A method for detecting and neutralizing nucleic acid in a sample

Abstract

The present invention provides a sample nucleic acid neutralization and detection method, the method comprising: extracting nucleic acid in a sample using a nucleic acid extracting solution to obtain a sample nucleic acid solution; mixing the obtained sample nucleic acid solution with a PCR reaction solution to obtain a mixed solution, performing an amplification reaction using the mixed solution to detect nucleic acid in the sample; the pH value of the PCR reaction solution is lower than that of the nucleic acid extracting solution. The present invention uses the PCR reaction solution to neutralize the extracted sample nucleic acid instead of neutralizing it during nucleic acid extraction, which can effectively improve the nucleic acid extraction efficiency and PCR amplification efficiency, and significantly improve the accuracy and sensitivity of sample nucleic acid detection.

Description

[0001] This application is a divisional application entitled A sample nucleic acid detection kit, reagents and their applications, with application number 201710291332.3. Technical Field

[0002] The invention belongs to the field of biological science and biotechnology, and particularly relates to a method for detecting sample nucleic acid and a method for neutralizing sample nucleic acid. Background Art

[0003] With the rapid development of molecular biology technology, it has penetrated into various fields such as biology, medicine, botany, genetics and zoology. As a major breakthrough in molecular biology technology, polymerase chain reaction (PCR) technology has the characteristics of high sensitivity, high specificity, time saving and rapidity. It has been widely used in human and animal disease diagnosis (such as prenatal diagnosis, newborn screening and genetic metabolic disease detection), forensic investigation, kinship analysis, seed purity identification, molecular marker-assisted breeding, gene positioning and genetically modified organism detection, etc.

[0004] The use of PCR technology for detection mainly involves three aspects: nucleic acid extraction, nucleic acid amplification, and amplification product detection. The quality and quantity of nucleic acid extraction usually directly determine the success or failure of the PCR reaction. Therefore, a large number of PCR-based test methods (such as fluorescent PCR, DNA sequencing, and RNA sequencing, etc.) require the extraction and purification of DNA and / or RNA nucleic acid molecules in biological samples (such as serum, plasma, whole blood, urine, vaginal secretions, saliva, feces, cotton swabs, epithelial exfoliated cells, fresh tissues, and paraffin tissue sections, etc.) before amplification.

[0005] For a long time, the extraction and purification of nucleic acids in biological samples has been a time-consuming and tedious process, which has seriously slowed down the detection speed. Especially for extremely small amounts of biological samples, the low recovery rate of conventional nucleic acid extraction methods for trace DNA in samples is the main reason for the poor subsequent PCR amplification effect. Therefore, establishing a rapid DNA extraction method is crucial to improving the efficiency of PCR detection.

[0006] According to the different properties of nucleic acids, there are many extraction methods. One of the more commonly used methods is the boiling method based on Chelex-100, namely the Chelex-100 boiling method. In this boiling method, Chelex-100 is a chemical ion chelating resin composed of styrene and styrene diethylene copolymer. Its suspension in an alkaline environment (pH 10-14) and 100°C can cause cell membrane rupture and release DNA from the cell nucleus after denaturation. In addition, Chelex-100 can highly selectively bind to multivalent cations, remove multivalent metal ions in samples and buffers, and avoid nucleic acid template degradation during boiling. In addition, it can effectively remove non-nucleic acid organic matter, with the advantages of economy, simplicity, and high efficiency, which can reduce the loss of nucleic acid during the extraction process. It has been widely used in the extraction of nucleic acid from forensic evidence materials such as trace blood, tissue plaques, semen stains, hair and bones.

[0007] The Chelex-100 boiling method is mainly divided into three steps: biological sample concentration; adding lysis solution and boiling at high temperature; high-speed centrifugation, and taking the supernatant, which is the extracted nucleic acid solution. In the boiling method, the lysis solution contains NaOH or KOH in addition to Chelex-100, so as to ensure that its pH value is maintained at 10-14. This also causes the extracted nucleic acid solution to maintain a strong alkalinity. If this strong alkalinity is not neutralized, it will be directly mixed with the PCR reaction solution for amplification reaction, which will lead to low PCR amplification reaction efficiency. In order to solve this problem, Chinese patent application CN201110295395.9 adds 200mM Tris-HCl buffer (pH 6.8-7.5) to neutralize after adding lysis solution for high-temperature boiling, thereby reducing the pH value in the extracted nucleic acid solution. However, when using this method to extract nucleic acids from biological samples, it is necessary to manually add neutralizing solution for neutralization, and the operation steps are somewhat cumbersome. If the operator forgets to add it, it will reduce the PCR amplification efficiency and affect the test results.

[0008] In addition, another commonly used method for extracting nucleic acids is alkaline lysis. Chinese patent application CN201210242862.6 uses a strong alkaline solution (such as NaOH or KOH solution) to lyse cells in a biological sample at high temperature to release nucleic acids, then adds an acidic buffer for neutralization, centrifuges, and obtains an extracted nucleic acid solution. Similarly, when using this method to extract nucleic acids from biological samples, it is necessary to manually add a neutralizing solution for neutralization. The operation steps are somewhat cumbersome. If the operator forgets to add it, it will reduce the PCR amplification efficiency and affect the test results.

[0009] Regardless of whether the Chelex-100 boiling method or the alkaline lysis method is used, there is always a problem when the nucleic acid in the sample is extracted and detected using the existing technology: taking the detection of HBV DNA that may exist in clinical blood or plasma samples as an example, the nucleic acid in the clinical blood or plasma sample is extracted using a strongly alkaline nucleic acid extraction solution to obtain an extracted nucleic acid solution, and then 2 to 5 μl of the extracted nucleic acid solution is added to a PCR reaction solution of about 35 μl for mixing. This is because the volume of the extracted nucleic acid solution added is small and has little effect on the mixed PCR reaction solution. However, when the volume of the extracted nucleic acid solution added is large, such as adding 20 μl of the extracted nucleic acid solution to 20 μl of the PCR reaction solution for mixing, although the total volume is still 40 μl, the volume ratio of the extracted nucleic acid solution increases significantly, which affects the mixed PCR reaction solution, resulting in a high pH value, thereby affecting the subsequent PCR amplification reaction efficiency and reducing the detection sensitivity and accuracy of the sample nucleic acid.

[0010] In addition, after the nucleic acid in the sample is extracted using a conventional weak alkaline nucleic acid lysis solution, when the volume of the extracted nucleic acid solution added to the PCR reaction solution is small (usually 2 to 5 μl), the nucleic acid in the sample can be detected, but the detection sensitivity is low; however, when the volume of the extracted nucleic acid solution added is large, such as adding 20 μl of the extracted nucleic acid solution to 20 μl of the PCR reaction solution for mixing, the weak alkaline nucleic acid lysis solution has a poor efficiency in extracting the sample nucleic acid, and the large volume of nucleic acid solution added is likely to contain more impurities and PCR amplification reaction inhibitors, which will cause the PCR amplification reaction to often fail to proceed normally, or although the PCR amplification reaction can be carried out, the detection sensitivity and accuracy of the sample nucleic acid will be reduced. Summary of the invention

[0011] In view of the deficiencies in the prior art, the present invention provides a reagent, method and kit for detecting sample nucleic acid. The reagent, method and kit all utilize a strongly alkaline nucleic acid extraction solution to extract nucleic acid from a sample, then add the extracted sample nucleic acid to a PCR reaction solution for neutralization, and simultaneously perform nucleic acid amplification and detection. In this way, there is no need to add a step of neutralizing the extracted sample nucleic acid during sample nucleic acid extraction, thereby greatly saving detection time, and also facilitating semi-automatic or fully automated processing, thereby improving PCR amplification reaction efficiency, increasing the sensitivity of sample nucleic acid detection and improving the accuracy of nucleic acid detection.

[0012] Because the components of samples such as serum, plasma, whole blood, sputum, urine, reproductive tract secretions, tears, feces, cerebrospinal fluid, fresh tissue, and paraffin tissue sections are very complex, direct detection of nucleic acids in samples will be interfered by other components. Therefore, some samples need to be pre-processed and preserved, and then the extracted sample nucleic acids need to be tested.

[0013] Different samples have different pretreatment methods. For example, whole blood contains red blood cells, so when extracting nucleic acid, it can be treated with a common red blood cell lysing solution, and then the nucleic acid extraction solution of the present invention is used to extract the sample nucleic acid; sputum contains a large amount of mucin and impurities, so when extracting nucleic acid, the sample is pretreated, usually using NaOH liquefaction, sodium citrate, 0.5% N-acetyl-L-cysteine ​​(NALC) or dithiothreitol (DTT) to remove mucin, and then the nucleic acid extraction solution of the present invention is used to extract the sample nucleic acid; the pretreatment method of fresh tissue is to first wash it with physiological saline, then mash or cut it into pieces, add proteinase K to digest it, and then use the nucleic acid extraction solution of the present invention to extract the sample nucleic acid; the pretreatment method of paraffin tissue sections is to first dewax with a dewaxing agent such as xylene, add proteinase K to digest it, and then use the nucleic acid extraction solution of the present invention to extract the sample nucleic acid.

[0014] The invention provides a sample nucleic acid detection reagent, which comprises a nucleic acid extract and a PCR reaction solution, wherein the nucleic acid extract comprises 10mM to 100mM of alkali metal hydroxide, 1mM to 25mM of Tris-HCl, 2% to 5% (w / v) of Chelex-100, and 0.1% to 0.4% (w / v) of EDTA, and the pH value of the nucleic acid extract is 10.5 to 12.5; the PCR reaction solution comprises 5mM to 75mM of Tris-HCl, and the pH value of the PCR reaction solution is 8.0 to 9.0.

[0015] Furthermore, the alkali metal hydroxide is NaOH or KOH.

[0016] Furthermore, the sample nucleic acid detection reagent also includes a concentrated solution.

[0017] Furthermore, the concentrated solution comprises 2.5% to 4.5% (w / w) of NaCl and 10% to 13% (w / v) of PEG6000.

[0018] Furthermore, the pH value of the PCR reaction solution is 8.1-8.7.

[0019] Furthermore, the pH value of the PCR reaction solution is 8.1-8.5.

[0020] Furthermore, the pH value of the PCR reaction solution is 8.1.

[0021] Furthermore, the sample nucleic acid detection reagent is used in detecting HBV nucleic acid.

[0022] Furthermore, the sample nucleic acid includes HBV, human B27 gene, HPV 6 / 11, TB or HCMV nucleic acid.

[0023] Furthermore, the sample nucleic acid includes HBV nucleic acid; in the nucleic acid extract, the concentration of alkali metal hydroxide is 25mM, the concentration of Tris-HCl is 1mM, the concentration of Chelex-100 is 2% (w / v), the concentration of EDTA is 0.1% (w / v), and the pH value of the nucleic acid extract is 11.5; in the PCR reaction solution, the concentration of Tris-HCl is 10mM, and the pH value of the PCR reaction solution is 8.1.

[0024] The present invention also provides a sample nucleic acid detection method, which comprises: (1) extracting nucleic acid in a sample using a nucleic acid extracting solution to obtain a sample nucleic acid solution; (2) mixing the sample nucleic acid solution obtained in (1) with a PCR reaction solution to obtain a mixed solution, and using the mixed solution to perform an amplification reaction to detect the nucleic acid in the sample, wherein the nucleic acid extracting solution comprises 10mM to 100mM alkali metal hydroxide, 1mM to 25mM Tris-HCl, 2% to 5% (w / v) Chelex-100, and 0.1% to 0.4% (w / v) EDTA, and the pH value of the nucleic acid extracting solution is 10.5 to 12.5; the PCR reaction solution comprises 5mM to 75mM Tris-HCl, and the pH value of the PCR reaction solution is 8.0 to 9.0; and the pH value of the mixed solution is 8.0 to 9.0.

[0025] Furthermore, the alkali metal hydroxide is NaOH or KOH.

[0026] Furthermore, before step (1), the sample is concentrated using a concentration solution.

[0027] Furthermore, the concentrated solution comprises 2.5% to 4.5% (w / w) of NaCl and 10% to 13% (w / v) of PEG6000.

[0028] Furthermore, the pH value of the PCR reaction solution is 8.1-8.7.

[0029] Furthermore, the pH value of the PCR reaction solution is 8.1-8.5.

[0030] Furthermore, the pH value of the PCR reaction solution is 8.1.

[0031] Furthermore, the sample nucleic acid includes HBV, human B27 gene, HPV 6 / 11, TB or HCMV nucleic acid.

[0032] Furthermore, the sample nucleic acid includes HBV nucleic acid; in the nucleic acid extract, the concentration of alkali metal hydroxide is 25mM, the concentration of Tris-HCl is 1mM, the concentration of Chelex-100 is 2% (w / v), the concentration of EDTA is 0.1% (w / v), and the pH value of the nucleic acid extract is 11.5; in the PCR reaction solution, the concentration of Tris-HCl is 10mM, and the pH value of the PCR reaction solution is 8.1.

[0033] The present invention also provides a sample nucleic acid detection kit, which includes a nucleic acid extract and a PCR reaction solution, wherein the nucleic acid extract includes 10mM to 100mM alkali metal hydroxide, 1mM to 25mM Tris-HCl, 2% to 5% (w / v) Chelex-100, and 0.1% to 0.4% (w / v) EDTA, and the pH value of the nucleic acid extract is 10.5 to 12.5; the PCR reaction solution includes 5mM to 75mM Tris-HCl, and the pH value of the PCR reaction solution is 8.0 to 9.0.

[0034] Furthermore, the alkali metal hydroxide is NaOH or KOH.

[0035] Furthermore, the nucleic acid detection kit also includes a concentrated solution.

[0036] Furthermore, the concentrated solution comprises 2.5% to 4.5% (w / w) of NaCl and 10% to 13% (w / v) of PEG6000.

[0037] Furthermore, the pH value of the PCR reaction solution is 8.1-8.7.

[0038] Furthermore, the pH value of the PCR reaction solution is 8.1-8.5.

[0039] Furthermore, the pH value of the PCR reaction solution is 8.1.

[0040] Furthermore, the sample nucleic acid includes HBV, human B27 gene, HPV 6 / 11, TB or HCMV nucleic acid.

[0041] Furthermore, the sample nucleic acid includes HBV nucleic acid; in the nucleic acid extract, the concentration of alkali metal hydroxide is 25mM, the concentration of Tris-HCl is 1mM, the concentration of Chelex-100 is 2% (w / v), the concentration of EDTA is 0.1% (w / v), and the pH value of the nucleic acid extract is 11.5; in the PCR reaction solution, the concentration of Tris-HCl is 10mM, and the pH value of the PCR reaction solution is 8.1.

[0042] The present invention also provides the use of the nucleic acid detection kit in detecting HBV, human B27 gene, HPV type 6 / 11, Mycobacterium tuberculosis or human cytomegalovirus in a sample.

[0043] The present invention also provides a method for neutralizing sample nucleic acid, comprising: (1) extracting nucleic acid in a sample using a nucleic acid extracting solution at a pH value of 10.5 to 12.5 to obtain a sample nucleic acid solution; (2) adding the sample nucleic acid solution obtained in (1) to a PCR reaction solution for neutralization; the PCR reaction solution having a lower pH value than that of the nucleic acid extracting solution.

[0044] Furthermore, the pH value of the PCR reaction solution is 8.0-9.0.

[0045] Furthermore, the nucleic acid extract comprises 10mM to 100mM alkali metal hydroxide, 1mM to 25mM Tris-HCl, 2% to 5% (w / v) Chelex-100, and 0.1% to 0.4% (w / v) EDTA; the PCR reaction solution comprises one of Tris-HCl, Tris-Acetate or Tris-Borate, and the concentration thereof is 5mM to 75mM. The pH value of the nucleic acid extract is 10.5 to 12.5.

[0046] Beneficial effects: first, a certain amount of sample is added to a strongly alkaline nucleic acid extraction solution to extract the nucleic acid in the sample, and then the extracted sample nucleic acid solution is added to a PCR reaction solution (whose pH value is lower than the pH value in the nucleic acid extraction solution) for neutralization. At the same time, the sample nucleic acid can be amplified and detected. In this way, the step of neutralizing the extracted sample nucleic acid solution during sample nucleic acid extraction can be omitted, thereby effectively saving detection time, reducing the chance of increased manual errors due to numerous steps, facilitating semi-automatic or fully automated processing, and this will also improve the PCR amplification reaction efficiency of the sample nucleic acid, and increase the detection sensitivity and accuracy of the sample nucleic acid. Taking the detection of HBV DNA that may exist in clinical blood or plasma samples as an example, the present invention improves the nucleic acid extract and PCR reaction solution in the prior art, uses the nucleic acid extract of the present invention (pH value is 10.5-12.5) to extract nucleic acid in clinical blood or plasma samples, obtains an extracted sample nucleic acid solution, and adds the extracted sample nucleic acid solution to the PCR reaction solution of the present invention (pH value is 8.0-9.0) for mixing. At this time, whether the added extracted sample nucleic acid solution is 2-5μl or 20μl, it will not affect the pH value of the mixed PCR reaction solution, that is, it will still remain in the appropriate pH range, so as not to affect the subsequent PCR amplification reaction efficiency, and at the same time, it will increase the detection sensitivity and accuracy of the sample nucleic acid. In addition, compared with the use of conventional weak alkaline nucleic acid lysing solution to extract nucleic acid in the sample, the use of the nucleic acid extract of the present invention can improve the extraction efficiency of the sample nucleic acid and reduce the impurities and PCR reaction inhibitors that may exist in the extracted sample nucleic acid solution. Moreover, when the nucleic acid extract of the present invention is used in combination with the PCR reaction solution of the present invention, when the volume of the extracted sample nucleic acid solution added to the PCR reaction solution is small (usually 2 to 5 μl), the nucleic acid in the sample can be detected with high detection sensitivity; when the volume of the extracted sample nucleic acid solution added is large, such as adding 20 μl of the extracted sample nucleic acid solution to 20 μl of the PCR reaction solution for mixing, the sample nucleic acid can also be detected with high sensitivity and high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 These are the fluorescence PCR test results of HBV positive samples of different concentrations when the pH value of the PCR reaction solution is 8.1.

[0048] Figure 2 These are the fluorescence PCR test results of HBV positive samples of different concentrations when the pH value of the PCR reaction solution is 8.9.

[0049] Figure 3The concentration of HBV positive samples was 20 IU / ml. The pH value of the PCR reaction solution was changed. The fluorescence PCR detection results were as follows when the pH values ​​were 8.1, 8.3, 8.5, 8.7 and 8.9 respectively. DETAILED DESCRIPTION

[0050] In order to extract nucleic acid from the sample, the present invention first adds a certain amount of sample to a strongly alkaline nucleic acid extraction solution, so that the cells or viruses in the sample are lysed, and the nucleic acid they carry is released, so that it is extracted. At this time, the pH value of the extracted sample nucleic acid solution is relatively high, which will affect the subsequent nucleic acid PCR amplification reaction, and even cause the nucleic acid amplification reaction to fail to start. Therefore, in order to reduce this effect, the inventor adds the extracted sample nucleic acid solution to a PCR reaction solution with a lower pH value for neutralization, and can also perform sample nucleic acid amplification and detection at the same time, so that the step of neutralizing the extracted sample nucleic acid during sample nucleic acid extraction can be omitted, which can effectively save detection time, reduce the chance of manual errors due to numerous steps, and facilitate semi-automatic or fully automated processing. Moreover, this will also improve the PCR amplification reaction efficiency of the sample nucleic acid and increase the detection sensitivity and accuracy of the sample nucleic acid.

[0051] In the present invention, the nucleic acid extracting solution used to extract nucleic acid from the sample can be selected from a high concentration strong alkaline solution or a strong alkaline solution containing Chelex-100, and the pH value of the nucleic acid extracting solution is ≥10, preferably 10.5-12.5, which can effectively extract the nucleic acid from the sample. Among them, the high concentration strong alkaline solution can be selected from a 0.05M-0.6M alkali metal hydroxide solution, preferably a 0.05M-0.6M NaOH solution or KOH solution; the strong alkaline solution containing Chelex-100 includes 10mM-100mM alkali metal hydroxide, 1mM-25mM Tris-HCl, 2%-5% Chelex-100, and 0.1%-0.4% EDTA, and the alkali metal hydroxide is preferably NaOH or KOH.

[0052] In addition, when the concentration of the target nucleic acid molecules to be detected in the sample is relatively low, it is preferred to add the sample to a concentrated solution for concentration before nucleic acid extraction, and then use the nucleic acid extracting solution of the present invention to extract the nucleic acid in the concentrated sample. As a first specific example of the concentrated solution, the concentrated solution may include NaCl 2.5% to 4.5%, PEG6000 10% to 13%; as a second specific example of the concentrated solution, the concentrated solution may include PEG8000 20%, NaCl 13.5%.

[0053] After the nucleic acid in the sample is extracted using the nucleic acid extract of the present invention, the extracted sample nucleic acid solution is added to a PCR reaction solution with a lower pH value at an appropriate volume ratio to reduce the pH value of the extracted sample nucleic acid solution to a level suitable for carrying out a PCR amplification reaction. In the present invention, the pH value of the PCR reaction solution is preferably 8.0 to 9.0. A value that is too high or too low will affect the efficiency of the PCR amplification reaction and reduce the detection sensitivity and accuracy. The PCR reaction solution contains Tris-HCl, KCl, dNTPs and Mg 2+ etc., wherein Tris-HCl can be replaced by a buffer system such as Tris-Acetate, Tris-Borate, etc., and when the PCR reaction solution is mixed with the extracted sample nucleic acid solution, ensure that the concentration of Tris-HCl is 5mM to 75mM. In addition, the PCR reaction solution may also include DNA polymerases such as Taq enzymes for nucleic acid amplification reactions, primers, and fluorescent dyes such as SYBRGreen, or fluorescent probes such as TaqMan probes, double hybridization probes, molecular beacon probes, or MGB probes. Of course, DNA polymerases, fluorescent dyes, or fluorescent probes may be added to the PCR reaction solution only when nucleic acid amplification reactions are required.

[0054] In the present invention, in order to further reduce the non-specific amplification that may occur during PCR amplification, anti-Taq enzyme antibodies are added to the PCR reaction solution. The antibodies can bind to the active site of the Taq enzyme, thereby making the Taq enzyme unable to function. Only in the denaturation stage, the anti-Taq enzyme antibodies are inactivated at high temperature and cannot bind to the active site of the Taq enzyme, thereby allowing the Taq enzyme to play a catalytic role and synthesize new DNA chains.

[0055] When the nucleic acid in the sample is extracted using the nucleic acid extracting solution of the present invention and the nucleic acid in the sample is detected using a PCR amplification reaction, a specific DNA or RNA fragment in the sample nucleic acid must be selected in advance, and then at least one pair of specific primers must be designed for the DNA or RNA fragment. If fluorescent PCR amplification is performed, a fluorescent dye such as SYBR Green can be added, or at least one fluorescent probe can be designed for the selected specific DNA or RNA fragment. In this way, the specific DNA or RNA fragment in the sample can be detected based on the fluorescence intensity generated during amplification.

[0056] The following examples further illustrate the present invention. These examples are not intended to limit the scope of the present invention, but to provide a further understanding of the present invention.

[0057] Example 1: Nucleic acid detection reagent

[0058] The sample nucleic acid detection reagent in this embodiment includes a nucleic acid extraction solution and a PCR reaction solution, and its composition is as follows:

[0059] Nucleic acid extraction solution: NaOH 10mM~100mM, Tris-HCl 1mM~25mM, Chelex-100 2%~5% (w / v), EDTA 0.1%~0.4% (w / v), pH=10.5~12.5.

[0060] PCR reaction solution (pH = 8.0 ~ 9.0), including Tris-HCl 5mM ~ 75mM, in addition to dNTPs, KCl, Mg 2+ and a pair of primers. When fluorescent PCR detection is adopted, the PCR reaction solution also includes fluorescent dyes such as SYBR Green, or fluorescent probes such as TaqMan, preferably TaqMan fluorescent probes. The PCR reaction solution may also include an enzyme mixture. Of course, the enzyme mixture and the PCR reaction solution may also be stored separately, and the enzyme mixture is added to the PCR reaction solution when nucleic acid amplification reaction is required. When the nucleic acid in the test sample is DNA, the enzyme mixture mainly includes a DNA polymerase, which may be selected from Taq DNA polymerase (referred to as Taq enzyme), Vent DNA polymerase, Deep DNA polymerase, Pfu DNA polymerase, Tgo DNA polymerase and KOD1 DNA polymerase, etc., preferably Taq enzyme. When the nucleic acid in the test sample is RNA, the enzyme mixture mainly includes MMLV reverse transcriptase, Taq enzyme and RNase inhibitor. When the selected DNA polymerase is Taq enzyme, no matter the nucleic acid in the test sample is DNA or RNA, the enzyme mixture may also include anti-Taq enzyme antibodies.

[0061] Furthermore, the sample nucleic acid detection reagent also includes a concentrated solution: NaCl 2.5% to 4.5% (w / v), PEG6000 10% to 13% (w / v).

[0062] When detecting hepatitis B virus (HBV) in serum or plasma samples, the concentrate: NaCl 2.5% (w / v), PEG6000 10% (w / v); nucleic acid extraction solution: NaOH 25mM, Tris-HCl 1mM, Chelex-100 2% (w / v), EDTA0.1% (w / v), pH = 11.5; PCR reaction solution, pH 8.1, wherein the PCR reaction solution includes Tris-HCl 10mM, and also includes KCl, dNTPs, Mg 2+ , BSA, primers and TaqMan probes, preferably also including Taq enzyme.

[0063] When detecting human B27 gene in whole blood sample, concentrate: NaCl 2.5% (w / v), PEG6000 13% (w / v); nucleic acid extraction solution: NaOH 25mM, Tris-HCl 10mM, Chelex-100 3% (w / v), EDTA 0.1% (w / v), pH = 12.0; PCR reaction solution, pH 8.8, wherein the PCR reaction solution includes Tris-HCl 5mM, and also includes KCl, dNTPs, Mg 2 + , BSA, primers and TaqMan probes, preferably also including Taq enzyme.

[0064] When detecting human papillomavirus 6 / 11 (HPV 6 / 11) in genital endocrine samples, nucleic acid extraction solution: NaOH 10mM, Tris-HCl 10mM, Chelex-100 2% (w / v), EDTA 0.1% (w / v), pH = 10.5; PCR reaction solution, pH 9.0, wherein the PCR reaction solution includes Tris-HCl 75mM, and also includes KCl, dNTPs, Mg 2+ , BSA, primers and TaqMan probes, preferably also including Taq enzyme solution.

[0065] When detecting Mycobacterium tuberculosis (TB) in sputum samples, the concentrate solution includes: NaCl 2.5% (w / v), PEG6000 10% (w / v); nucleic acid extraction solution includes: NaOH 25mM, Tris-HCl 25mM; Chelex-100 2% (w / v); EDTA 0.2% (w / v), pH = 11.5; PCR reaction solution, pH 8.0, wherein the PCR reaction solution includes Tris-HCl 50mM, and also includes KCl, dNTPs, Mg 2+ , BSA, primers and TaqMan probes, preferably also including Taq enzyme.

[0066] When detecting human cytomegalovirus (HCMV) in urine samples, the concentrate solution is: NaCl 4.5% (w / v), PEG600013% (w / v); nucleic acid extraction solution is: NaOH 100mM, Tris-HCl 10mM; Chelex-100 5% (w / v), EDTA 0.4% (w / v), pH = 12.5; PCR reaction solution, pH 8.9, wherein the PCR reaction solution includes Tris-HCl 10mM, and also includes KCl, dNTPs, Mg 2+ , BSA, primers and TaqMan probes, preferably also including Taq enzyme.

[0067] The NaOH in the nucleic acid extract can be replaced by other alkali metal hydroxides such as KOH.

[0068] Example 2: Sample nucleic acid extraction steps

[0069] The nucleic acid detection reagent in Example 1 is used to extract the sample nucleic acid as follows. Depending on the sample, the sample nucleic acid extraction method can be divided into the following two methods:

[0070] Scenario 1:

[0071] 1. Take 200 μl of the sample to be tested, add 200 μl of concentrate, shake and mix, and centrifuge at 12,000 rpm for 10 minutes;

[0072] 2. Discard the supernatant and add 50 μl of nucleic acid extraction solution to the precipitate;

[0073] 3. Oscillate and mix, place in a 100℃ water bath or dry bath for 10 minutes, then centrifuge at 12,000rpm for 10 minutes, and take the supernatant, which is the extracted sample nucleic acid solution, for PCR amplification, or store at -20℃±5℃ for future use.

[0074] Scenario 2:

[0075] 1. Take 100 μl of the sample to be tested, shake and mix, centrifuge at 12,000 rpm for 10 min, discard the supernatant, and add 50 μl of nucleic acid extraction solution to the precipitate;

[0076] 2. Oscillate and mix, place in a 100℃ water bath or dry bath for 10 minutes, then centrifuge at 12,000rpm for 10 minutes. Take the supernatant, which is the extracted sample nucleic acid solution, for PCR amplification, or store at -20℃±5℃ for future use.

[0077] In actual situations, the specific nucleic acid extraction steps are slightly different depending on the samples and test objects used for the test. For example, when detecting hepatitis B virus (HBV) in serum or plasma samples, the nucleic acid extraction steps are according to Scheme 1 of this embodiment; when detecting human B27 gene in whole blood samples, the nucleic acid extraction steps are according to Scheme 1 of this embodiment; when detecting human papillomavirus type 6 / 11 (HPV 6 / 11) in reproductive endocrine samples, the nucleic acid extraction steps are according to Scheme 2 of this embodiment; when detecting Mycobacterium tuberculosis (TB) in sputum samples, the nucleic acid extraction steps are according to Scheme 1 of this embodiment; when detecting human cytomegalovirus (HCMV) in urine samples, the nucleic acid extraction steps are according to Scheme 1 of this embodiment.

[0078] Example 3: Fluorescence PCR detection

[0079] PCR detection system 40μl, PCR reaction solution is divided into PCR reaction tubes according to the amount per person, and transferred to the sample processing area. Add 4-20μl of the sample nucleic acid solution extracted in Example 2 to the corresponding reaction tubes, cover the reaction tubes tightly, and transfer to the detection area for PCR amplification. Put each reaction tube into the PCR instrument in a certain order, set the detection PCR program and detection fluorescence, and perform fluorescent PCR amplification.

[0080] When detecting hepatitis B virus (HBV) in serum or plasma samples, the PCR detection system is 40 μl, including 20 μl of extracted sample nucleic acid solution and 20 μl of PCR reaction solution.

[0081] When detecting the human B27 gene in a whole blood sample, the PCR detection system is 40 μl, including 4 μl of the extracted sample nucleic acid solution and 36 μl of the PCR reaction solution.

[0082] When detecting human papillomavirus 6 / 11 (HPV 6 / 11) in reproductive tract endocrine samples, the PCR detection system is 40 μl, including 4 μl of extracted sample nucleic acid solution and 36 μl of PCR reaction solution.

[0083] When detecting Mycobacterium tuberculosis (TB) in sputum samples, the PCR detection system is 40 μl, including 10 μl of the extracted sample nucleic acid solution and 30 μl of the PCR reaction solution.

[0084] When detecting human cytomegalovirus (HCMV) in urine samples, the PCR detection system is 40 μl, including 4 μl of extracted sample nucleic acid solution and 36 μl of PCR reaction solution.

[0085] Example 4: HBV detection in clinical samples

[0086] The sample type is serum or plasma, and the detection of hepatitis B virus (HBV) is used as an example for description. According to the nucleic acid detection reagents, nucleic acid extraction methods and fluorescent PCR detection of Examples 1, 2 and 3, 20 clinical samples with confirmed positive or negative results are tested.

[0087] Control method: Take 50μl of the sample to be tested and add 50μl of nucleic acid extraction solution (NaCl 2M, Tris-HCl (pH 8.1) 50mM, mercaptoethanol 1% (v / v), Chelex-100 5% (w / v), pH 8.59), shake and mix for 15sec, place in a 100℃ water bath or dry bath for 10min, then centrifuge at 12,000rpm for 10min, and take 4μl and 20μl of the supernatant for PCR amplification respectively. The PCR reaction system includes PCR reaction solution (containing TAKARA PCR buffer, dNTPs, Mg 2+, primers and TaqMan probe, pH 9.05) and Taq enzyme. The detection sensitivity of this control method is 500 IU / ml.

[0088] Table 1 shows the results of HBV detection in serum or plasma samples, where “+” indicates a positive result and “-” indicates a negative result.

[0089] Table 1 HBV detection results in serum or plasma samples

[0090]

[0091]

[0092] The experimental results show that among these 20 test samples, 11 HBV positives and 9 HBV negatives were detected using the nucleic acid detection reagent, nucleic acid extraction method and fluorescent PCR test adopted by the present invention. For the control method, when 4μl of supernatant was taken for detection, 10 HBV positives and 10 HBV negatives were detected, and 1 control test was inconsistent with the clinical sample information; when 20μl of supernatant was taken for detection, the test results of these 20 test samples were all negative. This is because the pH value of the nucleic acid extraction reagent in the control method is non-strongly alkaline, and the nucleic acid extraction efficiency is not high compared with the method of the present invention. In addition, when the sample size is increased to 20ul, substances that inhibit PCR amplification may also be increased at the same time.

[0093] Example 5: Effect of pH value of PCR reaction solution on HBV detection sensitivity

[0094] According to the nucleic acid detection reagents, nucleic acid extraction methods and fluorescent PCR detection of Examples 1, 2 and 3, HBV-positive samples were detected: the high-concentration HBV-positive samples were gradiently diluted to obtain a series of low-concentration HBV-positive samples, with the concentrations from high to low being 1000 IU / ml, 500 IU / ml, 250 IU / ml, 100 IU / ml, 50 IU / ml and 20 IU / ml, respectively.

[0095] The pH value of the PCR reaction solution was adjusted to 8.1. The fluorescence PCR test results of these HBV positive samples were as follows: Figure 1 The results showed that the six low-concentration HBV-positive samples all had obvious S-shaped amplification curves and could be stably detected.

[0096] When the pH value of the PCR reaction solution was adjusted to 8.9, the fluorescence PCR test results for these HBV positive samples were as follows: Figure 2As shown. The results show that among the 6 low-concentration HBV positive samples, the positive samples with concentrations of 1000IU / ml, 500IU / ml and 250IU / ml can be stably detected, while the positive samples with concentrations of 100IU / ml, 50IU / ml and 20IU / ml cannot be stably detected. It can be seen that when the pH value of the PCR reaction solution of the present invention is 8.9, its detection sensitivity is 250IU / ml, which is also better than 500IU / ml of the control method.

[0097] In addition, for HBV positive samples with a concentration of 20 IU / ml, the pH values ​​of the PCR reaction solution were adjusted to 8.1, 8.3, 8.5, 8.7, and 8.9, respectively. The fluorescence PCR test results were as follows Figure 3 As shown. The results showed that as the pH value of the PCR reaction solution decreased, the amplification efficiency of HBV positive samples with a concentration of 20 IU / ml increased significantly, and the amplification efficiency was optimal when the pH value was 8.1. The results also showed that when the pH values ​​of the PCR reaction solution were 8.1, 8.3, 8.5 and 8.7, 20 IU / ml positive samples could be detected, but the amplification efficiency, linearity of the S-type amplification curve and fluorescence value when the pH values ​​were 8.1, 8.3 and 8.5 were significantly better than those when the pH value was 8.7; the amplification efficiency, linearity of the S-type amplification curve and fluorescence value when the pH value was 8.1 were significantly better than those when the pH values ​​were 8.3 and 8.5. It can be seen that, under the same other detection conditions, changing the pH value can have a very large impact on the amplification efficiency, the linearity of the S-type amplification curve and the fluorescence value, and improving the amplification efficiency and the fluorescence value of the S-type amplification curve can increase the sensitivity of the detection. Therefore, the detection sensitivity at pH values ​​of 8.1, 8.3 and 8.5 is significantly better than that at pH 8.7; the detection sensitivity at pH 8.1 is significantly better than that at pH 8.3 and 8.5. This means that as the pH value gradually decreases, the detection sensitivity gradually increases.

[0098] When detecting HBV DNA in serum or plasma, the present invention unexpectedly found that adjusting the pH value of the PCR reaction solution, that is, adjusting from 8.9 to 8.7, from 8.7 to 8.3-8.5, and from 8.3 to 8.1, achieved an unexpected amplification effect, thereby significantly improving the detection accuracy.

[0099] Example 6: Human B27 gene detection in clinical samples

[0100] The sample type is whole blood for clinical testing, taking human B27 gene testing as an example. According to the nucleic acid detection reagents, nucleic acid extraction methods and fluorescent PCR detection of Examples 1, 2 and 3, 13 clinical samples with confirmed positive or negative results were tested.

[0101] Control method: First, pre-treat 500μl of whole blood sample with red blood cell lysis buffer (NH4Cl 15mM, NaHCO3 1mM, EDTA-2Na 0.1mM), centrifuge, remove the supernatant, and retain the precipitate; add 100μl of nucleic acid extraction solution (Tris-HCl (pH 8.0) 100mM, Chelex-100 5%, EDTA (pH 8.0) 10mM, NaCl 50mM, SDS 2% (w / v), pH 8.2) to the precipitate, shake and mix for 15sec, place in a 100℃ water bath or dry bath for 10min, then centrifuge at 12,000rpm for 10min, and take 4μl of the supernatant for PCR amplification. The PCR reaction system includes PCR reaction solution (containing TAKARA PCR buffer, dNTPs, Mg 2+ , primers and TaqMan probe, pH 9.05) and Taq enzyme.

[0102] Table 2 shows the results of human B27 gene detection in whole blood samples, where “+” indicates a positive result and “-” indicates a negative result.

[0103] Table 2 Results of human B27 gene detection in whole blood samples

[0104]

[0105]

[0106] The experimental results showed that among the 13 test samples, 6 cases were detected to be human B27 gene positive and 7 cases were human B27 gene negative using the nucleic acid detection reagent, nucleic acid extraction method and fluorescent PCR detection adopted by the present invention; the control method detected 5 cases of human B27 gene positive and 8 cases of human B27 gene negative, and 1 case in the control test was inconsistent with the clinical sample information.

[0107] Example 7: HPV6 / 11 detection in clinical samples

[0108] The sample type is a clinical test of genital secretions, taking the detection of human papillomavirus 6 / 11 (HPV 6 / 11) as an example. According to the nucleic acid detection reagents, nucleic acid extraction methods and fluorescent PCR detection of Examples 1, 2 and 3, 15 clinical samples whose positive and negative have been clinically confirmed were tested.

[0109] Control method: Take 50μl of the sample to be tested and add 50μl of nucleic acid extraction solution (NaCl 2M, Tris-HCl (pH 8.0) 50mM, mercaptoethanol 1% (v / v), Chelex-100 5% (w / v), pH 8.59), shake and mix for 15sec, place in a 100℃ water bath or dry bath for 10min, then centrifuge at 10,000rpm for 5min, and take 4μl of the supernatant for PCR amplification. The PCR reaction system includes PCR reaction solution (containing TAKARA PCR buffer, dNTPs, Mg 2+ , primers and TaqMan probe, pH 9.05) and Taq enzyme.

[0110] Table 3 shows the HPV test results in genital secretion samples, where “+” indicates a positive result and “-” indicates a negative result.

[0111] Table 3 Results of HPV gene detection in genital secretion samples

[0112]

[0113]

[0114] The experimental results showed that among the 15 test samples, 10 HPV positives and 5 HPV negatives were detected using the nucleic acid detection reagent, nucleic acid extraction method and fluorescent PCR test adopted by the present invention; the control method detected 8 positives and 7 negatives, and the control results of 2 cases were inconsistent with the clinical sample information.

[0115] Example 8: TB detection in clinical samples

[0116] The sample type is sputum for clinical testing, taking Mycobacterium tuberculosis (TB) detection as an example. According to the nucleic acid detection reagents, nucleic acid extraction methods and fluorescent PCR detection of Examples 1, 2 and 3, 10 clinical samples that have been clinically confirmed to be positive or negative are tested.

[0117] Control method: 500μl sputum sample, add 5ml 1M NaOH solution, shake and liquefy for 30min, centrifuge at 14,000 for 5min, remove the supernatant, and retain the precipitate; add 50μl nucleic acid extraction solution (SDS 0.1% (w / v), NP40 1% (v / v), Tween-20 1% (v / v), Chelex-100 15% (w / v), pH 5.02) to the precipitate, shake and mix for 15sec, place in a 100℃ water bath or dry bath for 30min, then centrifuge at 12,000rpm for 10min, and take 10μl of the supernatant for PCR amplification. The PCR reaction system includes PCR reaction solution (containing TAKARA PCR buffer, dNTPs, Mg2+ , primers and TaqMan probe, pH 9.05) and Taq enzyme.

[0118] Table 4 shows the TB test results in sputum samples, where “+” indicates a positive result and “-” indicates a negative result.

[0119] Table 4 TB detection results in sputum samples

[0120] Sample No. The test results of the present invention Comparative method test results Clinical sample information 1 + + + 2 + - + 3 + + + 4 + + + 5 + + + 6 - - - 7 - - - 8 - - - 9 - - - 10 - - -

[0121] The experimental results showed that among the 10 test samples, 5 TB positive cases and 5 TB negative cases were detected using the nucleic acid detection reagent, nucleic acid extraction method and fluorescent PCR test adopted by the present invention; the control method detected 4 positive cases and 6 negative cases, and the control sample result of 1 case was inconsistent with the clinical sample information.

[0122] Example 9: HCMV detection in clinical samples

[0123] The sample type is a clinical test of urine, taking the human cytomegalovirus (HCMV) test as an example. According to the nucleic acid detection reagents, nucleic acid extraction methods and fluorescent PCR tests of Examples 1, 2 and 3, 10 clinical samples with confirmed positive or negative results were tested.

[0124] Control method: Collect 250μl of urine sample to be tested, centrifuge at 14,000 for 5 minutes, remove the supernatant, and retain the precipitate; add 250μl of nucleic acid extraction solution (Tris-HCl (pH 8.0) 100mM, Chelex-100 5%, EDTA (pH 8.0) 10mM, NaCl 50mM, SDS 2% (w / v), pH 8.2) to the precipitate, shake and mix for 15sec, place in a 100℃ water bath or dry bath for 10min, then centrifuge at 13,000rpm for 10min, and take 4μl of supernatant for PCR amplification. The PCR reaction system includes PCR reaction solution (containing TAKARA PCR buffer, dNTPs, Mg 2+ , primers and TaqMan probe, pH 9.05) and Taq enzyme.

[0125] Table 5 shows the results of HCMV detection in urine samples, where “+” indicates a positive result and “-” indicates a negative result.

[0126] Table 4 HCMV detection results in urine samples

[0127] Sample No. The test results of the present invention Comparative method test results Clinical sample information 1 + + + 2 + + + 3 + + + 4 + + + 5 + + + 6 - - - 7 - - - 8 - - - 9 - - - 10 - - -

[0128] The experimental results showed that among the 10 test samples, 5 HCMV-positive samples and 5 HCMV-negative samples were detected using the nucleic acid detection reagent, nucleic acid extraction method and fluorescent PCR detection adopted by the present invention; the control method detected 5 positive samples and 5 negative samples; the two test results were consistent.

[0129] Example 10: Sensitivity test results

[0130] According to the nucleic acid detection reagents, nucleic acid extraction methods and fluorescent PCR tests of Examples 1, 2 and 3, HBV, human B27 gene, HPV, TB and HCMV positive standard samples were detected: the positive standard samples were diluted respectively, and the concentrations were diluted according to Table 6, and each concentration was repeated 8 times. The test results are shown in Table 6, where "+" indicates a positive result and "-" indicates that it is below the detection limit of the reagent.

[0131] Table 6 Sensitivity test results

[0132]

[0133] The above results show that the HBV detection sensitivity of the present invention is 20 IU / ml, the human B27 gene detection sensitivity is 5 ng / reaction, the HPV 6 / 11 detection sensitivity is 250 copies / ml, the TB detection sensitivity is 5 bacteria / ml, and the HCMV detection sensitivity is 500 copies / ml.

Claims

1. A method for detecting sample nucleic acid for non-disease detection purposes, characterized in that: The method comprises: (1) extracting nucleic acid from a sample using a nucleic acid extracting solution to obtain a sample nucleic acid solution, wherein the nucleic acid extracting solution comprises 10 mM to 100 mM alkali metal hydroxide, 1 mM to 25 mM Tris-HCl, 2% to 5% (w / v) Chelex-100, and 0.1% to 0.4% (w / v) EDTA, and the pH value of the nucleic acid extracting solution is 10.5 to 12.5; (2) mixing the sample nucleic acid solution obtained in (1) with a PCR reaction solution to obtain a mixed solution, and performing an amplification reaction using the mixed solution to detect the nucleic acid in the sample, wherein the PCR reaction solution comprises one of Tris-HCl, Tris-Acetate, or Tris-Borate, and the concentration thereof is 5 mM to 75 mM; and the pH value of the PCR reaction solution is 8.0 to 9.

0.

2. The method according to claim 1, characterized in that The alkali metal hydroxide is NaOH or KOH.

3. The method according to claim 1, characterized in that The pH value of the mixed solution is 8.0-9.

0.

4. The method according to claim 1, characterized in that Prior to step (1), the sample is concentrated using a concentration solution.

5. The method according to claim 4, characterized in that The concentrated solution comprises NaCl 2.5% to 4.5% (w / w) and PEG6000 10% to 13% (w / v).

6. The method according to any one of claims 1 to 5, characterized in that: The nucleic acid in the sample includes HBV, human B27 gene, HPV 6 / 11, TB or HCMV nucleic acid.

7. A method for neutralizing sample nucleic acid, characterized in that: The method comprises: (1) extracting nucleic acid in a sample using a nucleic acid extracting solution having a pH value of 10.5 to 12.5 to obtain a sample nucleic acid solution, wherein the nucleic acid extracting solution comprises 10 mM to 100 mM alkali metal hydroxide, 1 mM to 25 mM Tris-HCl, 2% to 5% (w / v) Chelex-100, and 0.1% to 0.4% (w / v) EDTA; and (2) adding the sample nucleic acid solution obtained in (1) to a PCR reaction solution for neutralization, wherein the PCR reaction solution comprises one of Tris-HCl, Tris-Acetate or Tris-Borate, the concentration of which is 5 mM to 75 mM, and the pH value of the PCR reaction solution is 8.0 to 9.

0.

8. The method according to claim 7, characterized in that The alkali metal hydroxide is NaOH or KOH.

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