PCR (Polymerase Chain Reaction) amplification method, kit for PCR amplification and application

Abstract

The invention provides a PCR (Polymerase Chain Reaction) amplification method, a kit for PCR amplification and application. The PCR amplification method comprises the following steps: adding a PCR amplification enhancer into a PCR amplification system, and the PCR amplification enhancer comprises guanidine salt. The problem that a reagent for enhancing PCR reaction in the prior art is narrow in application range can be solved, and the reagent is suitable for the field of PCR amplification.

Description

Technical Field

[0001] The present invention relates to the field of PCR amplification, and in particular, to a PCR amplification method, a kit for PCR amplification and an application thereof. Background Art

[0002] In the research on PCR, people have gradually found that some substances can be used as PCR additives to enhance the reaction. For example, in some reports, betaine and dimethyl sulfoxide can reduce the secondary structure of DNA, lower the annealing temperature, and facilitate PCR amplification. In other reports, trehalose and polyethylene glycol can also change the annealing temperature of primers and templates and enhance PCR amplification. However, the effects of traditional enhancers are not stable. They have a synergistic effect on certain PCRs within a certain concentration range, but have an inhibitory effect on PCR reactions in other cases or within other concentration ranges. Summary of the Invention

[0003] The main object of the present invention is to provide a PCR amplification method, a kit for PCR amplification and an application thereof, so as to solve the problem of narrow application range of reagents for enhancing PCR reactions in the prior art.

[0004] To achieve the above object, according to the first aspect of the present invention, there is provided a PCR amplification method, which includes: adding a PCR amplification enhancer to a PCR amplification system, and the PCR amplification enhancer includes guanidine salt.

[0005] Further, the guanidine salt includes guanidine hydrochloride and / or guanidine isothiocyanate; preferably, the concentration of the guanidine salt in the PCR amplification system is 15 - 75 mM.

[0006] Further, the PCR amplification system further contains any one or more of the following: DNA polymerase, PCR buffer, amplification template, dNTP, primer or fluorescent probe; preferably, the primer is a degenerate primer.

[0007] Further, the amplification template includes a biological sample, and the biological sample includes one or more of blood, plasma, serum, ascites, saliva, throat swab, nasal swab, cerebrospinal fluid, synovial fluid, pleural fluid, peritoneal fluid, semen, vaginal secretion, prostatic fluid or amniotic fluid; the amplification template contains a target amplification fragment, and the target amplification fragment is DNA or RNA.

[0008] Further, when the target amplification fragment is RNA, the PCR amplification method includes: mixing reverse transcriptase, guanidine salt, DNA polymerase, amplification template and primer to form a PCR amplification system, and performing reverse transcription on the PCR amplification system and then performing PCR amplification.

[0009] To achieve the above object, according to the second aspect of the present invention, a kit for PCR amplification is provided, which contains a PCR amplification enhancer, and the PCR amplification enhancer includes guanidine salt.

[0010] Furthermore, the guanidine salt includes guanidine hydrochloride and / or guanidine isothiocyanate.

[0011] Furthermore, the kit further includes any one or more of the following: DNA polymerase, PCR buffer, primer or dNTP; preferably, the primer is a degenerate primer.

[0012] Furthermore, the kit further includes reverse transcriptase; preferably, the kit further includes a fluorescent probe.

[0013] To achieve the above object, according to the third aspect of the present invention, there is provided an application of the above PCR amplification method or the above kit for PCR amplification in PCR amplification; preferably, the guanidine salt includes guanidine hydrochloride and / or guanidine isothiocyanate; preferably, the PCR amplification includes PCR amplification using degenerate primers, and more preferably real-time fluorescence quantitative PCR amplification.

[0014] Applying the technical solution of the present invention, by setting guanidine salt as a PCR amplification enhancer in the kit for PCR amplification, the PCR amplification reaction carried out using such a kit can enhance the PCR reaction and improve the sensitivity and amplification efficiency of PCR amplification. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The specification drawings forming a part of this application are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0016] Figure 1 Shows the nucleic acid amplification result diagram of feline herpesvirus according to Example 2 of the present invention.

[0017] Figure 2 Shows the result diagram of nucleic acid amplification of feline calicivirus using primers containing degenerate bases according to Example 2 of the present invention.

[0018] Figure 3 Shows the result diagram of nucleic acid amplification of feline calicivirus using primers without degenerate bases according to Example 2 of the present invention.

[0019] Figure 4 Shows the nucleic acid amplification result diagram of feline immunodeficiency virus according to Example 2 of the present invention.

[0020] Figure 5 Shows the amplification result diagram of the influence of guanidine hydrochloride on the sensitivity of feline calicivirus nucleic acid according to Example 4 of the present invention. Detailed implementation manners

[0021] It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments may be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0022] As mentioned in the background art, there are only a few kinds of PCR amplification enhancers in the prior art, and the effects are not stable. There are many restrictions on the reaction conditions and reaction systems, and it is difficult to meet the actual needs. The application of PCR amplification enhancers in different reaction systems needs to be reasonably selected through specific experiments, and the same enhancer is not applicable to all PCR reaction systems. In the present application, the inventor attempts to develop a new type of kit for PCR amplification, which contains a new type of PCR amplification enhancer - guanidine salt, which has the effect of enhancing PCR in different reaction conditions and / or different reaction systems. On this basis, the applicant proposes a series of protection schemes for the present application.

[0023] In the first typical implementation manner of the present application, a PCR amplification method is provided. The PCR amplification method includes: adding a PCR amplification enhancer to the PCR amplification system, and the PCR amplification enhancer includes guanidine salt.

[0024] In the prior art, guanidine salt, as one of the components in the commonly used lysis solution for nucleic acid extraction, is considered a PCR amplification inhibitor. Therefore, there is no inspiration in the prior art to add guanidine salt to the PCR amplification system additionally during the PCR amplification stage. Most of the guanidine salt introduced during the sample lysis stage is also removed along with the nucleic acid purification after lysis. Even if a very small amount of guanidine salt remains in the template, since the volume of the template in the PCR amplification system is small, the guanidine salt present in the PCR amplification system and its effects can be ignored. However, in the present invention, the inventor unexpectedly found that guanidine salt can be used as a PCR enhancer to enhance the PCR amplification efficiency. Therefore, in the above-mentioned PCR amplification method, guanidine salt can be used as a PCR amplification enhancer and used in the PCR amplification step to achieve the effect of promoting PCR amplification. Moreover, the above-mentioned PCR amplification enhancer has low cost and simple composition, which is convenient for industrial production and use.

[0025] PCR or PCR amplification in the present application both refer to polymerase chain reaction, that is, amplification carried out through denaturation, annealing, and extension cycles, rather than amplification carried out in the manner of LAMP (loop-mediated isothermal amplification). The reaction principles, enzymes, primers, etc. used in PCR and LAMP are completely different.

[0026] Using the above PCR amplification method, a guanidine salt for enhancing PCR amplification is introduced into the PCR amplification system to promote the PCR amplification efficiency.

[0027] In a preferred embodiment, the guanidine salt includes guanidine hydrochloride and / or guanidine isothiocyanate; preferably, the concentration of the guanidine salt in the PCR amplification system is 15 - 75 mM, including but not limited to 15 - 50 mM, 15 - 30 mM or 30 - 50 mM, and further including but not limited to 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 mM.

[0028] In a preferred embodiment, the PCR amplification system further contains any one or more of the following: DNA polymerase, PCR buffer, amplification template, dNTP, primer or fluorescent probe; preferably, the primer is a degenerate primer.

[0029] A degenerate primer is a mixture of primers containing degenerate bases. PCR using such degenerate primers can simultaneously amplify multiple target fragments by PCR. There are many viruses in nature. Viral genes are divided into DNA and RNA. Most viruses are mainly RNA, and RNA mutates relatively frequently, with partial base mutations in conserved regions. This results in the need to design degenerate bases to cover the primer probes designed based on the RNA genome sequence. Due to the presence of degenerate bases, it will affect the stability of the binding between the primer and the target sequence, often greatly reducing the sensitivity and specificity of PCR detection, especially fluorescence detection (RT-PCR). Especially for some complex samples, the impact on PCR amplification is greater.

[0030] In a preferred embodiment, the amplification template includes a biological sample, and the biological sample includes one or more of blood, plasma, serum, ascites, saliva, throat swab, nasal swab, cerebrospinal fluid, synovial fluid, pleural fluid, semen, vaginal secretion, prostatic fluid or amniotic fluid, etc.; the amplification template contains a target amplification fragment, and the target amplification fragment is DNA or RNA.

[0031] In the above PCR amplification method, the amplification template containing the target fragment to be amplified added to the PCR amplification system includes biological samples, including but not limited to samples directly derived from organisms such as blood, plasma, serum, ascites, saliva or throat swabs. The components in such samples are complex and will interfere with and inhibit the amplification during the PCR amplification process, affecting the sensitivity, accuracy and PCR efficiency of the PCR amplification. By adding the above-mentioned guanidine salt to the PCR amplification system, not only can the amplification of primers (especially degenerate primers containing degenerate bases) be enhanced, promoting the binding of the probe to the target fragment, but also the interference of the components in such biological samples on the amplification can be inhibited, ensuring the normal progress of the PCR reaction. The above activities of the guanidine salt act together with its usual activity for sample lysis to achieve efficient PCR amplification of biological samples.

[0032] In a preferred embodiment, when the target amplification fragment is RNA, the PCR amplification method includes: mixing reverse transcriptase, guanidine salt, DNA polymerase, amplification template and primers to form a PCR amplification system, and performing reverse transcription on the PCR amplification system and then performing PCR amplification.

[0033] When the target amplification fragment is RNA, in the above PCR amplification method, the reverse transcription reaction system and the PCR amplification system are mixed in the same reaction system. First, use reverse transcriptase to reverse transcribe RNA into cDNA, and then directly perform subsequent PCR. The guanidine salt does not affect the progress of the reverse transcription reaction. And the amplification template in the reaction system can be directly added to the reaction system in the form of a biological sample. The reaction is convenient and fast, without the need for multiple purification steps, which can greatly reduce the time, reagents and sample loss required for detection, and is convenient for practical application.

[0034] In the second typical embodiment of the present application, a kit for PCR amplification is provided. The kit contains a PCR amplification enhancer, and the PCR amplification enhancer includes guanidine salt.

[0035] In a preferred embodiment, the guanidine salt includes guanidine hydrochloride and / or guanidine isothiocyanate.

[0036] In a preferred embodiment, the kit further includes any one or more of the following: DNA polymerase, PCR buffer, primers or dNTPs; preferably, the primers are degenerate primers.

[0037] In a preferred embodiment, the kit further includes reverse transcriptase; preferably, the kit further includes a fluorescent probe.

[0038] A degenerate primer is a mixture of primers containing degenerate bases. PCR using such degenerate primers can simultaneously amplify multiple target fragments by PCR. Due to the widespread existence of codon degeneracy in organisms, the above degenerate primers are particularly suitable for amplifying DNA obtained by reverse transcription of RNA. By reasonably designing degenerate primers, information on cDNA in a sample can be obtained. Further, a fluorescence probe for real-time fluorescence quantitative PCR (RT-PCR) is also provided in the kit, which releases a fluorescence signal by binding to the product obtained by PCR amplification, realizing the quantification of the PCR amplification product.

[0039] In the PCR amplification process of the above kit, the guanidine salt can not only enhance the amplification of primers, especially degenerate primers containing degenerate bases, but also promote the binding of the probe to the target fragment (i.e., the amplification product). The use of the above guanidine salt in RT-PCR can also significantly reduce the Ct value and improve the detection sensitivity.

[0040] In the third typical embodiment of the present application, an application of the above kit for PCR amplification or the above PCR amplification method in PCR amplification is provided; preferably, the guanidine salt includes guanidine hydrochloride and / or guanidine isothiocyanate; preferably, the PCR amplification includes PCR amplification using degenerate primers, and more preferably real-time fluorescence quantitative PCR amplification.

[0041] The beneficial effects of the present application will be further explained in detail below with specific examples.

[0042] Example 1

[0043] The amplification system described above includes the following components:

[0044] (I) qPCR premix: The main components are 2×RT-qPCR Mix (Runpeng Biotech), M-MLV reverse transcriptase (200U / μL) (Fepeng Biotech, product number: MD028), and AK Taq enzyme (5U / μL) (Fepeng Biotech, product number: MDAD006);

[0045] (II) Primer and probe sequences

[0046] Feline herpesvirus primer and probe sequences (without degenerate bases):

[0047] FHV1-F: 5’-TAAATCTGGAAGGAACCCCTGA-3’ (SEQ ID NO: 1).

[0048] FHV1-R: 5’-GTGCATATCACATGCGGCTATAA-3’ (SEQ ID NO: 2).

[0049] FHV1-P: 5'-FAM-CCGCCGCCTTGGGAGCACT-BHQ1-3' (SEQ ID NO: 3).

[0050] Feline calicivirus primer and probe sequences (containing degenerate bases):

[0051] FCV-F: 5'-CYAATCAACATGTGGTAACCG-3' (SEQ ID NO: 4).

[0052] FCV-R: 5'-AGCACATCATATGCGGCYCTGA-3' (SEQ ID NO: 5).

[0053] FCV-P: 5'-ROX-CGGTGTTTGATYTGGCCTGGGCT-BHQ2-3' (SEQ ID NO: 6).

[0054] Feline calicivirus primer and probe sequences (without degenerate bases):

[0055] FCV-F2: 5'-CCAATCAACATGTGGTAACCG-3' (SEQ ID NO: 7).

[0056] FCV-R2: 5'-AGCACATCATATGCGGCTCTGA-3' (SEQ ID NO: 8).

[0057] FCV-P2: 5'-ROX-CGGTGTTTGATTTGGCCTGGGCT-BHQ2-3' (SEQ ID NO: 9). Feline immunodeficiency virus primer and probe sequences (containing degenerate bases):

[0058] FIV-F: 5'-ACATTCCAGAACAGCCYACT-3' (SEQ ID NO: 10).

[0059] FIV-R: 5'-TGTACTTCYAGTCKAGCTTC-3' (SEQ ID NO: 11).

[0060] FIV-P: 5'-ROX-TGGGCTAGCCAAGCTATTCCAGAYTTG-BHQ2--3' (SEQ ID NO: 12). The "Y" and "K" in the above sequences are both degenerate bases, where Y represents C or T, and K represents G or T.

[0061] FAM and ROX in the probe sequences represent fluorescent groups, and BHQ1 and BHQ2 represent quenching groups.

[0062] The amplification system is shown in Table 1.

[0063] Table 1

[0064]

[0065]

[0066] The added volume of guanidine hydrochloride / guanidine isothiocyanate is X μL, and it can be flexibly adjusted according to the required concentration of guanidine salt in the amplification system.

[0067] Reaction procedure:

[0068] Reverse transcription treatment is at 50 °C for 5 min; pre-denaturation treatment is at 95 °C for 1 min; denaturation treatment is at 95 °C for 5 s, annealing / fluorescence signal collection treatment is at 60 °C for 10 s, for 40 cycles.

[0069] Example 2 Exploration and verification of guanidine hydrochloride concentration

[0070] Select final concentrations of 0 mM, 15 mM, 30 mM, 50 mM, 75 mM, and 100 mM guanidine hydrochloride and add them to the amplification system. The sample is feline whole blood containing feline herpesvirus and feline calicivirus nucleic acids, and the final concentration of feline whole blood is 2.5%. There are two replicates for each treatment. For each system, add the primer-probe sets for feline herpesvirus, feline immunodeficiency virus, and feline calicivirus.

[0071] The RT-PCR results are shown in Table 2, Figure 1 and Figure 2 and Figure 3 as shown, Figure 1 is the result diagram of feline herpesvirus nucleic acid amplification, Figure 2 is the result diagram of feline calicivirus nucleic acid amplification using primers containing degenerate bases, Figure 3 is the result diagram of feline calicivirus nucleic acid amplification using primers without degenerate bases. The results show that: in the amplification system with primer-probe containing degenerate bases, for feline calicivirus, the Ct values of 15 - 75 mM guanidine hydrochloride are all smaller than those of 0 mM guanidine hydrochloride, indicating that it has a promoting effect on amplification, while the Ct value of 100 mM guanidine hydrochloride is larger than that of 0 mM guanidine hydrochloride, indicating that 100 mM guanidine hydrochloride has an inhibitory effect. In the amplification system without degenerate bases, the Ct values of feline herpesvirus with 15 - 75 mM guanidine hydrochloride decrease, and the Ct values of feline calicivirus with 15 - 75 mM guanidine hydrochloride also decrease. Therefore, 15 - 75 mM guanidine hydrochloride has a promoting effect on the amplification of primer-probe with or without degenerate bases.

[0072] Table 2 Results of exploration of guanidine hydrochloride concentration in 2.5% feline whole blood

[0073]

[0074]

[0075] In addition, amplification systems with final concentrations of 30 mM guanidine hydrochloride and 0 mM guanidine hydrochloride were selected to detect 2.5% feline whole blood samples containing feline immunodeficiency virus nucleic acid from 2 cases. The RT-PCR results are as Figure 4 shown. The results indicate that the Ct value of amplification with 30 mM guanidine hydrochloride is earlier than that with 0 mM guanidine hydrochloride. Therefore, the addition of 30 mM guanidine hydrochloride promotes the amplification of feline immunodeficiency virus (with degenerate base primers).

[0076] Example 3 Applicability of Guanidine Hydrochloride in Different Sample Types

[0077] Select a final concentration of 30 mM guanidine hydrochloride and add it to the amplification system. The samples contain feline herpes and feline calicivirus nucleic acids, and 2.5% feline whole blood, 2.5% ascites, 2.5% saliva, and 5% throat swabs are set respectively, with two replicates for each sample.

[0078] The RT-PCR results are shown in Table 3. The results indicate that in the experiment without adding guanidine hydrochloride, the Ct values of the feline calicivirus samples in 2.5% feline whole blood, 2.5% ascites, 2.5% saliva, and 5% throat swabs are all higher than those of the water samples, indicating that feline calicivirus samples from organisms have an inhibitory effect on PCR amplification. In the experiment with the addition of 30 mM guanidine hydrochloride, for the samples containing 2.5% feline whole blood, 2.5% ascites, 2.5% saliva, 5% throat swabs, and water, the Ct values of feline calicivirus amplification do not change, indicating that guanidine hydrochloride does not affect amplification and can even reduce or completely eliminate the inhibitory effect of the sample itself on PCR.

[0079] Table 3 Applicability of 30 mM Guanidine Hydrochloride to 2.5% Feline Whole Blood, 2.5% Ascites, 2.5% Saliva, 5% Throat Swabs, and Water

[0080]

[0081] Example 4 Influence of Guanidine Hydrochloride on Amplification Sensitivity

[0082] Select amplification systems with final concentrations of 30 mM guanidine hydrochloride and 0 mM guanidine hydrochloride to detect 2.5% feline whole blood samples containing feline calicivirus nucleic acid at a final concentration of 500 copies / mL (copies / mL), and repeat the detection 4 times. The probe sequences used are the primers containing degenerate bases shown in SEQ ID NO: 4 - SEQ ID NO: 6.

[0083] The RT-PCR results are as Figure 5 shown. The results indicate that 30 mM guanidine hydrochloride detects 100% of the feline calicivirus in 2.5% feline whole blood, and 0 mM guanidine hydrochloride detects 75% of the feline calicivirus in 2.5% feline whole blood. Therefore, the addition of 30 mM guanidine hydrochloride improves the sensitivity.

[0084] Example 5 Influence of Guanidine Isothiocyanate on the Amplification of Feline Calicivirus

[0085] Amplification systems with a final concentration of 50 mM guanidine isothiocyanate and without guanidine isothiocyanate were selected to detect 5% ascites, 2.5% whole cat blood, and pure nucleic acid samples containing feline calicivirus nucleic acid, and the detection was repeated 2 times. The probe sequences used were the primers containing degenerate bases shown in SEQ ID NO: 4 - SEQ ID NO: 6.

[0086] The RT-PCR results are shown in Table 4. The results indicate that: 50 mM guanidine isothiocyanate advances the Ct values for the detection of feline calicivirus in 5% ascites, 2.5% whole cat blood, and inhibitor-free (water) samples. Therefore, adding 50 mM guanidine isothiocyanate has a promoting effect on the amplification of feline calicivirus.

[0087] Table 4

[0088]

[0089] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: By using the above PCR amplification method and adding guanidinium salts as PCR amplification enhancers to the PCR amplification system, the negative impact of biological samples on PCR amplification can be reduced or even overcome. In the PCR reaction using degenerate primers, the binding stability between the degenerate primers and the target sequence can be improved, thereby increasing the PCR amplification efficiency. Further, in the RT-PCR reaction, it can also promote the binding of the fluorescent probe and the amplification product, reduce the Ct value, and improve the detection sensitivity.

[0090] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, various modifications and changes can be made to the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A PCR amplification method, characterized in that, The PCR amplification method includes: adding a PCR amplification enhancer to the PCR amplification system, and the PCR amplification enhancer includes guanidine salt.

2. The PCR amplification method according to claim 1, wherein The guanidine salt includes guanidine hydrochloride and / or guanidine isothiocyanate; Preferably, the concentration of the guanidine salt in the PCR amplification system is 15 - 75 mM.

3. The PCR amplification method according to claim 1 or 2, characterized in that, The PCR amplification system further contains any one or more of the following: DNA polymerase, PCR buffer, amplification template, dNTP, primer or fluorescent probe; Preferably, the primer is a degenerate primer.

4. The PCR amplification method according to claim 3, wherein, The amplification template includes a biological sample, and the biological sample includes one or more of blood, plasma, serum, ascites, saliva, throat swab, nasal swab, cerebrospinal fluid, synovial fluid, pleural fluid, peritoneal fluid, semen, vaginal secretion, prostatic fluid or amniotic fluid; The amplification template contains a target amplification fragment, and the target amplification fragment is DNA or RNA.

5. The PCR amplification method according to claim 4, wherein When the target amplification fragment is RNA, the PCR amplification method includes: mixing reverse transcriptase, the guanidine salt, the DNA polymerase, the amplification template and the primer to form the PCR amplification system, and performing reverse transcription on the PCR amplification system and then performing PCR amplification.

6. A kit for PCR amplification, characterized in that, The kit contains a PCR amplification enhancer, and the PCR amplification enhancer includes guanidine salt.

7. The kit according to claim 6, characterized in that, The guanidine salt includes guanidine hydrochloride and / or guanidine isothiocyanate.

8. The kit according to claim 6 or 7, characterized in that, The kit further includes any one or more of the following: DNA polymerase, PCR buffer, primer or dNTP; Preferably, the primer is a degenerate primer.

9. The kit according to claim 8, wherein The kit further includes reverse transcriptase; Preferably, the kit further includes a fluorescent probe.

10. Use of the PCR amplification method according to any one of claims 1 to 5 or the kit for PCR amplification according to any one of claims 6 to 9 in PCR amplification; Preferably, the guanidine salt includes guanidine hydrochloride and / or guanidine isothiocyanate; Preferably, the PCR amplification includes PCR amplification using degenerate primers, and more preferably real-time fluorescence quantitative PCR amplification.

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