WO2020007042A1 - 乙肝病毒核酸的快速扩增方法 - Google Patents

乙肝病毒核酸的快速扩增方法 Download PDF

Info

Publication number
WO2020007042A1
WO2020007042A1 PCT/CN2019/074284 CN2019074284W WO2020007042A1 WO 2020007042 A1 WO2020007042 A1 WO 2020007042A1 CN 2019074284 W CN2019074284 W CN 2019074284W WO 2020007042 A1 WO2020007042 A1 WO 2020007042A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
pcr
hepatitis
amplification
denaturation
Prior art date
Application number
PCT/CN2019/074284
Other languages
English (en)
French (fr)
Inventor
戴立忠
范旭
Original Assignee
圣湘生物科技股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 圣湘生物科技股份有限公司 filed Critical 圣湘生物科技股份有限公司
Priority to EP19830704.3A priority Critical patent/EP3819390B1/en
Priority to US17/258,152 priority patent/US20210277490A1/en
Publication of WO2020007042A1 publication Critical patent/WO2020007042A1/zh

Links

Images

Classifications

    • 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
    • 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]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of biochemistry, in particular to a rapid amplification method for hepatitis B virus nucleic acid.
  • Hepatitis B virus is a DNA virus that belongs to the Hepadnaviridae family. About 257 million people are infected with HBV worldwide, and 93 million are infected with HBV in China. Therefore, research on the hepatitis B virus has received much attention worldwide. In the related scientific research of hepatitis B virus, it is often necessary to amplify the hepatitis B virus nucleic acid for non-disease diagnosis or treatment purposes in order to obtain a large amount of hepatitis B virus nucleic acid, thereby providing samples for various scientific experiments.
  • PCR Polymerase chain reaction
  • a method for rapidly amplifying hepatitis B virus nucleic acid includes the following steps:
  • a sample containing the hepatitis B virus is mixed with a nucleic acid release agent and added to a PCR premix solution to obtain a reaction solution.
  • the nucleic acid release agent includes savantin, potassium chloride, sodium dodecylsulfonate, and ethanol.
  • the PCR premix solution includes Deoxyribonucleoside triphosphate, upstream primer as shown in SEQ No. 1 sequence, downstream primer as shown in SEQ No. 2 sequence, DNA polymerase and amplification buffer;
  • reaction solution Placing the reaction solution in a PCR reaction tube so that the reaction solution has a film shape with a thickness of 0.1 mm or less;
  • the PCR reaction tube is placed in a PCR amplification instrument to set the following reaction conditions for PCR amplification: the pre-denaturation temperature is set to 90 to 100 ° C, the pre-denaturation time is set to 10s to 600s, and the denaturation temperature is set to 90 to 100 ° C The denaturation time is set to 0 to 1 s, the annealing extension temperature is set to 50 to 65 ° C, and the annealing extension time is set to 0 to 1 s.
  • the rapid amplification method of the hepatitis B virus nucleic acid of the present invention is mainly optimized from two aspects.
  • strong protein denaturants such as Savantin, potassium chloride and sodium dodecylsulfonate are used to rapidly Destroy the virus shell and completely release the viral nucleic acid, which is conducive to the rapid amplification of PCR.
  • There is no need to perform separate extractions such as heating, centrifugation, and supernatant. Only the sample is added to the reaction with the nucleic acid release agent and other components necessary for PCR. Mix well in the tube.
  • the upstream and downstream primers used have very excellent amplification efficiency, high sensitivity, and specificity, and can detect eight genotypes of HBV, further laying the foundation for rapid PCR amplification.
  • the heat transfer efficiency can be significantly improved, the difference in temperature change of each part of the reaction solution can be reduced, and the reaction solution can be improved.
  • the overall temperature consistency and temperature change rate provide another key element for rapid PCR amplification.
  • the present invention further uses the following short reaction conditions for PCR amplification: pre-denaturation at 90 to 100 ° C for 10s to 600s, denaturation at 90 to 100 ° C for 0 to 1s, and 50 to 65. Annealed at °C for 0 ⁇ 1s.
  • pre-denaturation at 90 to 100 ° C for 10s to 600s denaturation at 90 to 100 ° C for 0 to 1s
  • 50 to 65 Annealed at °C for 0 ⁇ 1s.
  • the following reaction conditions are set for PCR amplification: the pre-denaturation temperature is set to 93-95 ° C, the pre-denaturation time is set to 60s, the denaturation temperature is set to 93-95 ° C, the denaturation time is set to 0s, and annealing The elongation temperature was set to 56 to 58 ° C, and the annealing elongation time was set to 0s.
  • the receiving cavity of the PCR reaction tube is a flat receiving cavity having a thickness of 0.1 mm or less.
  • the PCR master mix further includes a first probe as shown in SEQ. No. 3 sequence.
  • the method further includes the step of performing fluorescence collection during a temperature change between the annealing extension and the denaturation.
  • the PCR master mix further includes a ROX reference dye.
  • the carboxyl terminus of the first probe is modified with a FAM fluorescent group and the hydroxyl terminus is modified with a BHQ1 quenching group.
  • the PCR master mix further includes an internal standard formed by inserting DNA shown in the sequence of SEQ No. 4 into the pUC18T vector and a second probe shown by the sequence of SEQ No. 5.
  • the carboxyl terminus of the second probe is modified with a HEX fluorescent group and the hydroxyl terminus is modified with a DABCYL quenching group.
  • the concentration of savantin in the nucleic acid release agent is 0.01 to 0.5 mmol / L
  • the concentration of potassium chloride is 50 to 200 mmol / L
  • the concentration of sodium dodecylsulfonate is 0.01.
  • the concentration of ethanol is 0.05 ⁇ 1mL / 100mL.
  • Figure 1 is a schematic diagram of traditional PCR reaction conditions
  • FIG. 2 is a schematic diagram of PCR reaction conditions according to an embodiment
  • FIG. 3 is a schematic structural diagram of a PCR reaction tube according to an embodiment
  • FIG. 4 is a schematic structural diagram of a conventional PCR reaction tube
  • FIG. 9 is an amplification curve chart of Comparative Examples 33 to 48.
  • FIG. 9 is an amplification curve chart of Comparative Examples 33 to 48.
  • a method for rapidly amplifying a hepatitis B virus nucleic acid includes the following steps S1 to S3:
  • the nucleic acid release agents include savantin, potassium chloride, sodium dodecylsulfonate, and ethanol.
  • the PCR premix includes deoxyribose Nucleoside triphosphate, upstream primer as shown in SEQ. No. 1 sequence, downstream primer as shown in SEQ. No. 2 sequence, DNA polymerase and amplification buffer.
  • the reaction solution is placed in a PCR reaction tube so that the reaction solution has a film shape with a thickness of 0.1 mm or less.
  • the pre-denaturation temperature is set to 90 to 100 ° C
  • the pre-denaturation time is set to 10s to 600s
  • the denaturation temperature is set to 90 to 100 ° C.
  • the denaturation time is set to 0 to 1 s
  • the annealing extension temperature is set to 50 to 65 ° C
  • the annealing extension time is set to 0 to 1 s.
  • the traditional PCR reaction conditions are: pre-denaturation at 94 ° C for about 5 minutes, denaturation at 94 ° C for 30s (or longer) in the cyclic amplification stage, and annealing for 45s (or longer) at 57 ° C for several times. cycle. Therefore, each cycle waits for a period of time at the denaturation temperature and the annealing extension temperature. Although it is only a few tens of seconds from a single cycle, the above processes all require continuous cycles to achieve the expansion of the target DNA or RNA. Increase, so as the number of cycles increases, the time required is also cumulatively increased, eventually leading to a large amount of time wasted.
  • the rapid amplification method of the hepatitis B virus nucleic acid of the present invention is mainly optimized from two aspects.
  • strong protein denaturants such as Savantin, potassium chloride and sodium dodecylsulfonate are used to rapidly Destroy the virus shell and completely release the viral nucleic acid, which is conducive to the rapid amplification of PCR.
  • There is no need to perform separate extractions such as heating, centrifugation, and supernatant. Only the sample is added to the reaction with the nucleic acid release agent and other components necessary for PCR. Mix well in the tube.
  • the upstream and downstream primers used have very excellent amplification efficiency, high sensitivity, and specificity, and can detect eight genotypes of HBV, further laying the foundation for rapid PCR amplification.
  • the heat transfer efficiency can be significantly improved, the difference in temperature change of each part of the reaction solution can be reduced, and the reaction solution can be improved.
  • the overall temperature consistency and temperature change rate provide another key element for rapid PCR amplification.
  • the present invention further uses the following short reaction conditions for PCR amplification: pre-denaturation at 90 to 100 ° C for 10s to 600s, denaturation at 90 to 100 ° C for 0 to 1s, and 50 to 65. Annealed at °C for 0 ⁇ 1s.
  • pre-denaturation at 90 to 100 ° C for 10s to 600s
  • denaturation at 90 to 100 ° C for 0 to 1s
  • 50 to 65 Annealed at °C for 0 ⁇ 1s.
  • the following reaction conditions are set for PCR amplification: the pre-denaturation temperature is set to 93-95 ° C, the pre-denaturation time is set to 60s, the denaturation temperature is set to 93-95 ° C, the denaturation time is set to 0s, and the annealing extension temperature is set to 56 to 58 ° C, and the annealing extension time was set to 0s.
  • the denaturation and annealing elongation time refers to the parameter conditions set on the PCR amplification instrument. It is set to 0 seconds, that is, the temperature always changes without a maintenance stage, as shown in Figure 2. Thus, the PCR reaction starts at the beginning of the process.
  • the temperature After the pre-denaturation is maintained, the temperature has always been a changing process. Therefore, the entire process has low requirements for the precision of temperature control and the corresponding measures for implementing temperature control (such as slowing down the heating and cooling rate in advance), which reduces the temperature.
  • the technical difficulty of control also saves the manufacturing cost of the PCR instrument.
  • the accommodating cavity of the PCR reaction tube is a flat-shaped accommodating cavity having a thickness of 0.1 mm or less, such as the accommodating cavity A of the PCR reaction tube in FIG. 3.
  • the commonly used PCR reaction vessels are conical blind hole vessels as shown in FIG. 4, and the bottom is an inverted cone structure.
  • the flat design of the PCR reaction tube facilitates the thickness of the cross-section of the liquid-phase object in the container to be much smaller than that of a traditional conical tube container.
  • the direct contact area of the liquid-phase object in the reaction container and the reaction container is greatly increased.
  • the phase object is formed into a thin film with a thickness of 0.1 mm or less, which significantly improves the heat transfer efficiency and helps accelerate the PCR amplification speed.
  • the specific structure of the PCR reaction tube is not limited to the structure shown in FIG. 3 as long as the reaction solution can be formed into a thin film having a thickness of 0.1 mm or less.
  • the PCR master mix further includes a first probe as shown in SEQ. No. 3 sequence.
  • a specific fluorescent probe is added at the same time as a pair of primers.
  • the probe is an oligonucleotide with two ends labeled with a reporter fluorescent group and a quencher fluorescent group.
  • the fluorescent signal emitted by the reporter group is absorbed by the quenching group.
  • the exonuclease activity of the Taq enzyme digests the probe to degrade the reporter fluorescent group and quenching fluorescent group.
  • the fluorescence monitoring system can receive a fluorescent signal, that is, each amplified DNA strand, a fluorescent molecule is formed, and the accumulation of the fluorescent signal is completely synchronized with the formation of the PCR product, which is conducive to the nucleic acid quantification by the fluorescence monitoring system.
  • the carboxyl end of the first probe is modified with a FAM fluorescent group
  • the hydroxyl end is modified with a BHQ1 quenching group.
  • the carboxyl end can be selected from TET, JOE, HEX and other fluorescent groups
  • the hydroxyl end can be selected from DABCYL
  • the quenching groups such as TAMRA, BHQ2, and BHQ3 are not limited thereto.
  • the method for rapid amplification of the hepatitis B virus nucleic acid further includes the step of performing fluorescence acquisition during a temperature change between annealing extension and denaturation. Due to the setting of the above-mentioned PCR reaction condition parameters, when the fluorescence quantification is required, the fluorescence collection can be performed during the temperature change, and it is not necessary to maintain a constant temperature for a long time, which can reduce the waste of energy and time.
  • the PCR master mix further includes a ROX reference dye.
  • a ROX reference dye When fluorescence quantification is required, errors due to various factors are difficult to avoid. Therefore, normalization correction can be performed by adding ROX reference dyes, which greatly improves the stability and repeatability of the test results.
  • the PCR master mix further includes an internal standard formed by inserting DNA shown in the sequence of SEQ No. 4 into the pUC18T vector and a second probe shown in the sequence of SEQ No. 5; The carboxyl terminus was modified with a HEX fluorescent group and the hydroxyl terminus was modified with a DABCYL quenching group.
  • an internal standard formed by inserting DNA shown in the sequence of SEQ No. 4 into the pUC18T vector and a second probe shown in the sequence of SEQ No. 5; The carboxyl terminus was modified with a HEX fluorescent group and the hydroxyl terminus was modified with a DABCYL quenching group.
  • carboxy terminus of the second probe can also be labeled with a fluorophore different from the first probe, such as TET, JOE, FAM, etc., and the hydroxyl terminus is selected with quenching groups such as BHQ1, TAMRA, BHQ2, BHQ3, etc., which is not limited to this.
  • the concentration of savantin in the nucleic acid release agent is 0.01 to 0.5 mmol / L
  • the concentration of potassium chloride is 50 to 200 mmol / L
  • the concentration of sodium dodecylsulfonate is 0.01 to 2 g / L.
  • 100mL the concentration of ethanol is 0.05 ⁇ 1mL / 100mL.
  • Ct value is the number of cycles that the fluorescence signal in each reaction tube goes through when it reaches a set domain value. Studies have shown that there is a linear relationship between the Ct value of each sample and the logarithm of the starting copy number of the sample. The larger the starting copy number, the smaller the Ct value.
  • a standard curve can be obtained by using a standard with a known starting copy number.
  • the abscissa represents the logarithm of the starting copy number, and the ordinate represents the Ct value. Therefore, as long as the Ct value of the sample is obtained, the initial copy number of the sample and its logarithm (LOG value) can be calculated according to the standard curve.
  • Enzyme solution containing amplification buffer, 0.2mmol / L deoxyribonucleoside triphosphate, 40mmol / L ⁇ 200mmol / L ROX reference dye, 0.2 ⁇ mol / L ⁇ 0.4 ⁇ mol / L upstream primer and downstream primer, 0.2 ⁇ mol /L ⁇ 0.4 ⁇ mol/L first probe.
  • Enzyme solution provided: Contains Taq enzyme at a concentration of 1U / ⁇ L.
  • 16 samples containing HBV were amplified.
  • the PCR reaction conditions were: 94 ° C pre-denaturation for 1 min; 94 ° C denaturation for 0 s, 57 ° C annealing extension for 0 s, 40 cycles and heating at 57 ° C for each cycle.
  • the fluorescence was collected during the process to 94 ° C, and the total time of the amplification program was 15 minutes.
  • the PCR instrument used was the GNM-C7-8 real-time quantitative PCR instrument produced by Kinome Biotechnology Co., Ltd.
  • the amplification curve is shown in FIG. 5. It can be seen that the amplification curves of Examples 1 to 16 maintain a good shape and have high amplification efficiency.
  • the logarithmic value (LOG value) is shown in Table 1.
  • the same 16 samples containing HBV were amplified.
  • the PCR reaction conditions were: pre-denaturation at 94 ° C for 5min; denaturation at 94 ° C for 15s, annealing extension at 57 ° C for 30s, and fluorescence collection, 45 cycles, amplification procedures The total time is 72min.
  • the PCR instrument used was the GNM-C7-8 real-time quantitative PCR instrument produced by Kinome Biotechnology Co., Ltd.
  • the amplification curve is shown in Figure 6, and the sample concentration and LOG values are shown in Table 1.
  • Reagent preparation Provide DNA extraction solution and HBV-PCR reaction solution for use.
  • the components of DNA extraction solution are chelex100, Tris-HCL, NaOH, Triton-100, NP-40 and EDTA.
  • the components of HBV-PCR reaction solution are primers.
  • Probe, dN (U) TP, buffer, DNA polymerase and UNG enzyme the sequences of their primers are different from SEQ No. 1 and SEQ No. 2.
  • Add the HBV-PCR reaction solution to the centrifuge tube and shake to mix. After instantaneous centrifugation, aliquot 45uL into each PCR reaction tube.
  • DNA extraction 100uL of sample was added with the same amount of DNA concentrate (PEG6000, NaCl) and shaken for 5 seconds, centrifuged at 10,000rpm for 10 minutes, the supernatant was removed, 30uL of DNA extract was added to the precipitate, vigorously shaken and mixed for 10 seconds, and centrifuged for a few seconds instantly , Constant temperature treatment at 100 ° C for 10 minutes, and centrifugation at 10,000 rpm for 5 minutes.
  • PCR amplification Add 5uL of the treated sample supernatant to each prepared PCR reaction tube, and centrifuge immediately for later use. The PCR reaction tube was put into a fluorescence quantitative PCR amplification instrument, and PCR amplification was performed according to the PCR reaction conditions of Examples 1 to 16. The amplification curve is shown in Figure 8 and the LOG values are shown in Table 2.
  • the same 16 HBV-containing samples were amplified.
  • the difference from Examples 1 to 16 is that the PCR reaction tube uses a conical blind hole container as shown in FIG. 4.
  • the amplification curve is shown in FIG. 9 and the LOG value is shown in Table 3.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Communicable Diseases (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

提供了一种乙肝病毒核酸的快速扩增方法，包括以下步骤：将含有乙肝病毒的样品与核酸释放剂混合后加入PCR预混液得到反应液，所述核酸释放剂包括莎梵婷、氯化钾、十二烷基磺酸钠和乙醇，所述PCR预混液包括脱氧核糖核苷三磷酸、如SEQ No.1序列所示的上游引物、如SEQ No.2序列所示的下游引物、DNA聚合酶和扩增缓冲液；将所述反应液置于PCR反应管中使所述反应液呈厚度小于等于0.1mm的薄膜状；将所述PCR反应管置于PCR扩增仪中按照以下反应条件进行PCR扩增：90～100℃预变性10s～600s，90～100℃变性0～1s，50～65℃退火延伸0～1s。所述方法在确保扩增的准确性和有效性的前提下，每一个循环所需要的时间都显著地缩短了，实现了快速简单地扩增乙肝病毒核酸的目的。

Description

乙肝病毒核酸的快速扩增方法 技术领域
本发明涉及生物化学领域，特别是涉及一种乙肝病毒核酸的快速扩增方法。
背景技术
乙型肝炎病毒(hepatitis B virus，缩写HBV)是一种DNA病毒，属于嗜肝DNA病毒科(Hepadnaviridae)。全世界约有2.57亿感染HBV，中国有9300万HBV感染者。因此，对乙型肝炎病毒的研究在世界范围内得到了重视。而在乙型肝炎病毒的相关科学研究中，常常需要对乙肝病毒核酸进行非疾病诊断或治疗目的的扩增，以获得大量的乙肝病毒核酸，从而为各种科学试验提供样品。
聚合酶链式反应(PCR)是一种主要的核酸体外扩增技术，近年来发展迅速。PCR技术的特点是模拟生物体内DNA的复制过程，在合适的温度条件下，利用扩增所需要的模板、引物、聚合酶等原料，使目标DNA或RNA片段经过变性、退火和延伸的不断循环得到目标DNA或者RNA片段的指数倍扩增。PCR作为分子生物学研究的基础技术，促进了生命科学的发展，但由于一般的PCR方法复杂且耗时，限制了技术的进一步发展，不利于快速获得大量的乙肝病毒核酸样品。
发明内容
基于此，有必要提供一种操作简单且耗时较短的乙型肝炎病毒核酸的快速扩增方法。
一种乙肝病毒核酸的快速扩增方法，包括以下步骤：
将含有乙肝病毒的样品与核酸释放剂混合后加入PCR预混液得到反应液， 所述核酸释放剂包括莎梵婷、氯化钾、十二烷基磺酸钠和乙醇，所述PCR预混液包括脱氧核糖核苷三磷酸、如SEQ No.1序列所示的上游引物、如SEQ No.2序列所示的下游引物、DNA聚合酶和扩增缓冲液；
将所述反应液置于PCR反应管中使所述反应液呈厚度小于等于0.1mm的薄膜状；
将所述PCR反应管置于PCR扩增仪中设定以下反应条件进行PCR扩增：预变性温度设为90～100℃，预变性时间设为10s～600s，变性温度设为90～100℃，变性时间设为0～1s，退火延伸温度设为50～65℃，退火延伸时间设为0～1s。
本发明的乙肝病毒核酸的快速扩增方法主要从两方面进行了优化，一方面在核酸的提取上，采用莎梵婷、氯化钾和十二烷基磺酸钠等强烈的蛋白变性剂快速破坏病毒外壳，彻底释放出病毒核酸，有利于PCR的快速扩增，且无需单独进行加热、离心、去上清等提取工作，只需将样品与核酸释放剂及其他PCR必须的组分加入反应管中混匀即可。而且所采用的上下游引物具有十分优异的扩增效率，灵敏度高，特异性强，能够检测出HBV的八个基因型，进一步为PCR的快速扩增奠定了基础。另一方面，通过将反应液置于PCR反应管中使反应液呈厚度小于等于0.1mm的薄膜状，可以显著地提高热传递效率，降低了反应液各部分的温度变化差异，提高了反应液整体的温度一致性和变温速度，为PCR的快速扩增提供了另一关键要素。在结合以上两方面的因素的情况下，本发明进而采用以下时间极短的的反应条件进行PCR扩增：90～100℃预变性10s～600s，90～100℃变性0～1s，50～65℃退火延伸0～1s。如此，在确保扩增的准确性和有效性的前提下，每一个循环所需要的时间都显著地缩短了，且随着循环数的增加，节省的时间和能耗愈加明显，实现了快速简单地扩增乙肝病毒核酸的目的，从而为各种科学研究提供足够多的核酸样品。
在其中一个实施例中，设定以下反应条件进行PCR扩增：预变性温度设为93～95℃，预变性时间设为60s，变性温度设为93～95℃，变性时间设为0s，退火延伸温度设为56～58℃，退火延伸时间设为0s。
在其中一个实施例中，所述PCR反应管的容纳腔为厚度小于等于0.1mm的扁平状容纳腔。
在其中一个实施例中，所述PCR预混液还包括如SEQ No.3序列所示的第一探针。
在其中一个实施例中，还包括以下步骤：在所述退火延伸和所述变性之间的升温变化过程中进行荧光采集。
在其中一个实施例中，所述PCR预混液还包括ROX参比染料。
在其中一个实施例中，所述第一探针的羧基端用FAM荧光基团修饰，羟基端用BHQ1淬灭基团修饰。
在其中一个实施例中，所述PCR预混液还包括如SEQ No.4序列所示的DNA插入到pUC18T载体而构成的内标和如SEQ No.5序列所示的第二探针。
在其中一个实施例中，所述第二探针的羧基端用HEX荧光基团修饰，羟基端用DABCYL淬灭基团修饰。
在其中一个实施例中，所述核酸释放剂中，莎梵婷的浓度为0.01～0.5mmol/L，氯化钾的浓度为50～200mmol/L，十二烷基磺酸钠的浓度为0.01～2g/100mL，乙醇的浓度为0.05～1mL/100mL。
附图说明
图1为传统的PCR反应条件示意图；
图2为一实施例的PCR反应条件示意图；
图3为一实施例的PCR反应管的结构示意图；
图4为传统的PCR反应管的结构示意图；
图5为实施例1～16的扩增曲线图；
图6为对比例1～16的扩增曲线图；
图7为实施例1～16与对比例1～16的相关性分析图；
图8为对比例17～32的扩增曲线图；
图9为对比例33～48的扩增曲线图。
具体实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述，并给出了本发明的较佳实施例。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
本发明一实施例的乙肝病毒核酸的快速扩增方法，包括以下步骤S1～S3：
S1、将含有乙肝病毒的样品与核酸释放剂混合后加入PCR预混液得到反应液，核酸释放剂包括莎梵婷、氯化钾、十二烷基磺酸钠和乙醇，PCR预混液包括脱氧核糖核苷三磷酸、如SEQ No.1序列所示的上游引物、如SEQ No.2序列所示的下游引物、DNA聚合酶和扩增缓冲液。
S2、将反应液置于PCR反应管中使反应液呈厚度小于等于0.1mm的薄膜状。
S3、将PCR反应管置于PCR扩增仪中设定以下反应条件进行PCR扩增：预变性温度设为90～100℃，预变性时间设为10s～600s，变性温度设为90～100℃，变性时间设为0～1s，退火延伸温度设为50～65℃，退火延伸时间设为0～1s。
如图1所示，传统的PCR反应条件是：94℃预变性5min左右，循环扩增阶段94℃变性30s(或更长时间)，57℃退火延伸45s(或更长时间)，进行若干个循环。因此，每个循环都在变性温度和退火延伸温度有一段时间的等待，虽然单独从一个循环来看只是几十秒的等待，但是以上过程都是需要经过不断循环来实现目标DNA或者RNA的扩增，所以随着循环的次数增大，所需要的时间也是累加增长，最终导致了大量时间的耗费。
本发明的乙肝病毒核酸的快速扩增方法主要从两方面进行了优化，一方面在核酸的提取上，采用莎梵婷、氯化钾和十二烷基磺酸钠等强烈的蛋白变性剂快速破坏病毒外壳，彻底释放出病毒核酸，有利于PCR的快速扩增，且无需单独进行加热、离心、去上清等提取工作，只需将样品与核酸释放剂及其他PCR必须的组分加入反应管中混匀即可。而且所采用的上下游引物具有十分优异的扩增效率，灵敏度高，特异性强，能够检测出HBV的八个基因型，进一步为PCR的快速扩增奠定了基础。另一方面，通过将反应液置于PCR反应管中使反应液呈厚度小于等于0.1mm的薄膜状，可以显著地提高热传递效率，降低了反应液各部分的温度变化差异，提高了反应液整体的温度一致性和变温速度，为PCR的快速扩增提供了另一关键要素。在结合以上两方面的因素的情况下，本发明进而采用以下时间极短的的反应条件进行PCR扩增：90～100℃预变性10s～600s，90～100℃变性0～1s，50～65℃退火延伸0～1s。如此，在确保扩增的准确性和有效性的前提下，每一个循环所需要的时间都显著地缩短了，且随着循环数的增加，节省的时间和能耗愈加明显，实现了快速简单地扩增乙肝病毒核酸的目的， 从而为各种科学研究提供足够多的核酸样品。
优选地，设定以下反应条件进行PCR扩增：预变性温度设为93～95℃，预变性时间设为60s，变性温度设为93～95℃，变性时间设为0s，退火延伸温度设为56～58℃，退火延伸时间设为0s。变性和退火延伸的时间指的是在PCR扩增仪上设定的参数条件，设为0秒即温度始终变化而不存在维持的阶段，如图2所示，如此，PCR反应在经过开始的预变性保持后，温度一直是一个变化的过程，因此整个过程对温控的精密度要求及实施温度控制的相应措施(如提前减慢升温、降温速度等)的要求低，即降低了对温度控制的技术难度，也节约了PCR仪的制造成本。
在一个实施例中，PCR反应管的容纳腔为厚度小于等于0.1mm的扁平状容纳腔，例如图3中PCR反应管的容纳腔A。目前通常用的PCR反应容器都为如图4所示的圆锥形盲孔容器，底部为倒锥形结构，虽然有利于最底部温度变化速度的提高，但对反应液整体的温度变化速度提高并不明显，在变温的过程中难以确保反应液边沿和中心温度的一致性，为了确保扩增的有效性和准确性，只能等待中心位置到达指定温度后再继续变温，降低了PCR扩增的速度，造成大量能耗和时间的浪费。本实施例通过PCR反应管的扁平化设计便于使液相物体在容器中的横切面厚度远远小于传统的圆锥管容器，反应容器中的液相物体与反应容器的直接接触面积大大增加，液相物体形成厚度小于等于0.1mm的薄膜状，显著提高了热传递效率，有助于加快PCR扩增速度。可以理解，PCR反应管的具体结构不限于图3所示的结构，只要能使反应液呈厚度小于等于0.1mm的薄膜状即可。
在一个实施例中，PCR预混液还包括如SEQ No.3序列所示的第一探针。在PCR扩增时，在加入一对引物的同时加入一个特异性的荧光探针，该探针为一 寡核苷酸，两端分别标记一个报告荧光基团和一个淬灭荧光基团。在探针完整时，报告基团发射的荧光信号被淬灭基团吸收，PCR扩增时，Taq酶的外切酶活性将探针酶切降解，使报告荧光基团和淬灭荧光基团分离，从而荧光监测系统可接收到荧光信号，即每扩增一条DNA链，就有一个荧光分子形成，实现了荧光信号的累积与PCR产物形成完全同步，有利于通过荧光监测系统进行核酸定量。优选地，第一探针的羧基端用FAM荧光基团修饰，羟基端用BHQ1淬灭基团修饰，可以理解，羧基端可以选用TET、JOE、HEX等荧光基团，羟基端可以选用DABCYL、TAMRA、BHQ2、BHQ3等淬灭基团，不限于此。
在一个实施例中，乙肝病毒核酸的快速扩增方法还包括以下步骤：在退火延伸和变性之间的温度变化过程中进行荧光采集。由于上述PCR反应条件参数的设置，在需要进行荧光定量时，在温度变化的过程中执行荧光采集即可，不需要保持长时间的恒定温度，可减少能量和时间的浪费。
在一个实施例中，PCR预混液还包括ROX参比染料。在需要进行荧光定量时，由于各种因素导致的误差难以避免，因此通过加入ROX参比染料可进行归一化校正，使检测结果的稳定性和重复性大大提高。
在一个实施例中，PCR预混液还包括如SEQ No.4序列所示的DNA插入到pUC18T载体而构成的内标和如SEQ No.5序列所示的第二探针，第二探针的羧基端用HEX荧光基团修饰，羟基端用DABCYL淬灭基团修饰。如此，通过加入内标，当样本中存在PCR干扰物质而导致扩增失败时能够快速地明确原因。可以理解，第二探针羧基端还可以选用不同于第一探针的荧光基团标记，如TET、JOE、FAM等，羟基端选用BHQ1、TAMRA、BHQ2、BHQ3等淬灭基团，不限于此。
在一个实施例中，核酸释放剂中，莎梵婷的浓度为0.01～0.5mmol/L，氯化 钾的浓度为50～200mmol/L，十二烷基磺酸钠的浓度为0.01～2g/100mL，乙醇的浓度为0.05～1mL/100mL。
以下为具体实施例，需要说明的是，为了体现PCR扩增是否准确有效，以下实施例均加入第一探针和ROX参比染料，并相应进行荧光采集，在实际进行乙肝病毒核酸的扩增时，可根据需要选择是否加入第一探针和ROX参比染料。在荧光定量PCR中，Ct值的含义是每个反应管内的荧光信号到达设定的域值时所经历的循环数。研究表明，每个样品的Ct值与该样品的起始拷贝数的对数存在线性关系，起始拷贝数越多，Ct值越小。利用已知起始拷贝数的标准品可得到标准曲线，横坐标代表起始拷贝数的对数，纵坐标代表Ct值。因此，只要获得样品的Ct值，即可根据标准曲线计算出该样品的起始拷贝数及其对数(LOG值)。
操作步骤如下：
提供储备液：包含扩增缓冲液、0.2mmol/L脱氧核糖核苷三磷酸、40mmol/L～200mmol/L ROX参比染料、0.2μmol/L～0.4μmol/L上游引物及下游引物、0.2μmol/L～0.4μmol/L第一探针。提供酶溶液：包含浓度为1U/μL的Taq酶。提供核酸释放剂：包含莎梵婷(surfactin)0.01mmol/L、氯化钾50mmol/L、十二烷基磺酸钠0.01g/100mL和乙醇0.05mL/100mL。
按38～44μL储备液与1～2μL酶溶液的比例混匀得到PCR预混液，瞬时离心后备用。使用如图3所示的容器作为PCR反应管，每个PCR反应管中加入核酸释放剂2～5μL和样品3～5μL，吸打3～5次混匀，每个PCR反应管加入PCR预混液40～45μL，吸打混匀2～3次，盖上管盖2000rpm离心30秒。将PCR反应管放入荧光定量PCR扩增仪，按照所设定的反应条件进行PCR扩增。反应结束后，仪器自动保存结果，获得Ct值、LOG值、扩增曲线等数据。
实施例1～16
按照操作步骤，对16份含有乙肝病毒的样品进行扩增，PCR反应条件为：94℃预变性1min；94℃变性0s，57℃退火延伸0s，40个循环并在每个循环的57℃升温至94℃的过程中进行荧光采集，扩增程序总时间为15min。使用的PCR仪为金诺美生物技术有限公司生产的GNM-C7-8实时荧光定量PCR仪。扩增曲线如图5所示，可见实施例1～16的扩增曲线保持了良好的形态，具有较高的扩增效率，对数值(LOG值)如表1所示。
此外，按照实施例1～16的方法对空白样品(不含乙肝病毒的样品)进行扩增，没有出现假阳性结果。
对比例1～16
按照操作步骤，对相同的16份含有乙肝病毒的样品进行扩增，PCR反应条件为：94℃预变性5min；94℃变性15s，57℃退火延伸30s及荧光采集，45个循环，扩增程序总时间为72min。使用的PCR仪为金诺美生物技术有限公司生产的GNM-C7-8实时荧光定量PCR仪。扩增曲线如图6所示，样品浓度和LOG值如表1所示。
表1
Figure PCTCN2019074284-appb-000001
Figure PCTCN2019074284-appb-000002
根据表1可知，实施例1～16的LOG值与采用传统的PCR反应条件的对比例1～16的LOG值相差很小，说明实施例1～16核酸扩增的有效性和准确性与对比例1～16相同，但是所花费的时间明显减少，每个样品的Ct值与该样品的起始浓度的对数保持良好的相关性。对实施例1～16的LOG值与对比例1～16的LOG值进行相关性分析，结果如图7所示，也可证明相关性较好。
对比例17～32
按照操作步骤，对相同的16份含有乙肝病毒的样品进行扩增，与实施例1～16的区别在于：
试剂准备：提供DNA提取液和HBV-PCR反应液备用，DNA提取液的组分为chelex100、Tris-HCL、NaOH、Triton-100、NP-40和EDTA，HBV-PCR反应液的组分为引物、探针、dN(U)TP、缓冲液、DNA聚合酶和UNG酶，其引物的序列与SEQ No.1和SEQ No.2不同。将HBV-PCR反应液加入离心管中震荡混匀，瞬时离心后，向每一个PCR反应管中分装45uL。
DNA提取：100uL样品加入等量的DNA浓缩液(PEG6000、NaCl)振荡混匀5秒，10000rpm离心10分钟，去上清，沉淀中加入30uL DNA提取液，剧烈振荡混匀10秒，瞬时离心数秒，100℃恒温处理10分钟，10000rpm离心5分钟备用。PCR扩增：向准备好的每个PCR反应液管中分别加入处理后的样品上清液5uL，瞬时离心备用。将PCR反应管放入荧光定量PCR扩增仪，按照实施例1～16的PCR反应条件进行PCR扩增。扩增曲线如图8所示，LOG值如表2所示。
表2
Figure PCTCN2019074284-appb-000003
根据图8和表2可知，对比例17～32大部分都无法呈现出扩增曲线，也就无法得到Ct值，少部分能够得到Ct值的对比例其LOG值与采用传统的PCR反应条件的对比例1～16的LOG值相差甚远，说明对比例17～32核酸扩增的有效性和准确性很差，扩增失败，每个样品的Ct值与该样品的起始浓度的对数无法保持良好的相关性。
对比例33～48
按照操作步骤，对相同的16份含有乙肝病毒的样品进行扩增，与实施例1～16的区别在于，PCR反应管使用如图4所示的圆锥形盲孔容器。扩增曲线如图9所示，LOG值如表3所示。
表3
Figure PCTCN2019074284-appb-000004
Figure PCTCN2019074284-appb-000005
根据图9和表3可知，对比例33～48都无法呈现出扩增曲线，也就无法得到Ct值，说明对比例33～48核酸扩增的有效性和准确性很差，扩增失败，每个样品的Ct值与该样品的起始浓度的对数无法保持良好的相关性。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种乙肝病毒核酸的快速扩增方法，其特征在于，包括以下步骤：
    将含有乙肝病毒的样品与核酸释放剂混合后加入PCR预混液得到反应液，所述核酸释放剂包括莎梵婷、氯化钾、十二烷基磺酸钠和乙醇，所述PCR预混液包括脱氧核糖核苷三磷酸、如SEQ No.1序列所示的上游引物、如SEQ No.2序列所示的下游引物、DNA聚合酶和扩增缓冲液；
    将所述反应液置于PCR反应管中使所述反应液呈厚度小于等于0.1mm的薄膜状；
    将所述PCR反应管置于PCR扩增仪中设定以下反应条件进行PCR扩增：预变性温度设为90～100℃，预变性时间设为10s～600s，变性温度设为90～100℃，变性时间设为0～1s，退火延伸温度设为50～65℃，退火延伸时间设为0～1s。
  2. 根据权利要求1所示的乙肝病毒核酸的快速扩增方法，其特征在于，设定以下反应条件进行PCR扩增：预变性温度设为93～95℃，预变性时间设为60s，变性温度设为93～95℃，变性时间设为0s，退火延伸温度设为56～58℃，退火延伸时间设为0s。
  3. 根据权利要求1所示的乙肝病毒核酸的快速扩增方法，其特征在于，所述PCR反应管的容纳腔为厚度小于等于0.1mm的扁平状容纳腔。
  4. 根据权利要求1所述的乙肝病毒核酸的快速扩增方法，其特征在于，所述PCR预混液还包括如SEQ No.3序列所示的第一探针。
  5. 根据权利要求4所述的乙肝病毒核酸的快速扩增方法，其特征在于，还包括以下步骤：在所述退火延伸和所述变性之间的升温变化过程中进行荧光采集。
  6. 根据权利要求4所述的乙肝病毒核酸的快速扩增方法，其特征在于，所 述PCR预混液还包括ROX参比染料。
  7. 根据权利要求4所述的乙肝病毒核酸的快速扩增方法，其特征在于，所述第一探针的羧基端用FAM荧光基团修饰，羟基端用BHQ1淬灭基团修饰。
  8. 根据权利要求4所述的乙肝病毒核酸的快速扩增方法，其特征在于，所述PCR预混液还包括如SEQ No.4序列所示的DNA插入到pUC18T载体而构成的内标和如SEQ No.5序列所示的第二探针。
  9. 根据权利要求8所述的乙肝病毒核酸的快速扩增方法，其特征在于，所述第二探针的羧基端用HEX荧光基团修饰，羟基端用DABCYL淬灭基团修饰。
  10. 根据权利要求1～9任一项所述的乙肝病毒核酸的快速扩增方法，其特征在于，所述核酸释放剂中，莎梵婷的浓度为0.01～0.5mmol/L，氯化钾的浓度为50～200mmol/L，十二烷基磺酸钠的浓度为0.01～2g/100mL，乙醇的浓度为0.05～1mL/100mL。
PCT/CN2019/074284 2018-07-05 2019-01-31 乙肝病毒核酸的快速扩增方法 WO2020007042A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP19830704.3A EP3819390B1 (en) 2018-07-05 2019-01-31 Rapid amplification method for nucleic acid of hepatitis b virus
US17/258,152 US20210277490A1 (en) 2018-07-05 2019-01-31 Rapid amplification methods fornucleic acid of hepatitis b virus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810728131.X 2018-07-05
CN201810728131.XA CN108913811B (zh) 2018-07-05 2018-07-05 乙肝病毒核酸的快速扩增方法

Publications (1)

Publication Number Publication Date
WO2020007042A1 true WO2020007042A1 (zh) 2020-01-09

Family

ID=64424096

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/074284 WO2020007042A1 (zh) 2018-07-05 2019-01-31 乙肝病毒核酸的快速扩增方法

Country Status (4)

Country Link
US (1) US20210277490A1 (zh)
EP (1) EP3819390B1 (zh)
CN (1) CN108913811B (zh)
WO (1) WO2020007042A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114959111A (zh) * 2022-05-07 2022-08-30 英科新创（苏州）生物科技有限公司 用于快速检测猫瘟病毒的试剂或试剂盒、其应用及检测方法

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108913811B (zh) * 2018-07-05 2020-06-16 圣湘生物科技股份有限公司 乙肝病毒核酸的快速扩增方法
CN110747278B (zh) * 2019-10-29 2023-02-21 圣湘生物科技股份有限公司 检测人乙醇代谢基因多态性的组合物、试剂盒及方法
CN111187828B (zh) * 2020-02-11 2023-09-15 圣湘生物科技股份有限公司 检测人叶酸代谢基因多态性的组合物、试剂盒及方法
CN111117869A (zh) * 2020-02-20 2020-05-08 珠海黑马生物科技有限公司 一种pcr试管
CN114250325A (zh) * 2021-12-31 2022-03-29 广西壮族自治区疾病预防控制中心 快速检测乙型肝炎病毒9个基因型的引物、探针、试剂盒和方法
CN116814397B (zh) * 2023-08-25 2023-11-28 新羿制造科技（北京）有限公司 Pcr反应管及多联排pcr管

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101802163A (zh) * 2007-05-23 2010-08-11 信诚医疗有限公司 反应液用容器，使用这种容器的反应促进装置，及其方法
CN102174660A (zh) * 2011-02-25 2011-09-07 湖南圣湘生物科技有限公司 乙型肝炎病毒荧光定量pcr检测试剂盒
CN106399053A (zh) * 2016-11-14 2017-02-15 湖南圣湘生物科技有限公司 一种pcr反应装置及其使用方法
CN107312874A (zh) * 2017-07-18 2017-11-03 广州市宝创生物技术有限公司 高灵敏实时荧光定量检测hbv核酸的引物、探针及试剂盒和方法
CN108913811A (zh) * 2018-07-05 2018-11-30 湖南圣湘生物科技有限公司 乙肝病毒核酸的快速扩增方法

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10222275A1 (de) * 2002-05-18 2003-12-04 Eppendorf Ag Verfahren zur Isolierung von Nukleinsäuren aus einer flüssigen Probe
JP2005328709A (ja) * 2004-05-18 2005-12-02 Toyobo Co Ltd 高速ｄｎａポリメラーゼを用いた高速ｐｃｒ
JP5700403B2 (ja) * 2009-11-04 2015-04-15 有限会社フルイド Ｐｃｒ方法及びｐｃｒ装置
CN201534842U (zh) * 2009-11-24 2010-07-28 四川飞阳科技有限公司 一种新型聚合酶链式反应容器
WO2014025924A1 (en) * 2012-08-07 2014-02-13 California Institute Of Technology Ultrafast thermal cycler
CN204848844U (zh) * 2015-06-05 2015-12-09 秦荣 一种pcr试管及其阵列

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101802163A (zh) * 2007-05-23 2010-08-11 信诚医疗有限公司 反应液用容器，使用这种容器的反应促进装置，及其方法
CN102174660A (zh) * 2011-02-25 2011-09-07 湖南圣湘生物科技有限公司 乙型肝炎病毒荧光定量pcr检测试剂盒
CN106399053A (zh) * 2016-11-14 2017-02-15 湖南圣湘生物科技有限公司 一种pcr反应装置及其使用方法
CN107312874A (zh) * 2017-07-18 2017-11-03 广州市宝创生物技术有限公司 高灵敏实时荧光定量检测hbv核酸的引物、探针及试剂盒和方法
CN108913811A (zh) * 2018-07-05 2018-11-30 湖南圣湘生物科技有限公司 乙肝病毒核酸的快速扩增方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114959111A (zh) * 2022-05-07 2022-08-30 英科新创（苏州）生物科技有限公司 用于快速检测猫瘟病毒的试剂或试剂盒、其应用及检测方法

Also Published As

Publication number Publication date
US20210277490A1 (en) 2021-09-09
CN108913811B (zh) 2020-06-16
EP3819390A4 (en) 2022-04-27
EP3819390B1 (en) 2023-10-18
CN108913811A (zh) 2018-11-30
EP3819390A1 (en) 2021-05-12
EP3819390C0 (en) 2023-10-18

Similar Documents

Publication Publication Date Title
WO2020007042A1 (zh) 乙肝病毒核酸的快速扩增方法
CN107058623B (zh) 一种高度灵敏和特异的血液HBV pgRNA荧光定量PCR检测体系和检测方法
US20210207204A1 (en) Directional polymerisation fluorescent probe pcr and test kit
JP5680078B2 (ja) ライゲーションに基づく核酸の正規化した定量化方法
WO2022007224A1 (zh) 用于荧光定量pcr的方法、组合物、试剂盒及其用途
CN107022651B (zh) 一种快速检测丙型肝炎病毒核酸的试剂盒及其检测方法
US20120028253A1 (en) Method for amplifying oligonucleotide and small rna by using polymerase-endonuclease chain reaction
WO2023246032A1 (zh) 一锅法单链DNA环化扩增和CRISPR/Cas介导的核酸分子检测方法
AU2019100992A4 (en) Primer set, kit, and method for detecting porcine epidemic diarrhea virus
CN109983138B (zh) 用于检测bk病毒的组合物和方法
CN114410836A (zh) 一种集样本处理、核酸提取及多重恒温扩增一体化的检测人细小病毒b19的试剂盒和方法
CN111826467B (zh) 用于快速检测新型冠状病毒核酸的组合物、试管装置和方法
US20230304081A1 (en) Primer and probe design method, detection composition, and kit for mirna detection
KR20230012467A (ko) 표적 핵산을 검출하기 위한 루프형 프라이머 및 루프-드-루프 방법
CN102634610B (zh) 麻疹病毒和风疹病毒的特异性检测用引物探针组合和试剂盒
CN107881257A (zh) 柯萨奇病毒a6型和a10型联合检测试剂盒及其应用
CN117004774A (zh) 超快速无需核酸提取的牛结节性皮肤病病毒检测试剂盒
US20070141559A1 (en) Methods for detecting and typing herpes simplex virus
CN108300804B (zh) 用于HBV miR-3荧光定量PCR检测的材料及方法
CN116426691A (zh) 多靶点检测HIV-1的crRNA、CRISPR-Cas12a系统和检测方法
CN112280832B (zh) 一种免抽提的核酸检测方法及试剂盒
CN109136353B (zh) 一种低序列依赖高阶恒温指数扩增检测microRNA的方法
JP2022540801A (ja) 試料中のＴｒｉｃｈｏｍｏｎａｓ ｖａｇｉｎａｌｉｓの存在を決定する際に使用するためのオリゴヌクレオチド
JP2021503285A (ja) Ｃ１ｏｒｆ４３核酸を検出するための組成物および方法
CN114250286B (zh) 用于核酸检测的组合、试剂盒及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19830704

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2019830704

Country of ref document: EP

Effective date: 20210205