymerase, HotStart Taq polymerase, PrimerStar HS DNA polymerase and AmpliTaqGold DNA polymerase.
4. The PCR premix according to any one of claims 1 to 3, characterized in that The surfactant is at least one of Tween 20, Tween 21, Tween 40, Tween 60, Tween 61, Tween 80, Span 20, Span 40, Span 60, Span 80, Span 65, Span 85, TritonX100 and NP40, preferably at least one of Tween 20, TritonX100 and NP40.
5. The PCR premix according to any one of claims 1 to 4, characterized in that: The antioxidant is at least one of dithiothreitol (DTT) and dithioerythritol (DTE).
6. The PCR premix according to any one of claims 1 to 5, characterized in that: The buffer is at least one of tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCL), phosphate buffered saline (PBS), 3-morpholinepropanesulfonic acid (MOPS) and 4-hydroxyethylpiperazineethanesulfonic acid (HEPES).
7. The PCR premix according to any one of claims 1 to 6, characterized in that In the monovalent cation component, the compound providing potassium ions is selected from one of potassium chloride, potassium sulfate, potassium nitrate, and potassium acetate, and the compound providing ammonium ions is selected from one of ammonium sulfate, ammonium chloride, ammonium nitrate, and ammonium acetate.
8. The PCR premix according to any one of claims 1 to 7, characterized in that: The metal ion chelating agent is at least one of ethylene glycol tetraacetic acid (EGTA) and ethylenediaminetetraacetic acid (EDTA).
9. The PCR premix according to any one of claims 1 to 8, characterized in that The DNA glycosylase is uracil DNA glycosylase or thermosensitive uracil DNA glycosylase.
10. The PCR premix according to any one of claims 1 to 9, characterized in that: The stabilizer is at least one of trehalose and betaine.
11. The PCR premix according to any one of claims 1 to 10, characterized in that: The amplification accelerator is at least one of PEG200-8000 and DMSO.
12. The PCR premix according to any one of claims 1 to 11, characterized in that: The PCR premix solution also includes a pH regulator to adjust the pH of the PCR premix solution to 7.5-8.5; preferably, the pH regulator is a sodium hydroxide solution.
13. The method for preparing a PCR premix according to any one of claims 1 to 12, characterized in that: The method comprises the following steps: preparing ingredients according to the components of the PCR premix solution and mixing them into a solution.
14. Use of the PCR premix according to any one of claims 1 to 12 in preparing a PCR kit.
15. The use according to claim 14, characterized in that: The PCR kit is used to identify the sex of giant pandas; preferably, the DNA sample used for PCR comes from the excrement, blood, hair, skin, cells and tissues of giant pandas.
16. A PCR kit for identifying the sex of a giant panda, characterized in that: The method comprises amplification primers and the PCR premix according to any one of claims 1 to 13.