QPCR (quantitative polymerase chain reaction) detection system suitable for throat swab nucleic acid direct expansion and application of qPCR detection system

Abstract

The invention relates to the technical field of nucleic acid detection, and particularly discloses a qPCR detection system suitable for throat swab nucleic acid direct expansion and application thereof.The detection system comprises a weakly alkaline buffer solution, DNA polymerase, dNTPs, metal salt and a reaction enhancer. The throat swab sample is directly used for qPCR amplification, the operation process is greatly simplified, the detection time and cost are reduced, the system is suitable for rapid detection and large-scale screening, and the system has excellent inhibition resistance and can effectively resist common inhibitors in the throat swab sample, so that the amplification efficiency and the detection sensitivity are ensured, and the detection cost is reduced. According to the system, the influence of an inhibitor on qPCR reaction is avoided, the occurrence rate of false negative results is reduced, the accuracy of detection results is ensured, and the detection system provides a faster, more accurate and more efficient solution for throat swab nucleic acid detection.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and in particular to a qPCR detection system suitable for direct amplification of throat swab nucleic acid and a use thereof. Background Art

[0002] As a highly sensitive method for detecting viruses or pathogens, nucleic acid amplification testing plays a key role in the diagnosis of infectious diseases. Quantitative PCR (qPCR) technology is one of the most commonly used methods in nucleic acid amplification testing. Among the many nucleic acid test samples, throat swab samples have become the most common choice because of their convenient sampling, high patient acceptance, and relative safety compared to sputum and other samples, and they are not prone to secondary infection of medical staff. Most respiratory pathogens, including influenza viruses, coronaviruses, etc., first replicate and spread in the upper respiratory tract. Therefore, throat swab samples can effectively capture the nucleic acids of these pathogens, improving the sensitivity and accuracy of the test. In the traditional qPCR detection process, complex sample processing steps are usually required, including nucleic acid extraction and purification operations, which is not only time-consuming, but also increases the laboratory's equipment requirements and detection costs.

[0003] In order to solve the complexity of the traditional qPCR detection process, nucleic acid direct amplification technology came into being. The throat swab nucleic acid direct amplification technology omits the step of nucleic acid extraction in traditional detection, and directly adds the sample to the qPCR reaction system for amplification detection. This method greatly improves the detection efficiency, shortens the overall process, reduces the demand for laboratory equipment and detection costs, and is particularly suitable for occasions with rapid detection needs and large-scale screening. However, there are often inhibitors in throat swab samples that affect subsequent qPCR detection. Common inhibitors include: 1) Mucus protein: There is a large amount of mucus in the throat, and the protein in the mucus may non-specifically bind to the enzyme in the qPCR reaction, inhibit the activity of the enzyme, and reduce the amplification efficiency. 2) Host cell DNA and RNA: Throat swab samples contain not only pathogen nucleic acids, but also a large amount of DNA and RNA from human cells. These non-target nucleic acids compete with primers for binding, interfering with the specificity and accuracy of amplification. 3) Phenolic compounds: Some sampling swabs may bring in phenols or other chemical inhibitors during sample collection. These substances may inhibit the activity of key enzymes in the qPCR reaction, resulting in failure or incomplete amplification reaction. The presence of these inhibitors makes nucleic acid direct amplification technology simpler and faster in operation, but the reliability of the test results may be interfered with, resulting in reduced sensitivity and even false negative results. Existing qPCR quantitative detection kits are often interfered with by inhibitors in throat swab samples and cannot provide accurate quantitative results for direct amplification of throat swab samples. Therefore, the development of a qPCR quantitative detection system with good anti-inhibition ability is crucial to improving the sensitivity and accuracy of sample detection. Summary of the invention

[0004] In order to solve the problems mentioned in the background technology, the purpose of the present invention is to provide a qPCR detection system suitable for direct amplification of throat swab nucleic acid.

[0005] The detection system comprises weak alkaline buffer, DNA polymerase, dNTPs, metal salt and reaction enhancer.

[0006] Furthermore, the weak alkaline buffer is selected from one or more of TES, Tricine, Tris, TAPS, HEPES, HEPPSO, and HEPPS, and the concentration of the weak alkaline buffer in the detection system is 10-100 mM.

[0007] Furthermore, the DNA polymerase is Taq DNA polymerase or Tth DNA polymerase, and the concentration of the DNA polymerase in the detection system is 0.1-5 U / μL.

[0008] Furthermore, the concentration of the dNTPs in the detection system is 0.1-0.5 mM.

[0009] Furthermore, the metal salt is selected from one or more of the following: MgCl2, with a concentration of 1-5 mM in the detection system; MgSO4, with a concentration of 1-5 mM in the detection system; KCl, with a concentration of 20-120 mM in the detection system; KOAc, with a concentration of 20-120 mM in the detection system; (NH4)2SO4, with a concentration of 2-50 mM in the detection system; and TMAC, with a concentration of 5-100 mM in the detection system.

[0010] Furthermore, the reaction enhancer is selected from one or more of the following: BSA, the concentration in the detection system is 5-300 μg / mL; SSB, the concentration in the detection system is 5-200 μg / mL; gelatin, the concentration in the detection system is 0.01-0.05%; DTT, the concentration in the detection system is 1-10 mM; DMSO, the concentration in the detection system is 1-5%; trehalose, the concentration in the detection system is 1-10%; mannitol, the concentration in the detection system is 1-10%; betaine, the concentration in the detection system is 0.1-1M; L-carnitine, the concentration in the detection system is 1-10%; Triton X-100, the concentration in the detection system is 0.01-0.1%; Tween 80, the concentration in the detection system is 0.01-0.1%; NP-40, the concentration in the detection system is 0.01-0.1%.

[0011] Furthermore, the detection system also includes dUTP and UDG, the concentration of dUTP in the detection system is 0.2-1 mM; the concentration of UDG in the detection system is 0.01-0.1 U / μL.

[0012] The application of the detection system in the field of nucleic acid detection is also within the protection scope of the present invention.

[0013] The beneficial effects of the present invention are as follows: the detection system provided by the present invention does not require complicated nucleic acid extraction steps, and directly uses throat swab samples for qPCR amplification, which greatly simplifies the operation process, reduces detection time and cost, and is suitable for rapid detection and large-scale screening. The system has excellent anti-inhibition ability and can effectively resist the inhibition of qPCR amplification by common inhibitors in throat swab samples, such as mucus proteins and host DNA, thereby ensuring amplification efficiency and detection sensitivity. This invention avoids the influence of inhibitors on qPCR reactions, reduces the incidence of false negative results, and ensures the accuracy of the test results. In addition, due to the optimized design of the detection system, the method can complete nucleic acid detection in a short time, greatly improving the detection efficiency. The invention provides a faster, more accurate and more efficient solution for throat swab nucleic acid detection. DETAILED DESCRIPTION

[0014] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention belong to the protection scope of the present invention.

[0015] Example 1

[0016] Ten people were randomly selected for throat swab sampling. After sampling, the samples were combined into swab preservation solution and ultrasonically treated for 2 minutes to fully lyse the cells in the sample. In order to verify the inhibitory effect of throat swab samples on qPCR amplification, the templates were selected to add A and B flow plasmids to throat swab samples and A and B flow plasmids without adding throat swab samples for amplification comparison test. Competitive qPCR reagents were tested simultaneously.

[0017] The 2x qPCR premix system used in the test is shown in Table 1:

[0018] Table 1. 2x qPCR premix system Mix 1-3

[0019]

[0020] The qPCR amplification reaction system is shown in Table 2:

[0021] Table 2. qPCR amplification reaction system

[0022]

[0023] The qPCR reaction program is shown in Table 3:

[0024] Table 3. qPCR reaction program

[0025]

[0026]

[0027] *NoCt indicates unsuccessful amplification.

[0028] The results showed that Mix 1-3 exhibited better inhibition ability against throat swab samples than competing reagents, among which the amplification efficiency of Mix 2 and Mix 3 was slightly higher than that of Mix 1.

[0029] Example 2

[0030] Based on the results of Example 1, the formula system was further optimized, and a new 2x qPCR premix system Mix4-6 was designed. The specific components are shown in Table 5, and the template in Example 1 was used for further screening:

[0031] Table 5. 2x qPCR premix system Mix 4-6

[0032]

[0033] The qPCR amplification reaction system and qPCR reaction procedure were the same as described in Example 1.

[0034] The test results are shown in Table 6:

[0035] Table 6. Ct values ​​of different qPCR systems (Mix 4-6)

[0036]

[0037]

[0038] The results showed that Mix 4-6 exhibited better inhibition ability against throat swab samples than competing reagents. Compared with Mix 1-3, the amplification efficiency of Mix 4 and Mix 6 was further improved after system optimization.

[0039] Example 3

[0040] Based on the results of Example 2, a syncytial plasmid template was further introduced to test the amplification efficiency of Mix 4 and Mix 6 for triple targets. The amplification system is shown in Table 7:

[0041] Table 7. qPCR amplification reaction system

[0042]

[0043] The qPCR reaction procedure was the same as that in Example 1.

[0044] The test results are shown in Table 8:

[0045] Table 8. Ct values ​​of triple qPCR system amplification

[0046]

[0047] Example 4

[0048] The Mix 4 and Mix 6 systems in the above example were stored at 37°C for 14 days. The stored systems were used to amplify the same template according to the method in Example 2, and the test results were compared with those of the freshly prepared Mix 4 and Mix 6 systems. The results are shown in Table 9. Both the Mix 4 and Mix 6 systems can be stably stored at 37°C for at least 14 days.

[0049] Table 9. Accelerated stability of qPCR system (Mix 4, 6)

[0050]

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. All modifications, equivalent substitutions, improvements, etc. within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A qPCR detection system suitable for direct amplification of throat swab nucleic acid, characterized in that: It includes weak alkaline buffer, DNA polymerase, dNTPs, metal salts and reaction enhancers.

2. A qPCR detection system suitable for direct amplification of throat swab nucleic acid according to claim 1, characterized in that: The weak alkaline buffer is selected from one or more of TES, Tricine, Tris, TAPS, HEPES, HEPPSO, and HEPPS, and the concentration of the weak alkaline buffer in the detection system is 10-100 mM.

3. A qPCR detection system suitable for direct amplification of throat swab nucleic acid according to claim 1, characterized in that: The DNA polymerase is Taq DNA polymerase or Tth DNA polymerase, and the concentration of the DNA polymerase in the detection system is 0.1-5 U / μL.

4. A qPCR detection system suitable for direct amplification of throat swab nucleic acid according to claim 1, characterized in that: The concentration of the dNTPs in the detection system is 0.1-0.5 mM.

5. A qPCR detection system suitable for direct amplification of throat swab nucleic acid according to claim 1, characterized in that: The metal salt is selected from one or more of the following: MgCl2, with a concentration of 1-5mM in the detection system; MgSO4, with a concentration of 1-5mM in the detection system; KCl, with a concentration of 20-120mM in the detection system; KOAc, with a concentration of 20-120mM in the detection system; (NH4)2SO4, with a concentration of 2-50mM in the detection system; and TMAC, with a concentration of 5-100mM in the detection system.

6. A qPCR detection system suitable for direct amplification of throat swab nucleic acid according to claim 1, characterized in that: The reaction enhancer is selected from one or more of the following: BSA, the concentration in the detection system is 5-300 μg / mL; SSB, the concentration in the detection system is 5-200 μg / mL; gelatin, the concentration in the detection system is 0.01-0.05%; DTT, the concentration in the detection system is 1-10 mM; DMSO, the concentration in the detection system is 1-5%; trehalose, the concentration in the detection system is 1-10%; mannitol, the concentration in the detection system is 1-10%; betaine, the concentration in the detection system is 0.1-1M; L-carnitine, the concentration in the detection system is 1-10%; Triton X-100, the concentration in the detection system is 0.01-0.1%; Tween 80, the concentration in the detection system is 0.01-0.1%; NP-40, the concentration in the detection system is 0.01-0.1%.

7. A qPCR detection system suitable for direct amplification of throat swab nucleic acid according to claim 1, characterized in that: The detection system also includes dUTP and UDG, wherein the concentration of dUTP in the detection system is 0.2-1 mM; the concentration of UDG in the detection system is 0.01-0.1 U / μL.

8. Application of the detection system according to any one of claims 1 to 7 in the field of nucleic acid detection.

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