WO2020125294A1 - Primers for hepatitis c virus nucleic acid detection, probe, kit, and detection method - Google Patents

Primers for hepatitis c virus nucleic acid detection, probe, kit, and detection method Download PDF

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WO2020125294A1
WO2020125294A1 PCT/CN2019/119046 CN2019119046W WO2020125294A1 WO 2020125294 A1 WO2020125294 A1 WO 2020125294A1 CN 2019119046 W CN2019119046 W CN 2019119046W WO 2020125294 A1 WO2020125294 A1 WO 2020125294A1
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nucleic acid
primer
hepatitis
probe
virus
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PCT/CN2019/119046
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French (fr)
Chinese (zh)
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张惠丹
戴敬
赵洪玉
节淳皓
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苏州绘真生物科技有限公司
苏州德思普生物科技有限公司
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Publication of WO2020125294A1 publication Critical patent/WO2020125294A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

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  • the present application relates to the field of molecular biology technology, and more specifically, to the field of RNA technology, in particular to a highly sensitive primer, probe and corresponding kit for detecting hepatitis C virus (HCV) nucleic acid, and the use of the reagent Method for detecting nucleic acid of hepatitis C virus by using a cassette.
  • HCV hepatitis C virus
  • Hepatitis C virus belongs to the Flaviviridae family, is a small single-stranded positive-strand RNA virus with a capsule structure. HCV is distributed worldwide, and humans are generally susceptible, which can be spread through blood, sex, mother and child. The onset of HCV infection is hidden, and it is more likely to develop into chronic infection than HBV. This is because most of the clinical manifestations of acute HCV infection are very light, and there is little severe hepatitis, and it is mostly in the chronic phase when it is diagnosed. The performance of chronic HCV infection varies, and about 20 to 30% can gradually develop to cirrhosis, and about 1 to 4% of patients with cirrhosis can develop to liver cancer every year. HCV-RNA detection can detect HCV infection as early as possible, and is of great significance for the selection of clinical treatment options and monitoring of efficacy.
  • the common detection methods of HCV include viral protein antibody detection, antigen detection, nucleic acid detection and other methods.
  • Viral antigen and antibody detection methods often use enzyme-linked immunosorbent assay (ELISA), that is, specific antigen or antibody is adsorbed on a solid phase carrier, so that it binds to the corresponding antibody or antigen in the sample to be tested, and then enzyme-labeled antigen or Antibody, add substrate to develop color, and finally calculate the content of the antigen or antibody to be tested according to the color depth.
  • ELISA enzyme-linked immunosorbent assay
  • the amount of virus that has just entered the body is very low.
  • the virus's antigen content is often lower than the detection limit of the ELISA method, and the body needs a period of time to produce antibodies. During this period, the blood antibody test is negative.
  • the antigen antibody is usually after infection. It can only be detected after 3-6 weeks.
  • gene detection technology has been popularized in various laboratories. Especially fluorescent quantitative PCR technology not only has good specificity, but also can increase the sensitivity of detection and shorten virus detection by amplifying viral nucleic acid. Window period.
  • virus detection kits based on fluorescent quantitative PCR technology at home and abroad, but the domestic detection kit has a high quantitative detection limit and poor sensitivity.
  • the imported high-sensitivity diagnostic kits are too expensive, which raises the cost of medical and health care.
  • the main purpose of the present application is to provide a primer and a probe for detecting hepatitis C virus nucleic acid in order to overcome the deficiencies in the prior art.
  • Another main objective of the present application is to provide a kit for detecting hepatitis C virus nucleic acid.
  • Another object of the present application is also to provide the use of the aforementioned kit in the detection of hepatitis C virus nucleic acid.
  • Another object of the present application is to provide a new method for detecting hepatitis C virus nucleic acid.
  • the embodiments of the present application provide a primer pair for detecting hepatitis C virus nucleic acid, which includes:
  • the sequence of the first primer is shown in SEQ ID NO.1;
  • the sequence of the second primer is shown in SEQ ID NO.2.
  • the embodiment of the present application further provides a probe for detecting hepatitis C virus nucleic acid, and the sequence of the probe is shown in SEQ ID NO. 3.
  • An embodiment of the present application further provides a kit for detecting hepatitis C virus nucleic acid, which includes at least one set of primer pairs and at least one probe, wherein one set of primer pairs includes a first primer and a second primer, the first The sequence of the primer is shown in SEQ ID NO. 1, and the sequence of the second primer is shown in SEQ ID NO. 2.
  • One of the probes is the aforementioned probe.
  • the embodiments of the present application also provide the use of the aforementioned kit in preparing products for detecting nucleic acid of hepatitis C virus.
  • the method for detecting nucleic acid of hepatitis C virus using the product includes:
  • a sample processing device is used to mix the nucleic acid sample to be detected with the PCR reaction solution, and the formed mixed solution is wrapped by the droplet generation solution to form droplets containing a single nucleic acid or not;
  • the embodiments of the present application also provide a product containing the aforementioned kit, which is applied to a method for detecting hepatitis C virus nucleic acid, the detection method includes:
  • a sample processing device is used to mix the nucleic acid sample to be detected with the PCR reaction solution, and the formed mixed solution is wrapped by the droplet generation solution to form droplets containing a single nucleic acid or not;
  • the highly sensitive method for detecting hepatitis C virus nucleic acid provided by this application has higher sensitivity and specificity of the detection result, and can effectively reduce the detection cost;
  • This application uses centrifugal force to make the nucleic acid solution and the PCR reaction solution wrapped in the droplet generation solution in the flow channel to form a single-molecule PCR reaction system, which increases the sensitivity of detection and lowers the lower limit of detection, which is conducive to early detection of viral infections;
  • the kit provided by this application optimizes primer design to ensure that the detection of hepatitis C virus nucleic acid can cover all known subtypes, while
  • the fragment length of the amplified product is between 100-150 bp, and the annealing temperature is between 56-62° C., and the mutual interaction between the designed primers is performed by the Thermo Fisher online primer analysis tool Analysis of the role to ensure that the interaction between primers will not affect the system;
  • This application optimizes the primer probes so that the primer probes for each virus can only detect the virus, and will not interact with the human genome or human hepatitis B virus (HBV) nucleic acid, human herpes virus (EBV) nucleic acid, Common viral nucleic acids such as cytomegalovirus (CMV) nucleic acids cross-react, ensuring the accuracy of positive results.
  • HBV hepatitis B virus
  • EBV human herpes virus
  • CMV cytomegalovirus
  • FIG. 1 is a schematic structural diagram of a sample processing device used in a typical embodiment of the present application.
  • Example 2 is a result of droplet fluorescence detection of the negative control in Example 1 of the present application.
  • Example 3 is a drop fluorescence detection result of No. 1 positive sample in Example 1 of the present application.
  • FIG. 5 is a result of detecting the fluorescence intensity of the droplet A of Reference A in Example 2 of the present application.
  • Example 6 is a result of detecting the fluorescence intensity of the droplet B of Reference B in Example 2 of the present application.
  • Example 7 is a result of detecting the fluorescence intensity of the droplet C of Reference C in Example 2 of the present application.
  • FIG. 8 is the result of detecting the fluorescence intensity of the droplet of Reference D in Example 2 of the present application.
  • Example 9 is a detection result of droplet fluorescence intensity of positive samples of HBV, CMV, EBV and HCV in Example 3 of the present application.
  • One aspect of the embodiments of the present application provides a primer pair for detecting hepatitis C virus nucleic acid, which includes:
  • the first primer the forward primer, whose sequence is shown in SEQ ID NO.1;
  • the second primer has the sequence shown in SEQ ID NO.2.
  • primer sequences contained in the primer pair are as follows:
  • HCV-F forward primer
  • HCV-R reverse primer
  • the kit provided by this application optimizes the primer design so that the detection of hepatitis C virus nucleic acid can cover all known subtypes.
  • this application By optimizing the primer design, the fragment length of the amplified product is between 100-150bp, and the annealing temperature is between 56-62°C, and the interaction between the designed primers is analyzed by the Thermo Fisher primer analysis online tool To ensure that the interaction between primers does not affect the system.
  • Another aspect of the embodiments of the present application further provides a probe for detecting hepatitis C virus nucleic acid, and the sequence of the probe is shown in SEQ ID NO. 3.
  • sequence of the probe is as follows:
  • This application optimizes the primer probe so that the primer probe for each virus can only detect the virus, and will not interact with the human genome or human hepatitis B virus (HBV) nucleic acid, human herpes virus (EBV) nucleic acid, megacell Common viral nucleic acids such as viral (CMV) nucleic acids cross-react, ensuring the accuracy of positive results.
  • HBV hepatitis B virus
  • EBV herpes virus
  • CMV viral nucleic acids
  • kits for detecting hepatitis C virus nucleic acid which includes at least one set of primer pairs and at least one probe, wherein one set of primer pairs includes a first primer and a second primer
  • one set of primer pairs includes a first primer and a second primer
  • the sequence of the first primer is shown in SEQ ID NO.1
  • the sequence of the second primer is shown in SEQ ID NO.2
  • the sequence of the probe is shown in SEQ ID NO.3.
  • the kit further includes a viral internal standard primer pair and an internal standard probe, wherein the viral internal standard primer pair includes a first internal standard primer (ie, internal standard forward primer) and a first Two internal standard primers (ie internal standard reverse primers), the sequence of the first internal standard primer is shown in SEQ ID NO.4, and the sequence of the second internal standard primer is shown in SEQ ID NO.5 The sequence of the internal standard probe is shown in SEQ ID NO.6.
  • the viral internal standard primer pair includes a first internal standard primer (ie, internal standard forward primer) and a first Two internal standard primers (ie internal standard reverse primers)
  • the sequence of the first internal standard primer is shown in SEQ ID NO.4
  • the sequence of the second internal standard primer is shown in SEQ ID NO.5
  • the sequence of the internal standard probe is shown in SEQ ID NO.6.
  • sequences of the internal standard forward primer and the reverse primer contained in the internal standard primer mixed solution in the kit of the present application are as follows:
  • sequence of the internal standard probe in the internal standard probe solution is: 5'-Cy5-5-CTGTATCGTCAAGGCACT-BHQ-3'.
  • the kit also includes conventional components for PCR amplification detection.
  • the conventional components for PCR amplification detection include a buffer solution for PCR reaction, a mixture of base deoxynucleotide triphosphates (2.5 mM dNTPs), DNA polymerase, RNA reverse transcriptase, and uracil DNA glycosylase , Water and liquid droplets.
  • the buffer for PCR reaction contains 20 mM potassium chloride, 50 mM PH8.0 Tris-HCl, and 1.5 mM magnesium ions.
  • dNTPs include dNTP and dUTP.
  • the kit specifically includes: PCR reaction buffer, 2.5 mM dNTPs, target nucleotide primer mixed solution, target nucleotide probe solution, internal standard primer mixed solution, internal Standard probe solution, 5U/ ⁇ L heat-resistant DNA polymerase, 5U/ ⁇ L RNA reverse transcriptase, 5U/ ⁇ L uracil DNA glycosylase, water, and droplet generation solution.
  • the target nucleotide primer mixed solution includes the aforementioned first primer and second primer.
  • the target nucleotide probe solution contains HCV specific probe.
  • the internal standard primer mixed solution includes an internal standard forward primer and an internal standard reverse primer.
  • Another aspect of the embodiments of the present application also provides the use of the aforementioned kit in preparing a product for detecting hepatitis C virus nucleic acid.
  • the method for detecting hepatitis C virus using the product includes:
  • a sample processing device is used to mix the nucleic acid sample to be detected with the PCR reaction solution, and the formed mixed solution is wrapped by the droplet generation solution to form droplets containing a single nucleic acid or not;
  • the method specifically includes:
  • the fluorescence intensity of the PCR amplified droplets is detected, and whether the nucleic acid sample contains hepatitis C virus nucleic acid is determined based on the fluorescence intensity.
  • the sample processing device includes a positioning hole, a sample injection hole, a reagent hole, a reaction hole, and a connecting flow channel.
  • the processing device is made of PC material, with a length of 40 mm, a width of 20 mm, and a thickness of 2 mm.
  • the volume of the injection well is 50 ⁇ L
  • the volume of the reagent well is 50 ⁇ L
  • the volume of the reaction well is 100 ⁇ L.
  • the detection method specifically includes: adding a droplet generation solution to the reagent well, adding the nucleic acid sample to be detected and the PCR reaction solution to the injection well, under the action of centrifugal force
  • the liquid in the injection well and the reagent well are mixed through the connecting flow channel, and the formed mixed liquid is wrapped by the droplet generation liquid to form droplets containing a single nucleic acid or not, and enters the reaction well.
  • the detection method may include: installing the sample processing device on the centrifuge through the positioning hole, adding the droplet generation solution to the reagent hole, and mixing the extracted nucleic acid solution and the PCR reaction solution in proportion to the injection hole. Centrifuge at 3000 rpm clockwise. Under the action of centrifugal force, the liquid in the sample well and the reagent well are mixed through the flow channel, so that the nucleic acid sample is wrapped into the PCR reaction solution to form droplets, and finally collected in the reaction well.
  • the droplet generation liquid includes oil phase substances such as mineral oil.
  • the method may specifically include the following steps:
  • the PCR reaction solution was removed from the refrigerator in advance and thawed, and after thawing, the mixture was vortexed and centrifuged. Take 10-19 ⁇ L of the reaction solution in a PCR tube, add 1-10 ⁇ L of the newly extracted nucleic acid sample to it, and mix with a pipette tip to ensure that the total amount of liquid is 20 ⁇ L. Pipette 20 ⁇ L of the mixed liquid into the injection well, and 20 ⁇ L of the droplet generation liquid into the reagent well. Fix the processing device on the centrifuge adapter through the positioning hole, and pay attention to keep the center symmetrical during installation.
  • the amplified products were analyzed using an 8-channel droplet reading system.
  • the background fluorescence intensity was determined according to the blank control without the template added. Droplets exceeding the background fluorescence intensity were set as positive droplets, and the fluorescence intensity of each reaction droplet was measured. And count the number of positive droplets.
  • the amount of hepatitis C virus nucleic acid in the nucleic acid sample can be calculated.
  • the present application uses centrifugal force to make the nucleic acid solution and the PCR reaction solution wrapped in the droplet generation solution in the flow channel to form a single-molecule PCR reaction system, which increases the sensitivity of detection and lowers the lower limit of detection, which is helpful for early detection of virus infection.
  • Another aspect of the embodiments of the present application also provides a product containing the aforementioned kit, which is applied to a method for detecting hepatitis C virus nucleic acid, the detection method includes:
  • a sample processing device is used to mix the nucleic acid sample to be detected with the PCR reaction solution, and the formed mixed solution is wrapped by the droplet generation solution to form droplets containing a single nucleic acid or not;
  • the product includes: performing droplet PCR amplification of the droplet using the primer pair and the probe contained in the kit;
  • the fluorescence intensity of the PCR amplified droplets is detected, and whether the nucleic acid sample contains hepatitis C virus nucleic acid is determined based on the fluorescence intensity.
  • the samples used in this example include 2 cases of HCV positive nucleic acid solution and 1 case of negative nucleic acid solution.
  • PCR amplifier polymerase chain reaction amplifier
  • the amplified products were analyzed using an 8-channel droplet reading system (Suzhou Haotong Biotechnology Co., Ltd.).
  • the fluorescence intensity of each droplet in the negative control was analyzed to determine the background fluorescence intensity.
  • the fluorescence intensity of each reaction droplet is measured, and the number of positive droplets is counted.
  • the detection result of each sample takes the order of the detection of each droplet as the abscissa and the fluorescence intensity of the droplet as the ordinate, and plots a scatter plot of the fluorescence intensity of each droplet.
  • the fluorescence intensity threshold line is determined by the background fluorescence value determined by the negative control, and the droplet whose fluorescence intensity exceeds the threshold line is the positive droplet.
  • the number of positive droplets detected in each sample represents the number of copies of HCV nucleic acid contained in each 1 ⁇ L of nucleic acid sample.
  • the result of the detection of the fluorescence intensity of the droplet of the positive sample No. 1 can be obtained that the HCV viral nucleic acid load in the nucleic acid sample is 668 copies/ ⁇ L.
  • the HCV viral nucleic acid load in the nucleic acid sample is 421 copies/ ⁇ L.
  • the samples used in this example include 4 kinds of plasmid quantitative reference products containing HCV-specific sequences, in which the plasmid concentrations are A (10copies/ ⁇ L), B (5copies/ ⁇ L), C (2copies/ ⁇ L) and D(1copies/ ⁇ L).
  • PCR amplifier polymerase chain reaction amplifier
  • the amplified products were analyzed using an 8-channel droplet reading system (Suzhou Haotong Biotechnology Co., Ltd.).
  • the fluorescence intensity of each droplet in the negative control was analyzed to determine the background fluorescence intensity.
  • the fluorescence intensity of each reaction droplet is measured, and the number of positive droplets is counted.
  • the detection result of each sample takes the order of the detection of each droplet as the abscissa and the fluorescence intensity of the droplet as the ordinate, and plots a scatter plot of the fluorescence intensity of each droplet.
  • the fluorescence intensity threshold line is determined by the background fluorescence value determined by the negative control, and the droplet whose fluorescence intensity exceeds the threshold line is the positive droplet.
  • the number of positive droplets detected in each sample represents the number of copies of HCV virus nucleic acid contained in each 10 ⁇ L of nucleic acid samples.
  • Fig. 5 is the result of the detection of the fluorescence intensity of the droplet of Reference A, it can be concluded that the nucleic acid load of HCV virus in this Reference is 10 copies/ ⁇ L.
  • nucleic acid load of HCV virus in this reference is 5 copies/ ⁇ L.
  • nucleic acid load of HCV virus in this Reference is 1 copies/ ⁇ L.
  • the samples used in this example included one sample of human hepatitis B virus (HBV) nucleic acid positive, human herpes virus (EBV) nucleic acid positive, cytomegalovirus (CMV) nucleic acid positive and HCV nucleic acid positive samples.
  • HBV human hepatitis B virus
  • EBV human herpes virus
  • CMV cytomegalovirus
  • PCR amplifier polymerase chain reaction amplifier
  • the amplified products were analyzed using an 8-channel droplet reading system (Suzhou Haotong Biotechnology Co., Ltd.).
  • the fluorescence intensity of each droplet in the negative control was analyzed to determine the background fluorescence intensity.
  • the fluorescence intensity of each reaction droplet is measured, and the number of positive droplets is counted.
  • the detection result of each sample takes the order of the detection of each droplet as the abscissa, and the fluorescence intensity of the droplet as the ordinate, and plots a scatter plot of the fluorescence intensity of each droplet.
  • the fluorescence intensity threshold line is determined by the background fluorescence value determined by the negative control, and the droplet whose fluorescence intensity exceeds the threshold line is the positive droplet.
  • the number of positive droplets detected in each sample represents the number of copies of HCV virus nucleic acid contained in each 10 ⁇ L of nucleic acid samples.
  • HBV, CMV, EBV and HCV positive sample droplet fluorescence intensity detection results it can be concluded that there is no cross-reaction with HBV, CMV, EBV viral nucleic acids, viral nucleic acid load in HCV positive samples 32.3copies/ ⁇ L.
  • the kit of the present application has a low detection limit, high sensitivity and good specificity
  • the detection method of the present application is simple and fast, the sensitivity and specificity of the test results are higher, and the detection can be effectively reduced cost.

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Abstract

Primers for hepatitis C virus nucleic acid detection, a probe, a kit, and a detection method. The primers for hepatitis C virus nucleic acid detection comprise a first primer and a second primer, of which the sequences are respectively as shown by SEQ ID NO.1 and SEQ ID NO.2. The sequence of the probe is as shown by SEQ ID NO.3. The kit comprises said primers and the probe. The hepatitis C virus nucleic acid detection method comprises: mixing a nucleic acid sample to be detected with a PCR liquid by using a sample treatment device, and coating the formed mixed liquid with a droplet generation liquid to form a droplet containing a single nucleic acid or containing no nucleic acid; and performing PCR amplification on the droplet by using the kit, and detecting a PCR amplification product. The kit is low in detection lower limit, high in sensitivity, and good in specificity. The detection method is simple and quick, high in detection efficiency, and low in cost.

Description

检测丙型肝炎病毒核酸的引物、探针、试剂盒及检测方法Primer, probe, kit and detection method for detecting hepatitis C virus nucleic acid 技术领域Technical field
本申请涉及分子生物学技术领域，更具体地，涉及用于RNA技术领域，特别是指一种高灵敏检测丙型肝炎病毒(HCV)核酸的引物、探针及相应试剂盒，以及利用该试剂盒检测丙型肝炎病毒核酸的方法。The present application relates to the field of molecular biology technology, and more specifically, to the field of RNA technology, in particular to a highly sensitive primer, probe and corresponding kit for detecting hepatitis C virus (HCV) nucleic acid, and the use of the reagent Method for detecting nucleic acid of hepatitis C virus by using a cassette.
背景技术Background technique
丙型肝炎病毒(Hepatitis virus C，HCV)属于黄病毒科，是一小的带有囊膜结构的单股正链RNA病毒。HCV呈世界性分布，人类普遍易感，可以通过血液、性以及母婴等途径传播。HCV感染起病隐匿，较HBV更易发展为慢性感染。这是由于HCV急性感染的临床表现大多很轻，少有重型肝炎发生，待确诊时已多为慢性期。慢性HCV感染的表现轻重不等，约20～30％可逐渐发展至肝硬化，肝硬化患者中每年约1～4％可发展至肝癌。HCV-RNA检测，可以尽早发现HCV感染，并且对于其临床治疗方案的选择与疗效监控有重要意义。Hepatitis C virus (HCV) belongs to the Flaviviridae family, is a small single-stranded positive-strand RNA virus with a capsule structure. HCV is distributed worldwide, and humans are generally susceptible, which can be spread through blood, sex, mother and child. The onset of HCV infection is hidden, and it is more likely to develop into chronic infection than HBV. This is because most of the clinical manifestations of acute HCV infection are very light, and there is little severe hepatitis, and it is mostly in the chronic phase when it is diagnosed. The performance of chronic HCV infection varies, and about 20 to 30% can gradually develop to cirrhosis, and about 1 to 4% of patients with cirrhosis can develop to liver cancer every year. HCV-RNA detection can detect HCV infection as early as possible, and is of great significance for the selection of clinical treatment options and monitoring of efficacy.
目前，HCV的常用检测方法包括病毒蛋白抗体检测、抗原检测、核酸检测等方法。病毒抗原抗体检测法常选用酶联免疫吸附试验(ELISA)，即将特异性抗原或抗体吸附于固相载体上，使其与待测样品中的相应抗体或抗原结合，然后加酶标记的抗原或抗体，再加底物显色，最后根据色泽深浅推算待测抗原或抗体的含量。但刚进入机体的病毒含量很低，病毒自身抗原含量往往低于ELISA法的检测下限，并且机体需要经过一段时间才会产生抗体，在此期间血液抗体检测呈阴性，抗原抗体通常是在感染后3-6周后才能被检测出来。近年来随着分子生物学的发展，基因检测技术已经在各实验室普及，尤其的荧光定量PCR技术，不仅有良好的特异性，还可以通过将病毒核酸扩增提高检测的灵敏度，缩短病毒检测的窗口期。At present, the common detection methods of HCV include viral protein antibody detection, antigen detection, nucleic acid detection and other methods. Viral antigen and antibody detection methods often use enzyme-linked immunosorbent assay (ELISA), that is, specific antigen or antibody is adsorbed on a solid phase carrier, so that it binds to the corresponding antibody or antigen in the sample to be tested, and then enzyme-labeled antigen or Antibody, add substrate to develop color, and finally calculate the content of the antigen or antibody to be tested according to the color depth. However, the amount of virus that has just entered the body is very low. The virus's antigen content is often lower than the detection limit of the ELISA method, and the body needs a period of time to produce antibodies. During this period, the blood antibody test is negative. The antigen antibody is usually after infection. It can only be detected after 3-6 weeks. In recent years, with the development of molecular biology, gene detection technology has been popularized in various laboratories. Especially fluorescent quantitative PCR technology not only has good specificity, but also can increase the sensitivity of detection and shorten virus detection by amplifying viral nucleic acid. Window period.
目前国内外已有多种基于荧光定量PCR技术的病毒检测试剂盒，但是国内的检测试剂盒定量检测下限较高，灵敏度较差。而国外进口的高灵敏诊断试剂盒价格过于昂贵，提高了医疗卫生成本。At present, there are a variety of virus detection kits based on fluorescent quantitative PCR technology at home and abroad, but the domestic detection kit has a high quantitative detection limit and poor sensitivity. The imported high-sensitivity diagnostic kits are too expensive, which raises the cost of medical and health care.
因此，目前迫切需要开发一种检测下限低，灵敏度高的检测丙型肝炎病毒的试剂盒及检测方法，以满足临床用药和检测诊断的要求。Therefore, there is an urgent need to develop a kit and a detection method for detecting hepatitis C virus with a low detection limit and a high sensitivity to meet the requirements of clinical medication and detection and diagnosis.
发明内容Summary of the invention
本申请的主要目的就是针对以上现状，提供一种检测丙型肝炎病毒核酸的引物、探针，以克服现有技术中的不足。The main purpose of the present application is to provide a primer and a probe for detecting hepatitis C virus nucleic acid in order to overcome the deficiencies in the prior art.
本申请的另一主要目的在于提供一种检测丙型肝炎病毒核酸的试剂盒。Another main objective of the present application is to provide a kit for detecting hepatitis C virus nucleic acid.
本申请的另一目的还在于提供前述试剂盒在检测丙型肝炎病毒核酸中的用途。Another object of the present application is also to provide the use of the aforementioned kit in the detection of hepatitis C virus nucleic acid.
本申请的另一目的还在于提供一种检测丙型肝炎病毒核酸的新方法。Another object of the present application is to provide a new method for detecting hepatitis C virus nucleic acid.
为实现前述发明目的，本申请采用的技术方案包括：In order to achieve the aforementioned object of the invention, the technical solutions adopted in this application include:
本申请实施例提供了一种检测丙型肝炎病毒核酸的引物对，其包括：The embodiments of the present application provide a primer pair for detecting hepatitis C virus nucleic acid, which includes:
第一引物，其序列如SEQ ID NO.1所示；The sequence of the first primer is shown in SEQ ID NO.1;
第二引物，其序列如SEQ ID NO.2所示。The sequence of the second primer is shown in SEQ ID NO.2.
本申请实施例还提供了一种检测丙型肝炎病毒核酸的探针，所述探针的序列如SEQ ID NO.3所示。The embodiment of the present application further provides a probe for detecting hepatitis C virus nucleic acid, and the sequence of the probe is shown in SEQ ID NO. 3.
本申请实施例还提供了一种检测丙型肝炎病毒核酸的试剂盒，其包括至少一组引物对和至少一条探针，其中一组引物对包括第一引物和第二引物，所述第一引物的序列如SEQ ID NO.1所示，所述第二引物的序列如SEQ ID NO.2所示，其中一条探针为前述的探针。An embodiment of the present application further provides a kit for detecting hepatitis C virus nucleic acid, which includes at least one set of primer pairs and at least one probe, wherein one set of primer pairs includes a first primer and a second primer, the first The sequence of the primer is shown in SEQ ID NO. 1, and the sequence of the second primer is shown in SEQ ID NO. 2. One of the probes is the aforementioned probe.
本申请实施例还提供了前述的试剂盒于制备检测丙型肝炎病毒核酸的产品中的用途。The embodiments of the present application also provide the use of the aforementioned kit in preparing products for detecting nucleic acid of hepatitis C virus.
进一步地，应用所述产品检测丙型肝炎病毒核酸的方法包括：Further, the method for detecting nucleic acid of hepatitis C virus using the product includes:
提供待检测的人丙型肝炎病毒核酸样本；Provide human hepatitis C virus nucleic acid samples to be tested;
采用样品处理装置，使所述待检测的核酸样本与PCR反应液混合，并使形成的混合液被液滴生成液包裹，形成包含单个核酸或者不包含核酸的液滴；A sample processing device is used to mix the nucleic acid sample to be detected with the PCR reaction solution, and the formed mixed solution is wrapped by the droplet generation solution to form droplets containing a single nucleic acid or not;
利用所述试剂盒所包含的引物对与探针对所述液滴进行PCR扩增；PCR amplification of the droplets by using the primer pair and the probe contained in the kit;
对PCR扩增产物进行检测。Detection of PCR amplification products.
本申请实施例还提供了一种包含前述试剂盒的产品，所述产品应用于丙型肝炎病毒核酸的检测方法，所述检测方法包括：The embodiments of the present application also provide a product containing the aforementioned kit, which is applied to a method for detecting hepatitis C virus nucleic acid, the detection method includes:
提供待检测的人丙型肝炎病毒核酸样本；Provide human hepatitis C virus nucleic acid samples to be tested;
采用样品处理装置，使所述待检测的核酸样本与PCR反应液混合，并使形成的混合液被液滴生成液包裹，形成包含单个核酸或者不包含核酸的液滴；A sample processing device is used to mix the nucleic acid sample to be detected with the PCR reaction solution, and the formed mixed solution is wrapped by the droplet generation solution to form droplets containing a single nucleic acid or not;
利用所述试剂盒所包含的引物对与探针对所述液滴进行PCR扩增；PCR amplification of the droplets by using the primer pair and the probe contained in the kit;
对PCR扩增产物进行检测。Detection of PCR amplification products.
与现有技术相比，本申请的优点包括：Compared with the prior art, the advantages of this application include:
1)本申请提供的高灵敏检测丙型肝炎病毒核酸的方法，检测结果的灵敏度及特异度更高，并可有效降低检测成本；1) The highly sensitive method for detecting hepatitis C virus nucleic acid provided by this application has higher sensitivity and specificity of the detection result, and can effectively reduce the detection cost;
2)本申请利用离心力使得核酸溶液与PCR反应液在流道中被包裹于液滴生成液中，形成单分子PCR反应体系，增加了检测的灵敏度，降低了检测下限，有利于尽早发现病毒感染；2) This application uses centrifugal force to make the nucleic acid solution and the PCR reaction solution wrapped in the droplet generation solution in the flow channel to form a single-molecule PCR reaction system, which increases the sensitivity of detection and lowers the lower limit of detection, which is conducive to early detection of viral infections;
3)在本申请中，由于经过处理后的液滴中仅包含单分子核酸模板，在扩增时受到的干扰减少，可以避免产生不确定的结果，提高反应特异性；3) In the present application, because the treated droplets contain only single-molecule nucleic acid templates, the interference received during amplification is reduced, which can avoid generating uncertain results and improve the specificity of the reaction;
4)与其他仅能检测部分常见丙型肝炎病毒亚型核酸的试剂盒不同，本申请提供的试剂盒通过优化引物设计，保证对于丙型肝炎病毒核酸的检测可以覆盖所有已知亚型，同时，本申请通过优化引物设计，使得扩增产物的片段长度在100-150bp之间，且退火温度在56-62℃之间，并通过赛默飞引物分析在线工具对所设计引物之间的相互作用进行分析，确保引物之间的相互作用不会对体系产生影响；4) Unlike other kits that can only detect some common hepatitis C virus subtype nucleic acids, the kit provided by this application optimizes primer design to ensure that the detection of hepatitis C virus nucleic acid can cover all known subtypes, while In this application, by optimizing the primer design, the fragment length of the amplified product is between 100-150 bp, and the annealing temperature is between 56-62° C., and the mutual interaction between the designed primers is performed by the Thermo Fisher online primer analysis tool Analysis of the role to ensure that the interaction between primers will not affect the system;
5)本申请通过优化引物探针，使得针对每种病毒的引物探针仅可检测该种病毒，不会与人基因组或者人乙型肝炎病毒(HBV)核酸、人疱疹病毒(EBV)核酸、巨细胞病毒(CMV)核酸等常见的病毒核酸发生交叉反应，保证了阳性结果的精确性。5) This application optimizes the primer probes so that the primer probes for each virus can only detect the virus, and will not interact with the human genome or human hepatitis B virus (HBV) nucleic acid, human herpes virus (EBV) nucleic acid, Common viral nucleic acids such as cytomegalovirus (CMV) nucleic acids cross-react, ensuring the accuracy of positive results.
附图说明BRIEF DESCRIPTION
图1是本申请一典型实施方案中采用的样品处理装置的结构示意图。FIG. 1 is a schematic structural diagram of a sample processing device used in a typical embodiment of the present application.
图2是本申请实施例1中阴性对照的液滴荧光检测结果。2 is a result of droplet fluorescence detection of the negative control in Example 1 of the present application.
图3是本申请实施例1中1号阳性样品的滴荧光检测结果。3 is a drop fluorescence detection result of No. 1 positive sample in Example 1 of the present application.
图4是本申请实施例1中2号阳性样品滴的荧光检测结果。4 is a result of fluorescence detection of the positive sample drop No. 2 in Example 1 of the present application.
图5是本申请实施例2中参考品A的液滴荧光强度检测结果。FIG. 5 is a result of detecting the fluorescence intensity of the droplet A of Reference A in Example 2 of the present application.
图6是本申请实施例2中参考品B的液滴荧光强度检测结果。6 is a result of detecting the fluorescence intensity of the droplet B of Reference B in Example 2 of the present application.
图7是本申请实施例2中参考品C的液滴荧光强度检测结果。7 is a result of detecting the fluorescence intensity of the droplet C of Reference C in Example 2 of the present application.
图8是本申请实施例2中参考品D的液滴荧光强度检测结果。FIG. 8 is the result of detecting the fluorescence intensity of the droplet of Reference D in Example 2 of the present application.
图9是本申请实施例3中HBV、CMV、EBV与HCV阳性样品的液滴荧光强度检测结果。9 is a detection result of droplet fluorescence intensity of positive samples of HBV, CMV, EBV and HCV in Example 3 of the present application.
具体实施方式detailed description
下文将对本申请的技术方案作更为详尽的解释说明。但是，应当理解，在本申请范围内，本申请的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。The technical solutions of the present application will be explained in more detail below. However, it should be understood that within the scope of the present application, the above technical features of the present application and the technical features specifically described in the following (eg, embodiments) can be combined with each other, thereby forming a new or preferred technical solution. Due to space limitations, I will not repeat them here.
本申请将根据具体事例做进一步的描述，但其仅为例证性的目的而不起到限制性作用。This application will be further described based on specific examples, but it is for illustrative purposes only and does not play a limiting role.
在对实例描述前，有必要提供一些备注说明：Before describing the example, it is necessary to provide some remarks:
采用不同厂家、不同批次的试剂会造成实验结果的差异，属于正常现象。The use of different manufacturers and different batches of reagents will cause differences in experimental results, which is normal.
在进行小规模实验时，为保证平行实验间的重复性，建议配置试剂后，充分混匀并分装，以保证每次实验试剂的均一性。When conducting small-scale experiments, in order to ensure the repeatability between parallel experiments, it is recommended that after the reagents are configured, they are thoroughly mixed and divided to ensure the uniformity of the reagents in each experiment.
本申请实施例的一个方面提供了一种检测丙型肝炎病毒核酸的引物对，其包括：One aspect of the embodiments of the present application provides a primer pair for detecting hepatitis C virus nucleic acid, which includes:
第一引物，即正向引物，其序列如SEQ ID NO.1所示；The first primer, the forward primer, whose sequence is shown in SEQ ID NO.1;
第二引物，即反向引物，其序列如SEQ ID NO.2所示。The second primer, the reverse primer, has the sequence shown in SEQ ID NO.2.
进一步地，所述引物对所含引物序列如下表：Further, the primer sequences contained in the primer pair are as follows:
HCV-F(正向引物)HCV-F (forward primer) 5'-ATTGCAGCAGTCAGAGCCCT-3'5'-ATTGCAGCAGTCAGAGCCCT-3'
HCV-R(反向引物)HCV-R (reverse primer) 5'-GCTCCTTTCTAAGCCTTCTTCACT-3'5'-GCTCCTTTCTAAGCCTTCTTCACT-3'
与其他仅能检测部分常见丙型肝炎病毒亚型核酸的试剂盒不同，本申请提供的试剂盒通过优化引物设计，使丙型肝炎病毒核酸的检测可以覆盖所有已知亚型，同时，本申请通过优化引物设计，使得扩增产物的片段长度在100-150bp之间，且退火温度在56-62℃之间，并通过赛默飞引物分析在线工具对所设计引物之间的相互作用进行分析，确保引物之间的相互作用不会对体系产生影响。Unlike other kits that can only detect some common hepatitis C virus subtype nucleic acids, the kit provided by this application optimizes the primer design so that the detection of hepatitis C virus nucleic acid can cover all known subtypes. At the same time, this application By optimizing the primer design, the fragment length of the amplified product is between 100-150bp, and the annealing temperature is between 56-62℃, and the interaction between the designed primers is analyzed by the Thermo Fisher primer analysis online tool To ensure that the interaction between primers does not affect the system.
本申请实施例的另一个方面还提供了一种检测丙型肝炎病毒核酸的探针，所述探针的序列如SEQ ID NO.3所示。Another aspect of the embodiments of the present application further provides a probe for detecting hepatitis C virus nucleic acid, and the sequence of the probe is shown in SEQ ID NO. 3.
进一步地，所述探针的序列如下表：Further, the sequence of the probe is as follows:
HCV-ProbeHCV-Probe 5'-HEX-CATCCTGCTGCTATGCCTCATCTTCTT-BHQ-3'5'-HEX-CATCCTGCTGCTATGCCTCATCTTCTT-BHQ-3'
本申请通过优化引物探针，使得针对每种病毒的引物探针仅可检测该种病毒，不会与人基因组或者人乙型肝炎病毒(HBV)核酸、人疱疹病毒(EBV)核酸、巨细胞病毒(CMV)核酸等常见的病毒核酸发生交叉反应，保证了阳性结果的精确性。This application optimizes the primer probe so that the primer probe for each virus can only detect the virus, and will not interact with the human genome or human hepatitis B virus (HBV) nucleic acid, human herpes virus (EBV) nucleic acid, megacell Common viral nucleic acids such as viral (CMV) nucleic acids cross-react, ensuring the accuracy of positive results.
本申请实施例的另一个方面还提供了一种检测丙型肝炎病毒核酸的试剂盒，其包括至少一组引物对和至少条一探针，其中一组引物对包括第一引物和第二引物，所述第一引物的序列如SEQ ID NO.1所示，所述第二引物的序列如SEQ ID NO.2所示，其中所述探针的序列如SEQ ID NO.3所示。Another aspect of the embodiments of the present application further provides a kit for detecting hepatitis C virus nucleic acid, which includes at least one set of primer pairs and at least one probe, wherein one set of primer pairs includes a first primer and a second primer The sequence of the first primer is shown in SEQ ID NO.1, the sequence of the second primer is shown in SEQ ID NO.2, and the sequence of the probe is shown in SEQ ID NO.3.
在一些实施例中，所述试剂盒还包括病毒内标引物对、内标探针，其中所述病毒内标引物对包括第一内标引物(即内标正向引物)和第二内标引物(即内标反向引物)，所述第一内标引物的序列如SEQ ID NO.4所示，所述第二内标引物的序列如SEQ ID NO.5所示，其中内标探针的序列如SEQ ID NO.6所示。In some embodiments, the kit further includes a viral internal standard primer pair and an internal standard probe, wherein the viral internal standard primer pair includes a first internal standard primer (ie, internal standard forward primer) and a first Two internal standard primers (ie internal standard reverse primers), the sequence of the first internal standard primer is shown in SEQ ID NO.4, and the sequence of the second internal standard primer is shown in SEQ ID NO.5 The sequence of the internal standard probe is shown in SEQ ID NO.6.
进一步地，本申请试剂盒中内标引物混合液中包含的内标正向引物及反向引物的序列如下表：Further, the sequences of the internal standard forward primer and the reverse primer contained in the internal standard primer mixed solution in the kit of the present application are as follows:
内标-F(内标正向引物)Internal standard-F (internal standard forward primer) 5'-ATTAACCTTATGTGTGACAT-3'5'-ATTAACCTTATGTGTGACAT-3'
内标-R(内标反向引物)Internal standard-R (internal standard reverse primer) 5'-CATATTCGTCCACAAAATGATTCTG-3'5'-CATATTCGTCCACAAAATGATTCTG-3'
进一步地，内标探针溶液中内标探针的序列为：5'-Cy5-5-CTGTATCGTCAAGGCACT-BHQ-3'。Further, the sequence of the internal standard probe in the internal standard probe solution is: 5'-Cy5-5-CTGTATCGTCAAGGCACT-BHQ-3'.
在一些实施例中，所述试剂盒还包括PCR扩增检测的常规组件。In some embodiments, the kit also includes conventional components for PCR amplification detection.
进一步地，所述PCR扩增检测的常规组件包括PCR反应用缓冲液、三磷酸碱基脱氧核苷酸混合液(2.5mMdNTPs)、DNA聚合酶、RNA逆转录酶、尿嘧啶DNA糖基化酶、水以及液滴生成液。Further, the conventional components for PCR amplification detection include a buffer solution for PCR reaction, a mixture of base deoxynucleotide triphosphates (2.5 mM dNTPs), DNA polymerase, RNA reverse transcriptase, and uracil DNA glycosylase , Water and liquid droplets.
更进一步地，所述PCR反应用缓冲液包含20mM氯化钾、50mM的PH8.0Tris-HCl、1.5mM镁离子。Furthermore, the buffer for PCR reaction contains 20 mM potassium chloride, 50 mM PH8.0 Tris-HCl, and 1.5 mM magnesium ions.
更进一步地，dNTPs中包含dNTP与dUTP。Furthermore, dNTPs include dNTP and dUTP.
在一些更为具体的实施例中，所述试剂盒具体包括：PCR反应用缓冲液、2.5mMdNTPs、靶核苷酸引物混合液、靶核苷酸探针溶液、内标引物混合液、内标探针溶液、5U/μL耐热DNA聚合酶、5U/μLRNA逆转录酶、5U/μL尿嘧啶DNA糖基化酶、水以及液滴生成液。In some more specific embodiments, the kit specifically includes: PCR reaction buffer, 2.5 mM dNTPs, target nucleotide primer mixed solution, target nucleotide probe solution, internal standard primer mixed solution, internal Standard probe solution, 5U/μL heat-resistant DNA polymerase, 5U/μL RNA reverse transcriptase, 5U/μL uracil DNA glycosylase, water, and droplet generation solution.
其中，所述靶核苷酸引物混合液包含前述的第一引物和第二引物。Wherein, the target nucleotide primer mixed solution includes the aforementioned first primer and second primer.
其中，所述靶核苷酸探针溶液包含HCV特异性探针。Wherein, the target nucleotide probe solution contains HCV specific probe.
其中，所述内标引物混合液中包含内标正向引物及内标反向引物。Wherein, the internal standard primer mixed solution includes an internal standard forward primer and an internal standard reverse primer.
本申请实施例的另一个方面还提供了前述试剂盒于制备检测丙型肝炎病毒核酸的产品中的用途。Another aspect of the embodiments of the present application also provides the use of the aforementioned kit in preparing a product for detecting hepatitis C virus nucleic acid.
进一步地，应用所述产品检测丙型肝炎病毒的方法包括：Further, the method for detecting hepatitis C virus using the product includes:
提供待检测的人丙型肝炎病毒核酸样本；Provide human hepatitis C virus nucleic acid samples to be tested;
采用样品处理装置，使所述待检测的核酸样本与PCR反应液混合，并使形成的混合液被液滴生成液包裹，形成包含单个核酸或者不包含核酸的液滴；A sample processing device is used to mix the nucleic acid sample to be detected with the PCR reaction solution, and the formed mixed solution is wrapped by the droplet generation solution to form droplets containing a single nucleic acid or not;
利用所述试剂盒所包含的引物对与探针对所述液滴进行PCR扩增；PCR amplification of the droplets by using the primer pair and the probe contained in the kit;
对PCR扩增产物进行检测。Detection of PCR amplification products.
在一些实施例中，所述方法具体包括：In some embodiments, the method specifically includes:
利用所述的试剂盒所包含的引物对与探针对所述液滴进行液滴PCR扩增；Using the primer pair and the probe contained in the kit to perform droplet PCR amplification on the droplet;
对PCR扩增后的液滴的荧光强度进行检测，根据该荧光强度判断核酸样本是否包含丙型肝炎病毒核酸。The fluorescence intensity of the PCR amplified droplets is detected, and whether the nucleic acid sample contains hepatitis C virus nucleic acid is determined based on the fluorescence intensity.
进一步地，所述样品处理装置包括定位孔、进样孔、试剂孔、反应孔以及连接流道。本处理装置为PC材料制成，长40mm，宽20mm，厚度2mm，其中进样孔容积为50μL，试剂孔容积为50μL，反应孔容积为100μL。Further, the sample processing device includes a positioning hole, a sample injection hole, a reagent hole, a reaction hole, and a connecting flow channel. The processing device is made of PC material, with a length of 40 mm, a width of 20 mm, and a thickness of 2 mm. The volume of the injection well is 50 μL, the volume of the reagent well is 50 μL, and the volume of the reaction well is 100 μL.
在一些更为具体的实施例中，所述检测方法具体包括：将液滴生成液加入所述试剂孔，将待检测的核酸样本与PCR反应液加入所述进样孔，在离心力的作用下使进样孔与试剂孔中液体通过连接流道混合，并使形成的混合液被液滴生成液包裹，形成包含单个核酸或者不包含核酸的液滴，并进入反应孔。In some more specific embodiments, the detection method specifically includes: adding a droplet generation solution to the reagent well, adding the nucleic acid sample to be detected and the PCR reaction solution to the injection well, under the action of centrifugal force The liquid in the injection well and the reagent well are mixed through the connecting flow channel, and the formed mixed liquid is wrapped by the droplet generation liquid to form droplets containing a single nucleic acid or not, and enters the reaction well.
进一步地，所述检测方法可以包括：将样品处理装置通过定位孔安装在离心机上，将液滴生成液加入试剂孔，将提取好的核酸溶液与PCR反应液按比例混匀加入进样孔。以3000rpm顺 时针离心，在离心力的作用下进样孔与试剂孔中液体通过流道混合，让核酸样品被包裹进PCR反应液中形成液滴，最终收集到反应孔中。Further, the detection method may include: installing the sample processing device on the centrifuge through the positioning hole, adding the droplet generation solution to the reagent hole, and mixing the extracted nucleic acid solution and the PCR reaction solution in proportion to the injection hole. Centrifuge at 3000 rpm clockwise. Under the action of centrifugal force, the liquid in the sample well and the reagent well are mixed through the flow channel, so that the nucleic acid sample is wrapped into the PCR reaction solution to form droplets, and finally collected in the reaction well.
进一步地，所述液滴生成液包括矿物油等油相物质。Further, the droplet generation liquid includes oil phase substances such as mineral oil.
在一些较为具体的实施方案中，所述方法具体可以包括以下步骤：In some more specific embodiments, the method may specifically include the following steps:
将PCR反应液预先从冰箱中取出解冻，完全融化后涡旋混匀并瞬时离心。取10-19μL反应液置于PCR管中，向其中加入1-10μL新提取的核酸样品，用枪头混匀，保证总液体量为20μL。吸取20μL混合液体加入进样孔中，吸取20μL液滴生成液加入试剂孔中。通过定位孔，将处理装置固定于离心机适配器上，需注意在安装时保持中心对称。以3000rpm离心3分钟，使得核酸样品与PCR反应液在离心力的作用下流入流道中，并在流道中被液滴生成液包裹，形成仅包含单个核酸或者不包含核酸的液滴。The PCR reaction solution was removed from the refrigerator in advance and thawed, and after thawing, the mixture was vortexed and centrifuged. Take 10-19 μL of the reaction solution in a PCR tube, add 1-10 μL of the newly extracted nucleic acid sample to it, and mix with a pipette tip to ensure that the total amount of liquid is 20 μL. Pipette 20 μL of the mixed liquid into the injection well, and 20 μL of the droplet generation liquid into the reagent well. Fix the processing device on the centrifuge adapter through the positioning hole, and pay attention to keep the center symmetrical during installation. Centrifuge at 3000 rpm for 3 minutes, so that the nucleic acid sample and the PCR reaction solution flow into the flow channel under the action of centrifugal force, and are surrounded by the droplet generation liquid in the flow channel, forming droplets containing only a single nucleic acid or not.
将反应孔中生成的液滴转移到PCR管中，放入聚合酶链式反应扩增仪(PCR扩增仪)中，按下列表1的条件进行扩增：Transfer the droplets generated in the reaction well to a PCR tube, put it into a polymerase chain reaction amplifier (PCR amplifier), and perform amplification according to the conditions in Table 1:
表1：PCR扩增条件Table 1: PCR amplification conditions
Figure PCTCN2019119046-appb-000001
Figure PCTCN2019119046-appb-000001
将扩增产物使用8通道液滴阅读***进行分析，首先根据未加入模板的空白对照确定背景荧光强度，设定超过背景荧光强度的液滴为阳性液滴，测量每个反应液滴的荧光强度，并计数阳性液滴个数。The amplified products were analyzed using an 8-channel droplet reading system. First, the background fluorescence intensity was determined according to the blank control without the template added. Droplets exceeding the background fluorescence intensity were set as positive droplets, and the fluorescence intensity of each reaction droplet was measured. And count the number of positive droplets.
通过计数阳性液滴个数与确定加入核酸样品体积，则可以计算核酸样品中丙型肝炎病毒核酸数量。By counting the number of positive droplets and determining the volume of the nucleic acid sample added, the amount of hepatitis C virus nucleic acid in the nucleic acid sample can be calculated.
本申请利用离心力使得核酸溶液与PCR反应液在流道中被包裹于液滴生成液中，形成单分子PCR反应体系，增加了检测的灵敏度，降低了检测下限，有利于尽早发现病毒感染。The present application uses centrifugal force to make the nucleic acid solution and the PCR reaction solution wrapped in the droplet generation solution in the flow channel to form a single-molecule PCR reaction system, which increases the sensitivity of detection and lowers the lower limit of detection, which is helpful for early detection of virus infection.
本申请实施例的另一个方面还提供了一种包含前述试剂盒的产品，所述产品应用于丙型肝 炎病毒核酸的检测方法，所述检测方法包括：Another aspect of the embodiments of the present application also provides a product containing the aforementioned kit, which is applied to a method for detecting hepatitis C virus nucleic acid, the detection method includes:
提供待检测的人丙型肝炎病毒核酸样本；Provide human hepatitis C virus nucleic acid samples to be tested;
采用样品处理装置，使所述待检测的核酸样本与PCR反应液混合，并使形成的混合液被液滴生成液包裹，形成包含单个核酸或者不包含核酸的液滴；A sample processing device is used to mix the nucleic acid sample to be detected with the PCR reaction solution, and the formed mixed solution is wrapped by the droplet generation solution to form droplets containing a single nucleic acid or not;
利用所述试剂盒所包含的引物对与探针对所述液滴进行PCR扩增；PCR amplification of the droplets by using the primer pair and the probe contained in the kit;
对PCR扩增产物进行检测。Detection of PCR amplification products.
进一步地，所述产品包括：利用所述的试剂盒所包含的引物对与探针对所述液滴进行液滴PCR扩增；Further, the product includes: performing droplet PCR amplification of the droplet using the primer pair and the probe contained in the kit;
对PCR扩增后的液滴的荧光强度进行检测，根据该荧光强度判断核酸样本是否包含丙型肝炎病毒核酸。The fluorescence intensity of the PCR amplified droplets is detected, and whether the nucleic acid sample contains hepatitis C virus nucleic acid is determined based on the fluorescence intensity.
以下结合附图和若干较佳实施例对本申请的技术方案作进一步的解释说明，但其中的实验条件和设定参数不应视为对本申请基本技术方案的局限。并且本申请的保护范围不限于下述的实施例。The technical solution of the present application will be further explained below with reference to the drawings and several preferred embodiments, but the experimental conditions and setting parameters therein should not be considered as limitations on the basic technical solution of the present application. And the protection scope of the present application is not limited to the following embodiments.
实施例1Example 1
1.本实施例所用样品包括HCV阳性核酸溶液2例，以及阴性核酸溶液1例。1. The samples used in this example include 2 cases of HCV positive nucleic acid solution and 1 case of negative nucleic acid solution.
2.PCR反应体系制备2. Preparation of PCR reaction system
(1)将PCR反应液预先从冰箱中取出解冻，完全融化后涡旋混匀并瞬时离心；(1) Remove the PCR reaction solution from the refrigerator in advance and thaw it, after completely thawing, vortex to mix and centrifuge instantly;
(2)取19μL PCR反应液置于PCR管中，向其中加入1μL核酸样品，用枪头混匀；(2) Take 19 μL of PCR reaction solution in a PCR tube, add 1 μL of nucleic acid sample to it, and mix with a pipette tip;
(3)吸取20μL混合液体加入进样孔中，吸取20μL液滴生成液加入试剂孔中；(3) Pipette 20μL of mixed liquid into the injection well, and pipette 20μL of droplet generation liquid into the reagent well;
(4)通过定位孔，将样品处理装置固定于离心机适配器上，需注意在安装处理装置时保持中心对称；(4) Fix the sample processing device on the centrifuge adapter through the positioning hole, pay attention to keep the center symmetry when installing the processing device;
(5)以3000rpm离心3分钟，使得核酸样品与PCR反应液在离心力的作用下流入流道中，并在流道中被液滴生成液包裹，形成仅包含单个核酸或者不包含核酸的液滴；(5) Centrifuge at 3000 rpm for 3 minutes, so that the nucleic acid sample and the PCR reaction solution flow into the flow channel under the action of centrifugal force, and are surrounded by the droplet generation liquid in the flow channel to form droplets containing only a single nucleic acid or no nucleic acid;
(6)将反应孔中生成的液滴转移到PCR管中。(6) Transfer the droplets generated in the reaction well to the PCR tube.
3.PCR扩增3. PCR amplification
扩增在聚合酶链式反应扩增仪(PCR扩增仪)中进行，反应程序如下表所示：The amplification is performed in a polymerase chain reaction amplifier (PCR amplifier), and the reaction procedure is shown in the following table:
Figure PCTCN2019119046-appb-000002
Figure PCTCN2019119046-appb-000002
Figure PCTCN2019119046-appb-000003
Figure PCTCN2019119046-appb-000003
4.结果分析4. Results analysis
将扩增产物使用8通道液滴阅读***(苏州昊通生物科技有限公司)进行分析，首先分析阴性对照中各个液滴的荧光强度，以此确定背景荧光强度。再通过设定超过背景荧光强度的液滴为阳性液滴，测量每个反应液滴的荧光强度，并计数阳性液滴个数。每个样品的检测结果以每个液滴检测时的次序为横坐标，以该液滴的荧光强度为纵坐标，绘制每个液滴荧光强度的散点图。通过阴性对照确定的背景荧光值确定荧光强度阈值线，荧光强度超过阈值线的液滴即为阳性液滴。The amplified products were analyzed using an 8-channel droplet reading system (Suzhou Haotong Biotechnology Co., Ltd.). First, the fluorescence intensity of each droplet in the negative control was analyzed to determine the background fluorescence intensity. Then, by setting the droplets that exceed the background fluorescence intensity as positive droplets, the fluorescence intensity of each reaction droplet is measured, and the number of positive droplets is counted. The detection result of each sample takes the order of the detection of each droplet as the abscissa and the fluorescence intensity of the droplet as the ordinate, and plots a scatter plot of the fluorescence intensity of each droplet. The fluorescence intensity threshold line is determined by the background fluorescence value determined by the negative control, and the droplet whose fluorescence intensity exceeds the threshold line is the positive droplet.
由于本次实验中均加入1μL核酸样品，所以每个样品检测的阳性液滴个数，就代表了每1μL核酸样品中包含的HCV病毒核酸拷贝数。Since 1 μL of nucleic acid samples are added in this experiment, the number of positive droplets detected in each sample represents the number of copies of HCV nucleic acid contained in each 1 μL of nucleic acid sample.
如图2为阴性对照液滴荧光强度检测结果。As shown in Figure 2 is the negative control droplet fluorescence intensity detection results.
如图3为1号阳性样品液滴荧光强度检测结果，可以得出该核酸样品中HCV病毒核酸载量为668copies/μL。As shown in Fig. 3, the result of the detection of the fluorescence intensity of the droplet of the positive sample No. 1 can be obtained that the HCV viral nucleic acid load in the nucleic acid sample is 668 copies/μL.
如图4为2号阳性样品液滴荧光强度检测结果，可以得出该核酸样品中HCV病毒核酸载量为421copies/μL。As shown in Fig. 4 for the detection result of the fluorescence intensity of droplet No. 2 positive sample, it can be concluded that the HCV viral nucleic acid load in the nucleic acid sample is 421 copies/μL.
实施例2Example 2
1.本实施例所用样品包括，4种浓度的含有HCV特异性序列的质粒定量参考品，其中质粒浓度分别为A(10copies/μL)、B(5copies/μL)、C(2copies/μL)和D(1copies/μL)。1. The samples used in this example include 4 kinds of plasmid quantitative reference products containing HCV-specific sequences, in which the plasmid concentrations are A (10copies/μL), B (5copies/μL), C (2copies/μL) and D(1copies/μL).
2.PCR反应体系制备2. Preparation of PCR reaction system
(1)将PCR反应液预先从冰箱中取出解冻，完全融化后涡旋混匀并瞬时离心；(1) Remove the PCR reaction solution from the refrigerator in advance and thaw it, after completely thawing, vortex to mix and centrifuge instantly;
(2)取10μLPCR反应液置于PCR管中，向其中加入10μL核酸样品，用枪头混匀；(2) Place 10 μL of PCR reaction solution in a PCR tube, add 10 μL of nucleic acid sample to it, and mix with a pipette tip;
(3)吸取20μL混合液体加入进样孔中，吸取20μL液滴生成液加入试剂孔中；(3) Pipette 20μL of mixed liquid into the injection well, and pipette 20μL of droplet generation liquid into the reagent well;
(4)通过定位孔，将样品处理装置固定于离心机适配器上，需注意在安装处理装置时保持中心对称；(4) Fix the sample processing device on the centrifuge adapter through the positioning hole, pay attention to keep the center symmetry when installing the processing device;
(5)以3000rpm离心3分钟，使得核酸样品与PCR反应液在离心力的作用下流入流道中，并在流道中被液滴生成液包裹，形成仅包含单个核酸或者不包含核酸的液滴；(5) Centrifuge at 3000 rpm for 3 minutes, so that the nucleic acid sample and the PCR reaction solution flow into the flow channel under the action of centrifugal force, and are surrounded by the droplet generation liquid in the flow channel to form droplets containing only a single nucleic acid or no nucleic acid;
(6)将反应孔中生成的液滴转移到PCR管中。(6) Transfer the droplets generated in the reaction well to the PCR tube.
3.PCR扩增3. PCR amplification
扩增在聚合酶链式反应扩增仪(PCR扩增仪)中进行，反应程序如下表所示：The amplification is performed in a polymerase chain reaction amplifier (PCR amplifier), and the reaction procedure is shown in the following table:
Figure PCTCN2019119046-appb-000004
Figure PCTCN2019119046-appb-000004
4.结果分析4. Results analysis
将扩增产物使用8通道液滴阅读***(苏州昊通生物科技有限公司)进行分析，首先分析阴性对照中各个液滴的荧光强度，以此确定背景荧光强度。再通过设定超过背景荧光强度的液滴为阳性液滴，测量每个反应液滴的荧光强度，并计数阳性液滴个数。每个样品的检测结果以每个液滴检测时的次序为横坐标，以该液滴的荧光强度为纵坐标，绘制每个液滴荧光强度的散点图。通过阴性对照确定的背景荧光值确定荧光强度阈值线，荧光强度超过阈值线的液滴即为阳性液滴。The amplified products were analyzed using an 8-channel droplet reading system (Suzhou Haotong Biotechnology Co., Ltd.). First, the fluorescence intensity of each droplet in the negative control was analyzed to determine the background fluorescence intensity. Then, by setting the droplets that exceed the background fluorescence intensity as positive droplets, the fluorescence intensity of each reaction droplet is measured, and the number of positive droplets is counted. The detection result of each sample takes the order of the detection of each droplet as the abscissa and the fluorescence intensity of the droplet as the ordinate, and plots a scatter plot of the fluorescence intensity of each droplet. The fluorescence intensity threshold line is determined by the background fluorescence value determined by the negative control, and the droplet whose fluorescence intensity exceeds the threshold line is the positive droplet.
由于本次实验中均加入10μL核酸样品，所以每个样品检测的阳性液滴个数，就代表了每10μL核酸样品中包含的HCV病毒核酸拷贝数。Since 10 μL of nucleic acid samples are added in this experiment, the number of positive droplets detected in each sample represents the number of copies of HCV virus nucleic acid contained in each 10 μL of nucleic acid samples.
如图5为参考品A的液滴荧光强度检测结果，可以得出该参考品中HCV病毒核酸载量为10copies/μL。As shown in Fig. 5 is the result of the detection of the fluorescence intensity of the droplet of Reference A, it can be concluded that the nucleic acid load of HCV virus in this Reference is 10 copies/μL.
如图6为参考品B的液滴荧光强度检测结果，可以得出该参考品中HCV病毒核酸载量为5copies/μL。As shown in Figure 6 for the detection result of the fluorescence intensity of the droplet of reference B, it can be concluded that the nucleic acid load of HCV virus in this reference is 5 copies/μL.
如图7为参考品C的液滴荧光强度检测结果，可以得出该参考品中HCV病毒核酸载量为2copies/μL。As shown in Figure 7 is the result of the detection of the fluorescence intensity of the droplet of the reference C, it can be concluded that the nucleic acid load of the HCV virus in the reference is 2copies/μL.
如图8为参考品D的液滴荧光强度检测结果，可以得出该参考品中HCV病毒核酸载量为 1copies/μL。As shown in Fig. 8 for the results of the detection of the fluorescence intensity of the droplet of Reference D, it can be concluded that the nucleic acid load of HCV virus in this Reference is 1 copies/μL.
实施例3Example 3
1.本实施例所用样品包含人乙型肝炎病毒(HBV)核酸阳性、人疱疹病毒(EBV)核酸阳性、巨细胞病毒(CMV)核酸阳性以及HCV核酸阳性样品各1例。1. The samples used in this example included one sample of human hepatitis B virus (HBV) nucleic acid positive, human herpes virus (EBV) nucleic acid positive, cytomegalovirus (CMV) nucleic acid positive and HCV nucleic acid positive samples.
2.PCR反应体系制备2. Preparation of PCR reaction system
(1)将PCR反应液预先从冰箱中取出解冻，完全融化后涡旋混匀并瞬时离心；(1) Remove the PCR reaction solution from the refrigerator in advance and thaw it, after completely thawing, vortex to mix and centrifuge instantly;
(2)取10μL PCR反应液置于PCR管中，向其中加入10μL核酸样品，用枪头混匀；(2) Take 10 μL of PCR reaction solution in a PCR tube, add 10 μL of nucleic acid sample to it, and mix with a pipette tip;
(3)吸取20μL混合液体加入进样孔中，吸取20μL液滴生成液加入试剂孔中；(3) Pipette 20μL of mixed liquid into the injection well, and pipette 20μL of droplet generation liquid into the reagent well;
(4)通过定位孔，将样品处理装置固定于离心机适配器上，需注意在安装处理装置时保持中心对称；(4) Fix the sample processing device on the centrifuge adapter through the positioning hole, pay attention to keep the center symmetry when installing the processing device;
(5)以3000rpm离心3分钟，使得核酸样品与PCR反应液在离心力的作用下流入流道中，并在流道中被液滴生成液包裹，形成仅包含单个核酸或者不包含核酸的液滴；(5) Centrifuge at 3000 rpm for 3 minutes, so that the nucleic acid sample and the PCR reaction solution flow into the flow channel under the action of centrifugal force, and are surrounded by the droplet generation liquid in the flow channel to form droplets containing only a single nucleic acid or no nucleic acid;
(6)将反应孔中生成的液滴转移到PCR管中。(6) Transfer the droplets generated in the reaction well to the PCR tube.
3.PCR扩增3. PCR amplification
扩增在聚合酶链式反应扩增仪(PCR扩增仪)中进行，反应程序如下表所示：The amplification is performed in a polymerase chain reaction amplifier (PCR amplifier), and the reaction procedure is shown in the following table:
Figure PCTCN2019119046-appb-000005
Figure PCTCN2019119046-appb-000005
4.结果分析4. Results analysis
将扩增产物使用8通道液滴阅读***(苏州昊通生物科技有限公司)进行分析，首先分析阴性对照中各个液滴的荧光强度，以此确定背景荧光强度。再通过设定超过背景荧光强度的液滴为阳性液滴，测量每个反应液滴的荧光强度，并计数阳性液滴个数。每个样品的检测结果以每个液滴检测时的次序为横坐标，以该液滴的荧光强度为纵坐标，绘制每个液滴荧光强度的散 点图。通过阴性对照确定的背景荧光值确定荧光强度阈值线，荧光强度超过阈值线的液滴即为阳性液滴。The amplified products were analyzed using an 8-channel droplet reading system (Suzhou Haotong Biotechnology Co., Ltd.). First, the fluorescence intensity of each droplet in the negative control was analyzed to determine the background fluorescence intensity. Then, by setting the droplets that exceed the background fluorescence intensity as positive droplets, the fluorescence intensity of each reaction droplet is measured, and the number of positive droplets is counted. The detection result of each sample takes the order of the detection of each droplet as the abscissa, and the fluorescence intensity of the droplet as the ordinate, and plots a scatter plot of the fluorescence intensity of each droplet. The fluorescence intensity threshold line is determined by the background fluorescence value determined by the negative control, and the droplet whose fluorescence intensity exceeds the threshold line is the positive droplet.
由于本次实验中均加入10μL核酸样品，所以每个样品检测的阳性液滴个数，就代表了每10μL核酸样品中包含的HCV病毒核酸拷贝数。Since 10 μL of nucleic acid samples are added in this experiment, the number of positive droplets detected in each sample represents the number of copies of HCV virus nucleic acid contained in each 10 μL of nucleic acid samples.
如图9从左到右依次为HBV、CMV、EBV与HCV阳性样品的液滴荧光强度检测结果，可以得出与HBV、CMV、EBV病毒核酸未发生交叉反应，HCV阳性样品中病毒核酸载量为32.3copies/μL。As shown in Figure 9 from left to right, HBV, CMV, EBV and HCV positive sample droplet fluorescence intensity detection results, it can be concluded that there is no cross-reaction with HBV, CMV, EBV viral nucleic acids, viral nucleic acid load in HCV positive samples 32.3copies/μL.
综上所述，藉由上述技术方案，本申请的试剂盒检测下限低，灵敏度高，特异性好，本申请的检测方法简单快速，检测结果的灵敏度及特异度更高，并可有效降低检测成本。In summary, with the above technical solutions, the kit of the present application has a low detection limit, high sensitivity and good specificity, the detection method of the present application is simple and fast, the sensitivity and specificity of the test results are higher, and the detection can be effectively reduced cost.
应当理解，上述实施例仅为说明本申请的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本申请的内容并据以实施，并不能以此限制本申请的保护范围。凡根据本申请精神实质所作的等效变化或修饰，都应涵盖在本申请的保护范围之内。It should be understood that the above embodiments are only to illustrate the technical concept and characteristics of the present application, and the purpose thereof is to enable those familiar with the technology to understand the content of the present application and implement it accordingly, but not to limit the scope of protection of the present application. All equivalent changes or modifications based on the spirit of this application should be covered within the scope of protection of this application.
Figure PCTCN2019119046-appb-000006
Figure PCTCN2019119046-appb-000006
Figure PCTCN2019119046-appb-000007
Figure PCTCN2019119046-appb-000007

Claims (10)

  1. 一种检测丙型肝炎病毒核酸的引物对，其特征在于包括：A primer pair for detecting hepatitis C virus nucleic acid, which is characterized by comprising:
    第一引物，其序列如SEQ ID NO.1所示；The sequence of the first primer is shown in SEQ ID NO.1;
    第二引物，其序列如SEQ ID NO.2所示。The sequence of the second primer is shown in SEQ ID NO.2.
  2. 一种检测丙型肝炎病毒核酸的探针，其特征在于，所述探针的序列如SEQ ID NO.3所示。A probe for detecting hepatitis C virus nucleic acid, characterized in that the sequence of the probe is shown in SEQ ID NO.3.
  3. 一种检测丙型肝炎病毒核酸的试剂盒，其特征在于包括至少一组引物对和至少一条探针，其中一组引物对包括第一引物和第二引物，所述第一引物的序列如SEQ ID NO.1所示，所述第二引物的序列如SEQ ID NO.2所示，其中一条探针为权利要求2所述的探针。A kit for detecting hepatitis C virus nucleic acid, characterized by comprising at least one set of primer pairs and at least one probe, wherein one set of primer pairs includes a first primer and a second primer, the sequence of the first primer is as SEQ As shown in ID NO.1, the sequence of the second primer is shown in SEQ ID NO.2, and one of the probes is the probe of claim 2.
  4. 如权利要求3所述的试剂盒，其特征在于还包括病毒内标引物对、内标探针，其中所述病毒内标引物对包括第一内标引物和第二内标引物，所述第一内标引物的序列如SEQ ID NO.4所示，所述第二内标引物的序列如SEQ ID NO.5所示，其中内标探针的序列如SEQ ID NO.6所示。The kit according to claim 3, further comprising a viral internal standard primer pair and an internal standard probe, wherein the viral internal standard primer pair includes a first internal standard primer and a second internal standard primer The sequence of the first internal standard primer is shown in SEQ ID NO.4, the sequence of the second internal standard primer is shown in SEQ ID NO.5, and the sequence of the internal standard probe is shown in SEQ ID NO .6 shown.
  5. 如权利要求3或4所述的试剂盒，其特征在于还包括PCR扩增检测的常规组件；优选的，所述PCR扩增检测的常规组件包括PCR反应用缓冲液、三磷酸碱基脱氧核苷酸混合液、DNA聚合酶、RNA逆转录酶、尿嘧啶DNA糖基化酶、水以及液滴生成液；尤其优选的，所述PCR反应用缓冲液包含氯化钾、Tris-HCl和镁离子。The kit according to claim 3 or 4, further comprising a conventional component for PCR amplification detection; preferably, the conventional component for PCR amplification detection includes a buffer for PCR reaction, a base deoxyribonucleic acid triphosphate Glycoside mixture, DNA polymerase, RNA reverse transcriptase, uracil DNA glycosylase, water and droplet generation liquid; particularly preferably, the PCR reaction buffer contains potassium chloride, Tris-HCl and magnesium ion.
  6. 权利要求3-5中任一项所述的试剂盒于制备检测丙型肝炎病毒核酸的产品中的用途。Use of the kit according to any one of claims 3-5 in the preparation of products for detecting nucleic acid of hepatitis C virus.
  7. 如权利要求6所述的用途，其特征在于，应用所述产品检测丙型肝炎病毒核酸的方法包括：The use according to claim 6, characterized in that the method for detecting nucleic acid of hepatitis C virus using the product includes:
    提供待检测的人丙型肝炎病毒核酸样本；Provide human hepatitis C virus nucleic acid samples to be tested;
    采用样品处理装置，使所述待检测的核酸样本与PCR反应液混合，并使形成的混合液被液滴生成液包裹，形成包含单个核酸或者不包含核酸的液滴；A sample processing device is used to mix the nucleic acid sample to be detected with the PCR reaction solution, and the formed mixed solution is wrapped by the droplet generation solution to form droplets containing a single nucleic acid or not;
    利用所述试剂盒所包含的引物对与探针对所述液滴进行PCR扩增；PCR amplification of the droplets by using the primer pair and the probe contained in the kit;
    对PCR扩增产物进行检测。Detection of PCR amplification products.
  8. 根据权利要求7所述的检测方法，其特征在于包括：利用前述的试剂盒所包含的引物对与探针对所述液滴进行液滴PCR扩增；The detection method according to claim 7, comprising: performing droplet PCR amplification of the droplet using the primer pair and the probe contained in the aforementioned kit;
    对PCR扩增后的液滴的荧光强度进行检测，根据该荧光强度判断核酸样本是否包含丙型肝炎病毒核酸；Detect the fluorescence intensity of the PCR amplified droplets, and determine whether the nucleic acid sample contains hepatitis C virus nucleic acid according to the fluorescence intensity;
    和/或，所述液滴生成液包括油相物质；优选的，所述油相物质包括矿物油；And/or, the droplet generation liquid includes an oil phase substance; preferably, the oil phase substance includes a mineral oil;
    和/或，所述样品处理装置包括进样孔、试剂孔、反应孔以及连接流道；优选的，所述检测方法包括：将液滴生成液加入所述试剂孔，将待检测的核酸样本与PCR反应液加入所述进样孔，在离心力的作用下使进样孔与试剂孔中液体通过连接流道混合，并使形成的混合液被液滴生成液包裹，形成包含单个核酸或者不包含核酸的液滴，并进入反应孔。And/or, the sample processing device includes a sample inlet, a reagent well, a reaction well, and a connecting flow channel; preferably, the detection method includes: adding a droplet generation liquid to the reagent well, and adding the nucleic acid sample to be detected Add the reaction solution with PCR into the injection well, mix the liquid in the injection well and the reagent well through the connecting flow channel under the action of centrifugal force, and make the formed mixed solution be wrapped by the droplet generation liquid to form a single nucleic acid or not The droplets containing nucleic acid enter the reaction well.
  9. 一种包含权利要求3-5中任一项所述的试剂盒的产品，所述产品应用于丙型肝炎病毒核酸的检测方法，所述检测方法包括：A product comprising the kit according to any one of claims 3-5, which is applied to a method for detecting hepatitis C virus nucleic acid, the detection method comprising:
    提供待检测的人丙型肝炎病毒核酸样本；Provide human hepatitis C virus nucleic acid samples to be tested;
    采用样品处理装置，使所述待检测的核酸样本与PCR反应液混合，并使形成的混合液被液滴生成液包裹，形成包含单个核酸或者不包含核酸的液滴；A sample processing device is used to mix the nucleic acid sample to be detected with the PCR reaction solution, and the formed mixed solution is wrapped by the droplet generation solution to form droplets containing a single nucleic acid or not;
    利用所述试剂盒所包含的引物对与探针对所述液滴进行PCR扩增；PCR amplification of the droplets by using the primer pair and the probe contained in the kit;
    对PCR扩增产物进行检测。Detection of PCR amplification products.
  10. 如权利要求9的产品，其特征在于包括：利用所述的试剂盒所包含的引物对与探针对所述液滴进行液滴PCR扩增；The product according to claim 9, characterized by comprising: performing droplet PCR amplification of the droplet using the primer pair and the probe contained in the kit;
    对PCR扩增后的液滴的荧光强度进行检测，根据该荧光强度判断核酸样本是否包含丙型肝炎病毒核酸。The fluorescence intensity of the PCR amplified droplets is detected, and whether the nucleic acid sample contains hepatitis C virus nucleic acid is determined based on the fluorescence intensity.
PCT/CN2019/119046 2018-12-18 2019-11-18 Primers for hepatitis c virus nucleic acid detection, probe, kit, and detection method WO2020125294A1 (en)

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