WO2019228449A1 - 癌症相关的eb病毒核酸含量检测试剂盒及方法 - Google Patents

癌症相关的eb病毒核酸含量检测试剂盒及方法 Download PDF

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WO2019228449A1
WO2019228449A1 PCT/CN2019/089246 CN2019089246W WO2019228449A1 WO 2019228449 A1 WO2019228449 A1 WO 2019228449A1 CN 2019089246 W CN2019089246 W CN 2019089246W WO 2019228449 A1 WO2019228449 A1 WO 2019228449A1
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virus
reagent tube
nucleic acid
acid content
cancer
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PCT/CN2019/089246
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English (en)
French (fr)
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陈琦
梁昊原
谷东风
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深圳市圣必智科技开发有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • C12M1/38—Temperature-responsive control
    • 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
    • C12Q1/6851—Quantitative amplification
    • 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
    • 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
    • C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster

Definitions

  • the invention relates to the technical field of EB virus detection, in particular to a cancer-related EB virus nucleic acid content detection kit and method.
  • Epstein-Barr virus is a DNA virus of the human herpes virus ⁇ subfamily lymphotropic virus. It has an infection rate of more than 90% in our country and can affect all organs and systems throughout the body. Epstein-Barr virus specifically infects humans and some in vivo Biological characteristics of primate B cells; humans are the host of EB virus infection, mainly transmitted through saliva; EB virus proliferates in oropharyngeal epithelial cells, and then infects B lymphocytes.
  • EBV infection can be expressed as proliferative infection and latent infection; different antigens are expressed in different infection states, and antigens expressed during proliferative infection include EBV early antigen, EBV capsid protein, and EBV membrane antigen, EBV nuclear antigen and latent membrane protein are expressed during the latent infection period.
  • EBV early antigen EBV capsid protein
  • EBV membrane antigen EBV nuclear antigen and latent membrane protein are expressed during the latent infection period.
  • the clinical manifestations of the virus infection are diverse, and they are easily missed and misdiagnosed.
  • PCR Polymerase chain reaction
  • kits have problems such as tedious steps to use, mutual detection reagent contamination, difficult to distinguish between reagent tubes, etc., and because the reagent storage temperature in the kit cannot be kept at a constant temperature, the accuracy of the detection of EB virus content is affected. low.
  • the main purpose of the present invention is to provide a cancer-related EB virus nucleic acid content detection kit and method, which aims to solve the existing problem that the accuracy of EB virus content detection is affected because the reagent storage temperature in the kit cannot be kept at a constant temperature.
  • the present invention provides a cancer-related EB virus nucleic acid content detection kit, which includes a shell, an inner cavity, and an inner holder.
  • the inner cavity is provided with two slide rails, and four corners of the inner holder.
  • a pulley is set at the bottom, and the inner holder is movably arranged in the inner cavity through the pulley and two slide rails.
  • the inner holder is provided with eight detection recesses arranged in a 2 ⁇ 4 array, of which seven Reagent tubes are inserted into each of the detection wells, and the other detection well is a spare well, of which:
  • the reagent tube includes a reaction reagent tube containing an RT-PCR reaction solution, an enzyme reagent tube containing an enzyme mixed solution, a negative control reagent tube containing an EB virus negative control, and an EB virus positive control.
  • Positive control reagent tube double distilled water reagent tube with sterilized double distilled water, ROX reagent tube with ROX calibration solution, and primer probe solution reagent tube;
  • a temperature-adjustable semiconductor thermostat is installed in the inner cavity and below the inner support.
  • the power input end of the semiconductor thermostat is electrically connected to a battery.
  • the semiconductor thermostat is used to connect the environment in the inner cavity. The temperature was adjusted to the temperature required for EB virus detection to achieve reagent incubation.
  • the pore diameter of the detection recesses of the reagent tube of the EB-PCR reaction solution is larger than that of the other seven detection recesses, and the pore diameters of the other seven detection recesses are equal, and each reagent tube is a sterilized screw. Mouth glass tube.
  • the inner cavity is further provided with a driver, and a power line input end of the driver is electrically connected to a battery, and the battery provides driving power for the driver.
  • the driver includes a driving motor and an electrically driven free telescopic rod.
  • One end of the free telescopic rod is mechanically connected to one side of the inner support, and the other end of the free telescopic rod is electrically connected to the driving motor.
  • the inner support is driven to slide freely along the two slide rails in the inner cavity.
  • the housing connected to the side of the opposite side of the inner support is provided with a sliding slot, and the two ends of the sliding slot match the positions of the two sliding rails, and the height of the sliding slot is greater than the thickness of the inner support. , The length is greater than the width of the inner support.
  • the surface of the housing is provided with a warehouse exit button and a warehouse entry button, and the warehouse exit button and the warehouse entry button are both electrically connected to the control terminal of the driver; when the inspector presses the warehouse exit button, the The driver drives the inner tray to slide out of the inner cavity through the chute to the outside of the reagent box; when the inspector presses the enter button, the driver drives the inner tray to automatically advance through the chute along the two slide rails into the inner cavity.
  • the enzyme mixture contained in the enzyme reagent tube is a reverse transcriptase and an amplification enzyme.
  • the inner wall of the shell is coated with a heat insulation layer, and the heat insulation layer is made of a heat insulation material.
  • the present invention also provides a cancer-related EB virus nucleic acid content detection method.
  • the method includes the steps of: using a primer probe solution reagent tube to aspirate a sample to be tested; Viral RNA is extracted from the sample, and viral RNA is amplified.
  • the reaction reagent tube containing the EB virus RT-PCR reaction solution is used for PCR reaction to configure the EB virus reaction solution.
  • the configured EB virus reaction solution is placed in a fluorescent PCR detector.
  • Fluorescent quantitative RT-PCR detection After the quantitative quantitative RT-PCR detection is over, the fluorescent PCR detector will automatically record and save the fluorescent signal of each cycle; select the fluorescent channel to adjust the threshold of the fluorescent signal and observe the EB virus amplification The results of the curve and Ct value were used to determine the EB virus nucleic acid content.
  • the detection result of the EB virus nucleic acid content is judged as follows: When the Ct value is ⁇ 30.0 or no Ct value, the result indicates that the EB virus-free nucleic acid content or the nucleic acid content in the sample to be detected is lower than the detection limit of the detection kit When the Ct value is less than 30, the result indicates that the EB virus nucleic acid is contained in the sample to be tested, which is positive.
  • the cancer-related EB virus nucleic acid content detection kit and method according to the present invention adopt the above technical scheme, and achieve the following technical effects: it can quickly and accurately detect cancer-related EB virus with strong specificity and high sensitivity , Stable, convenient, no contamination between detection reagents, and can accurately detect the EB virus content; coated with insulation material on the inner wall of the kit, and the inner support is a rigid foam;
  • the semiconductor temperature controller is convenient for the reagent cartridge to be taken out to ensure that the temperature of the reagent in the reagent cartridge is kept at a constant temperature and avoids affecting the detection accuracy due to the temperature exceeding the storage temperature.
  • FIG. 1 is a schematic structural diagram of a cancer-related EB virus nucleic acid content detection kit of the present invention
  • FIG. 2 is a flowchart of a preferred embodiment of a method for detecting a nucleic acid content of EB virus related to cancer according to the present invention.
  • FIG. 1 is a schematic structural diagram of a cancer-related EB virus nucleic acid content detection kit of the present invention.
  • the cancer-related EB virus content detection kit includes a kit body 10, and the kit body 10 has a rectangular parallelepiped shape with a pull-out structure.
  • the kit body 10 includes a casing 1 and an inner cavity 2. As well as Neto 3.
  • the inner wall of the casing 1 is coated with a heat-insulating layer 11 made of a heat-insulating material, which realizes heat preservation of reagents, improves the detection accuracy of EB virus, and avoids affecting detection accuracy due to temperature factors.
  • the inner cavity 2 is provided with two slide rails 12, the inner support 3 is a rectangular parallelepiped structure, and four corners of the inner support 3 are respectively provided with a pulley 13, and each of the pulleys 13 can slide the rail 12. Free sliding on.
  • the inner support 3 is movably disposed in the inner cavity 2 through the pulley 13 and two slide rails 12. The distance between the two slide rails 12 provided in the inner cavity 2 is equal to the length of the inner support 3, so that the inner support 3 can pass the pulleys 13 provided at the four corner bottoms along the two slide rails 12 along the inner cavity 2. Free sliding inside.
  • the inner cavity 2 is further provided with a driver 14 and a storage battery 15.
  • a power line input end of the driver 14 is electrically connected to the storage battery 15, and the storage battery 15 provides driving power for the driver 14.
  • the driver 14 includes a driving motor 140 and an electrically driven free telescopic rod 141.
  • One end of the free telescopic rod 141 is mechanically connected to one side of the inner support 3, and the other end is electrically connected to the driving motor 140.
  • the driving motor 140 drives The free telescopic rod 141 drives the inner support 3 to slide freely along the two slide rails 12 in the inner cavity 2 when the free telescopic rod 141 moves up and down.
  • the inner support 3 is made of rigid foam.
  • the inner support 3 is provided with eight detection recesses arranged in a 2 ⁇ 4 array, and seven of the eight detection recesses are provided.
  • a reagent tube is inserted into the detection recess; the reagent tube includes a reaction reagent tube 31 containing an EB virus RT-PCR reaction solution, an enzyme reagent tube 32 containing an enzyme mixture solution, and a negative of an EB virus negative control Control reagent tube 33, positive control reagent tube 34 containing EB virus positive control, double distilled water reagent tube 35 containing sterilized double distilled water, ROX reagent tube 36 containing ROX calibration solution, and primer probe solution Reagent tube 37.
  • the other detection well is a spare well 38.
  • the diameter of the detection well with the EB-PCR reaction solution reagent tube 31 inserted is larger than that of the other seven detection wells.
  • the other seven detection wells are equal in size.
  • Each reagent An electronic label is attached to the outside of the tube to distinguish the EB virus detection reagent in each reagent tube.
  • the EB virus RT-PCR reaction solution contained in the reaction reagent tube 31 is used to perform a PCR reaction on the sample to be tested to configure the EB virus reaction solution
  • the enzyme mixed solution contained in the enzyme reagent tube 32 is Reverse transcriptase and amplification enzyme are used to extract viral nucleic acid (RNA) from EB virus reaction solution and perform viral RNA amplification.
  • the EB virus negative control contained in the negative control reagent tube 33 and the EB virus positive control contained in the positive control reagent tube 34 can detect the EB virus nucleic acid or its nucleic acid content from the test sample.
  • the sterilized double-distilled water contained in the double-distilled water reagent tube 35 is used to configure the concentration of the EB virus reaction solution, and the ROX correction solution contained in the ROX reagent tube 36 is used to correct the concentration of the EB virus reaction solution.
  • the primer probe solution reagent tube 37 is used to suck a sample to be measured.
  • each reagent tube is a sterilized screw-mouth glass tube
  • a temperature-adjustable semiconductor temperature controller 4 is installed in the inner cavity 2 and below the inner support 3, and the semiconductor temperature control
  • the power cord input end of the device 4 is electrically connected to the battery 15, the semiconductor temperature controller 4 can adjust the ambient temperature in the inner cavity 2 to the temperature required for the EB virus detection, to achieve reagent insulation, improve the accuracy of the EB virus detection, and avoid Temperature factors affect detection accuracy.
  • the driver 14 is connected to the housing 1 on the opposite side of the inner support 3 and is provided with a sliding groove 16.
  • the two ends of the sliding groove 16 are matched with the two ends of the two sliding rails 12.
  • the height of 16 is greater than the thickness of the inner support 3 and the length is greater than the width of the inner support 3. Therefore, the inner support 3 can slide out of the reagent box body 10 from the inner cavity 2 through the chute 16.
  • the surface of the housing 1 of the reagent cartridge body 10 is provided with a warehouse exit button 17 and a warehouse advance button 18, and the warehouse exit button 17 and the warehouse advance button 18 are both electrically connected to the control end of the driver 14. .
  • the driver 14 can drive the inner tray 3 through the chute 16 and slide out from the inner cavity 2 to the outside of the reagent box body 10, so that the tester can add a test solution (such as saliva or Sweat, etc.);
  • a test solution such as saliva or Sweat, etc.
  • the driver 14 can automatically advance the inner holder 3 along the two slide rails 12 through the slide groove 16 into the inner cavity 2.
  • the cancer-related EB virus nucleic acid content detection kit can quickly and accurately detect cancer-related EB virus, has strong specificity, high sensitivity, stability, and convenience, does not contaminate between detection reagents, and can detect EB virus content.
  • a semiconductor thermostat in the kit, it is easy to ensure that the kit is in the kit when it is taken out
  • the reagent temperature is kept at a constant temperature to avoid affecting the detection accuracy due to the temperature exceeding the storage temperature.
  • FIG. 2 is a flowchart of a preferred embodiment of a method for detecting a cancer-related EB virus nucleic acid content of the present invention.
  • the method for detecting a cancer-related EB virus nucleic acid content includes the following steps: Step S21: using a primer probe solution reagent tube 37 to aspirate a sample to be measured; step S22: passing an enzyme reagent tube containing an enzyme mixture solution 32 extract viral RNA from the sample to be tested and perform viral RNA amplification; step S23: the reaction reagent tube containing the EB virus RT-PCR reaction solution 31 performs a PCR reaction to configure an EB virus reaction solution, wherein the total reaction system is 25 ⁇ L, Includes 12 ⁇ L of 2XRT-PCR buffer, 7.5 ⁇ L of primer probe pickup, 0.5 ⁇ l of amplification enzyme, and 5 ⁇ L of sample nucleic acid.
  • Step S24 Put the configured EB virus reaction solution into a fluorescent PCR detector for fluorescent quantitative RT-PCR detection.
  • the PCR (Polymerase Chain Reaction) instrument is an existing pathogen quantitative nucleic acid molecular diagnostic instrument, which is widely used for rapid quantitative detection and early diagnosis of infectious diseases such as hepatitis B HBV, hepatitis C HCV, AIDS HIV, sexually transmitted diseases STD, and neoplastic cancer. .
  • the reaction cycle parameters are: 50 ° C 2min, 1 cycle; 95 ° C 2min, 1 cycle; 91 ° C, 15s ⁇ 58 ° C 1min, 45 cycles, fluorescence collection at 58 ° C; wherein the upstream primer is The sequence shown in Seq ID No.1, the downstream primer is shown in Seq ID No.2, the EB virus TaqMan fluorescent probe is shown in Seq ID No.3:
  • Seq ID No. 1 5'- CCTTCAGAGGAACCAGGGA-3 ';
  • Seq ID No. 2 5'- GTTTAACGGGGCTCAGAGG-3 ';
  • Seq ID No. 3 5'-FAM-CGCCCTCCTGGTCTCCGCTC-BHQ1-3 ';
  • the 5 'end of the EB virus TaqMan fluorescent probe is FAM, and the 3' end is labeled with the fluorescent quencher BHQ1.
  • Step S25 After the quantitative RT-PCR test is completed, the fluorescence PCR detector will automatically record and save the fluorescence signal of each cycle; step S26: select the fluorescence channel (FAM) to adjust the fluorescence signal threshold, and observe the EB virus by observing The results of the amplification curve and the Ct value were used to determine the EB virus nucleic acid content; when the negative control showed no logarithmic increase in fluorescence, no Ct value was detected; the positive control showed a logarithmic increase in fluorescence, and the fluorescence channel FAM showed a typical amplification curve; In this example, the detection result of the EB virus nucleic acid content is judged: when the Ct value is ⁇ 30.0 or there is no Ct value, the result indicates that the EB virus nucleic acid content or the nucleic acid content in the sample to be detected is lower than the detection limit of the detection kit, which is negative ; When the Ct value is less than 30, the result indicates that the EB virus nucleic acid is contained
  • the cancer-related EB virus nucleic acid content detection method can quickly and accurately distinguish EB virus, and can accurately detect the EB virus content; can more fully understand the incidence of EB virus, and improve the diagnosis efficiency of EB virus , And put forward corresponding prevention and control measures to reduce unnecessary economic losses; coated with thermal insulation material on the inner wall of the kit, and the inner support is a rigid foam; in addition, the semiconductor thermostat is placed in the kit to facilitate the kit When the body is taken out, it can ensure that the temperature of the reagent in the kit is kept at a constant temperature to avoid affecting the detection accuracy because the temperature exceeds the storage temperature.
  • the cancer-related EB virus nucleic acid content detection kit and method according to the present invention adopt the above technical scheme, and achieve the following technical effects: it can quickly and accurately detect cancer-related EB virus with strong specificity and high sensitivity , Stable, convenient, no contamination between detection reagents, and can accurately detect the EB virus content; coated with insulation material on the inner wall of the kit, and the inner support is a rigid foam;
  • the semiconductor temperature controller is convenient for the reagent cartridge to be taken out to ensure that the temperature of the reagent in the reagent cartridge is kept at a constant temperature and avoids affecting the detection accuracy due to the temperature exceeding the storage temperature.

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Abstract

本发明提供一种癌症相关的EB病毒核酸含量检测试剂盒及方法，该试剂盒包括外壳、内腔和内托。内托通过滑轮和两条滑轨活动设置于内腔中，内托设置以2×4阵列排布的八个检测凹孔，其中七个检测凹孔内插有试剂管，另一个检测凹孔为备用孔。试剂管包括装有RT-PCR反应液的反应试剂管、装有酶混合液的酶试剂管、装有EB病毒阴性质控品的阴性对照试剂管、装有EB病毒阳性质控品的阳性对照试剂管、装有灭菌双蒸水的双蒸水试剂管、装有ROX校正液的ROX试剂管以及引物探针液试剂管。在内腔中且位于内托的下方安装有半导体温控器，用于将内腔的环境温度调节至EB病毒检测所需温度。本发明能够快速准确的检测出EB病毒含量、特异性强、灵敏度高、稳定、方便。

Description

癌症相关的EB病毒核酸含量检测试剂盒及方法 技术领域
本发明涉及EB病毒检测的技术领域，尤其涉及一种癌症相关的EB病毒核酸含量检测试剂盒及方法。
 背景技术
EB病毒属人类疱疹病毒γ亚科嗜淋巴细胞病毒的DNA病毒，在我国人群感染率达90％以上，可累及全身各个器官和系统；EB病毒具有在体内外专一性地感染人类及某些灵长类B细胞的生物学特性；人是EB病毒感染的宿主，主要通过唾液传播；EB病毒在口咽部上皮细胞内增殖，然后感染B淋巴细胞，这些细胞大量进入血液循环而造成全身性感染，并可长期潜伏在人体淋巴组织中；EBV感染可表现为增殖性感染和潜伏性感染；不同感染状态表达不同的抗原，增殖性感染期表达的抗原有EBV早期抗原、EBV衣壳蛋白和EBV膜抗原，潜伏感染期表达的抗原有EBV核抗原和潜伏膜蛋白；该病毒感染临床表现多样，且容易漏诊、误诊。
聚合酶链式反应（PCR）技术已广泛应用于动物疫病检测，该技术主要是基于病原微生物核酸的特异性，针对靶标设计特异性引物对DNA或RNA片段进行指数扩增；因此，应用荧光PCR技术建立EB病毒检测方法对于快速、准确的检测EB病毒具有重要的意义。目前国外已经上市的检测EBV-DNA的试剂盒有几种，检测的方法主要是荧光免疫杂交(HIGH)，国内检测EBV主要采用ELISA方法，未见有批准上市检测EBV-DNA的试剂盒。基于EBV感染情况如此严重，开发一种简单、易行、成本较低的检测试剂盒就显得尤为重要。现有的试剂盒大多存在使用起来步骤繁琐，检测试剂相互污染，试剂管之间难以区分等问题，且因试剂盒内的试剂保存温度不能处于恒温保存状态而影响EB病毒含量检测精度，灵敏度较低。
 技术问题
本发明的主要目的在于提供一种癌症相关的EB病毒核酸含量检测试剂盒及方法，旨在解决现有因试剂盒内的试剂保存温度不能处于恒温保存状态而影响EB病毒含量检测精度的问题。
 技术解决方案
为实现上述目的，本发明提供一种癌症相关的EB病毒核酸含量检测试剂盒，包括外壳、内腔和内托，所述内腔内设置有两条滑轨，所述内托的四个角底部分别设置一个滑轮，所述内托通过所述滑轮和两条滑轨活动设置于内腔中，所述内托上设置有以2×4阵列排布的八个检测凹孔，其中有七个检测凹孔内插有试剂管，另外一个检测凹孔为备用孔，其中：
所述试剂管包括装有RT-PCR反应液的反应试剂管、装有酶混合液的酶试剂管、装有EB病毒阴性质控品的阴性对照试剂管、装有EB病毒阳性质控品的阳性对照试剂管、装有灭菌双蒸水的双蒸水试剂管、装有ROX校正液的ROX试剂管以及引物探针液试剂管；
在所述内腔中且位于内托的下方安装有温度可调的半导体温控器，所述半导体温控器的电源线输入端电连接蓄电池，该半导体温控器用于将内腔内的环境温度调节至EB病毒检测所需的温度以实现试剂保温。
优选的，所述EB-PCR反应液试剂管的检测凹孔的孔径大于另外七个检测凹孔的孔径，所述另外七个检测凹孔的孔径大小相等，每一个试剂管为灭菌的螺口玻璃管。
优选的，所述内腔还设置有驱动器，该驱动器的电源线输入端电连接至蓄电池，所述蓄电池为驱动器提供驱动电能。
优选的，所述驱动器包括驱动电机以及电驱动的自由伸缩杆，该自由伸缩杆的一端机械连接至内托的一侧，该自由伸缩杆的另一端电连接至驱动电机，当所述驱动电机驱动自由伸缩杆上下伸缩时带动内托沿着两条滑轨在内腔中自由滑动。
优选的，所述驱动器连接内托相对面一侧的外壳开设有滑槽，该滑槽的两端位置和两条滑轨的两端位置相匹配，所述滑槽的高度大于内托的厚度，长度大于内托的宽度。
优选的，所述外壳的表面设置有出仓按钮和进仓按钮，所述出仓按钮和进仓按钮均电连接至所述驱动器的控制端；当检测者按下出仓按钮时，所述驱动器驱动内托穿过滑槽从内腔滑出至试剂盒体外；当检测者按下进仓按钮时，所述驱动器驱动内托沿着两条滑轨穿过滑槽自动推进内腔内。
优选的，所述酶试剂管内装有的酶混合液为逆转录酶和扩增酶。
优选的，所述外壳的内壁涂覆有隔热层，所述隔热层由保温材料制成。
另一方面，本发明还提供一种癌症相关的EB病毒核酸含量检测方法，该方法包括步骤：采用引物探针液试剂管吸取待测样品；通过装有酶混合液的酶试剂管从待测样品提取病毒RNA，并进行病毒RNA扩增；装有EB病毒RT-PCR反应液的反应试剂管进行PCR反应配置EB病毒反应溶液；将配置完成的EB病毒反应溶液放入荧光PCR检测仪中进行荧光定量RT-PCR检测；荧光定量RT-PCR检测结束后，荧光PCR检测仪将自动记录保存每个循环的荧光信号；选定荧光通道将荧光信号进行阈值调整，并通过观察EB病毒的扩增曲线及Ct值进行结果判断得到EB病毒核酸含量。
优选的，所述EB病毒核酸含量检测结果按照如下方式判断：当Ct值≥30.0或无Ct值时，结果表明待检测样品中无EB病毒核酸含量或其核酸含量低于检测试剂盒的检测限值，呈阴性；当Ct值<30时，结果表明待检测样品中含有EB病毒核酸，呈阳性。
 有益效果
相较于现有技术，本发明所述癌症相关的EB病毒核酸含量检测试剂盒及方法采用上述技术方案，取得如下技术效果：能够快速准确的检测出癌症相关EB病毒，特异性强，灵敏度高，稳定、方便，检测试剂之间不会污染，并能够对EB病毒含量做出准确检测；在试剂盒体内壁涂覆保温材料，且内托为硬质泡沫；另外，通过在试剂盒体内放置半导体温控器，便于试剂盒体被外带时，能够保证试剂盒体内的试剂温度处于恒温保存状态，避免因温度超过保存温度而影响检测精度。
 附图说明
图1是本发明癌症相关的EB病毒核酸含量检测试剂盒的结构示意图；
图2是本发明癌症相关的EB病毒核酸含量检测方法优选实施例的流程图。
本发明目的的实现、功能特点及优点将结合实施例，参照附图做进一步说明。
 本发明的实施方式
为更进一步阐述本发明为达成预定发明目的所采取的技术手段及功效，以下结合附图及较佳实施例，对本发明的具体实施方式、结构、特征及其功效，详细说明如下。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
参照图1所示，图1是本发明癌症相关的EB病毒核酸含量检测试剂盒的结构示意图。在本实施例中，所述癌症相关的EB病毒含量检测试剂盒包括试剂盒体10，所述试剂盒体10为抽拉式结构的长方体形状，该试剂盒体10包括外壳1、内腔2以及内托3。所述外壳1的内壁涂覆有隔热层11，所述隔热层11由保温材料制成，实现试剂保温，提高EB病毒检测精度，避免因温度因素影响检测准确度。
作为优选实施例，所述内腔2内设置有两条滑轨12，所述内托3为长方体结构，所内托3的四个角底部分别设置一个滑轮13，每一个滑轮13可以滑轨12上自由滑动。所述内托3通过所述滑轮13和两条滑轨12活动设置于内腔2中。设置在内腔2中的两条滑轨12之间的距离与内托3的长度相等，从而可以使内托3通过四个角底部设置的滑轮13沿着两条滑轨12在内腔2内自由滑动。所述内腔2还设置有驱动器14和蓄电池15，所述驱动器14的电源线输入端电连接至蓄电池15，所述蓄电池15为驱动器14提供驱动电能。所述驱动器14包括驱动电机140以及电驱动的自由伸缩杆141，该自由伸缩杆141的一端机械连接至内托3的一侧，另一端电连接至驱动电机140，当所述驱动电机140驱动自由伸缩杆141上下伸缩时带动内托3沿着两条滑轨12在内腔2内自由滑动。
作为优选实施例，所述内托3由硬质泡沫制成，所述内托3上设置有以2×4阵列排布的八个检测凹孔，八个检测凹孔之中其中有七个检测凹孔内插有试剂管；所述试剂管包括装有EB病毒RT-PCR反应液的反应试剂管31、装有酶混合液的酶试剂管32、装有EB病毒阴性质控品的阴性对照试剂管33、装有EB病毒阳性质控品的阳性对照试剂管34、装有灭菌双蒸水的双蒸水试剂管35、装有ROX校正液的ROX试剂管36以及引物探针液试剂管37。另外一个检测凹孔为备用孔38，插有EB-PCR反应液试剂管31的检测凹孔的孔径大于其他七个检测凹孔的孔径，其他七个检测凹孔的孔径大小相等，每一个试剂管外部贴有电子标签，用于区分每一试剂管内的EB病毒检测试剂。
在本实施例中，所述反应试剂管31装有的EB病毒RT-PCR反应液用于将待测样品进行PCR反应配置EB病毒反应溶液，所述酶试剂管32装有的酶混合液为逆转录酶和扩增酶，用于从EB病毒反应溶液提取病毒核酸（RNA），并进行病毒RNA扩增。所述阴性对照试剂管33装有的EB病毒阴性质控品和阳性对照试剂管34装有的EB病毒阳性质控品能够从待测样品中检测出EB病毒核酸或其核酸含量。所述双蒸水试剂管35装有的灭菌双蒸水用于配置EB病毒反应溶液的浓度，所述ROX试剂管36装有的ROX校正液用于对修正EB病毒反应溶液的浓度，所述引物探针液试剂管37用于吸取待测样品。
作为优选的实施方案，每一个试剂管为灭菌的螺口玻璃管，在所述内腔2中且位于内托3的下方安装有温度可调的半导体温控器4，所述半导体温控器4的电源线输入端电连接蓄电池15，该半导体温控器4可以将内腔2内的环境温度调节至EB病毒检测所需的温度内，实现试剂保温，提高EB病毒检测精度，避免因温度因素影响检测准确度。
作为优选的实施方案，所述驱动器14连接内托3相对面一侧的外壳1开设有滑槽16，该滑槽16的两端位置和两条滑轨12的两端位置相匹配，滑槽16的高度大于内托3的厚度，长度大于内托3的宽度，因此，内托3可以透过滑槽16从内腔2滑出试剂盒体10外。在本实施例中，所述试剂盒体10的外壳1表面设置有出仓按钮17和进仓按钮18，所述出仓按钮17和进仓按钮18均电连接至所述驱动器14的控制端。当检测者按下出仓按钮17时，驱动器14可以驱动内托3穿过滑槽16从内腔2滑出至试剂盒体10外，便于检测者在试剂管内添加待测溶液（例如唾液或者汗液等）；当检测者在试剂管内添加待测溶液完毕并按下进仓按钮18时，驱动器14可以将内托3沿着两条滑轨12穿过滑槽16自动推进内腔2内。
本发明所述癌症相关的EB病毒核酸含量检测试剂盒能够快速准确的检测出癌症相关EB病毒，特异性强，灵敏度高，稳定、方便，检测试剂之间不会污染，并能够对EB病毒含量做出准确检测；在试剂盒体内壁涂覆保温材料，且内托为硬质泡沫；另外，通过在试剂盒体内放置半导体温控器，便于试剂盒体被外带时，能够保证试剂盒体内的试剂温度处于恒温保存状态，避免因温度超过保存温度而影响检测精度。
如图2所示，图2是本发明癌症相关的EB病毒核酸含量检测方法优选实施例的流程图。在本实施例中，所述癌症相关的EB病毒核酸含量检测方法包括如下步骤：步骤S21：采用引物探针液试剂管37吸取待测样品；步骤S22：通过装有酶混合液的酶试剂管32从待测样品提取病毒RNA，并进行病毒RNA扩增；步骤S23：装有EB病毒RT-PCR反应液的反应试剂管31进行PCR反应配置EB病毒反应溶液，其中，反应总体系为25μL，包括2XRT-PCR缓冲液12μL，引物探针吸取量7.5μL，扩增酶0.5μl，样本核酸5μL。
步骤S24：将配置完成的EB病毒反应溶液放入荧光PCR检测仪中进行荧光定量RT-PCR检测。所述PCR（聚合酶链式反应）仪器为现有一种病原体定量核酸分子诊断仪器，广泛应用于乙肝HBV、丙肝HCV、艾滋病HIV、性病STD等传染病及肿瘤性癌症的快速定量检测及早期诊断。作为优选的实施方案，反应循环参数：50℃ 2min，1个循环；95℃ 2min，1个循环；91℃，15s→58℃ 1min，45个循环，在58℃收集荧光；其中，上游引物如Seq ID No.1所示序列，下游引物如Seq ID No.2所示序列，EB病毒TaqMan荧光探针如Seq ID No.3所示序列：
Seq ID No.1：5'- CCTTCAGAGGAACCAGGGA-3'；
Seq ID No.2：5'- GTTTAACGGGGCTCAGAGG-3'；
Seq ID No.3：5'-FAM-CGCCCTCCTGGTCTCCGCTC-BHQ1-3'；
EB病毒TaqMan荧光探针的5'端荧光报告基团是FAM，3'端标记的是荧光淬灭基团BHQ1。
步骤S25：荧光定量RT-PCR检测结束后，荧光PCR检测仪将自动记录保存每个循环的荧光信号；步骤S26：选定荧光通道（FAM）将荧光信号进行阈值调整，并通过观察EB病毒的扩增曲线及Ct值进行结果判断得到EB病毒核酸含量；当阴性对照无荧光对数增长，未检测到Ct值；阳性对照有荧光对数增长，且荧光通道FAM出现典型的扩增曲线；在本实施例中，EB病毒核酸含量检测结果判断：当Ct值≥30.0或无Ct值时，结果表明待检测样品中无EB病毒核酸或其核酸含量低于检测试剂盒的检测限值，呈阴性；当Ct值<30时，结果表明待检测样品中含有EB病毒核酸，呈阳性。
本发明所述癌症相关的EB病毒核酸含量检测方法能够快速、准确的区分EB病毒，并能对EB病毒含量做出准确检测；能够更全面地了解EB病毒的发病情况，提高EB病毒的诊断效率，并提出相应的防控措施，减少不必要的经济损失；在试剂盒体内壁涂覆保温材料，且内托为硬质泡沫；另外，通过在试剂盒体内放置半导体温控器，便于试剂盒体被外带时，能够保证试剂盒内的试剂温度处于恒温保存状态，避免因温度超过保存温度而影响检测精度。
以上仅为本发明的优选实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书及附图内容所作的等效结构或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。
 工业实用性
相较于现有技术，本发明所述癌症相关的EB病毒核酸含量检测试剂盒及方法采用上述技术方案，取得如下技术效果：能够快速准确的检测出癌症相关EB病毒，特异性强，灵敏度高，稳定、方便，检测试剂之间不会污染，并能够对EB病毒含量做出准确检测；在试剂盒体内壁涂覆保温材料，且内托为硬质泡沫；另外，通过在试剂盒体内放置半导体温控器，便于试剂盒体被外带时，能够保证试剂盒体内的试剂温度处于恒温保存状态，避免因温度超过保存温度而影响检测精度。

Claims (10)

  1. 一种癌症相关的EB病毒核酸含量检测试剂盒，包括外壳、内腔以及内托，其特征在于，所述内腔内设置有两条滑轨，所述内托的四个角底部分别设置一个滑轮，所述内托通过所述滑轮和两条滑轨活动设置于内腔中，所述内托上设置有以2×4阵列排布的八个检测凹孔，其中有七个检测凹孔内插有试剂管，另外一个检测凹孔为备用孔，其中：
    所述试剂管包括装有RT-PCR反应液的反应试剂管、装有酶混合液的酶试剂管、装有EB病毒阴性质控品的阴性对照试剂管、装有EB病毒阳性质控品的阳性对照试剂管、装有灭菌双蒸水的双蒸水试剂管、装有ROX校正液的ROX试剂管以及引物探针液试剂管；
    在所述内腔中且位于内托的下方安装有温度可调的半导体温控器，所述半导体温控器的电源线输入端电连接蓄电池，该半导体温控器用于将内腔内的环境温度调节至EB病毒检测所需温度以实现试剂保温。
  2. 如权利要求1所述的癌症相关的EB病毒核酸含量检测试剂盒，其特征在于，所述EB-PCR反应液试剂管的检测凹孔的孔径大于另外七个检测凹孔的孔径，所述另外七个检测凹孔的孔径大小相等，每一个试剂管为灭菌的螺口玻璃管。
  3. 如权利要求1所述的癌症相关的EB病毒核酸含量检测试剂盒，其特征在于，所述内腔还设置有驱动器，该驱动器的电源线输入端电连接至蓄电池，所述蓄电池为驱动器提供驱动电能。
  4. 如权利要求3所述的癌症相关的EB病毒核酸含量检测试剂盒，其特征在于，所述驱动器包括驱动电机以及电驱动的自由伸缩杆，该自由伸缩杆的一端机械连接至内托的一侧，该自由伸缩杆的另一端电连接至驱动电机，当所述驱动电机驱动自由伸缩杆上下伸缩时带动内托沿着两条滑轨在所述内腔中自由滑动。
  5. 如权利要求3所述的癌症相关的EB病毒核酸含量检测试剂盒，其特征在于，所述驱动器连接内托相对面一侧的外壳开设有滑槽，该滑槽的两端位置和两条滑轨的两端位置相匹配，所述滑槽的高度大于内托的厚度，长度大于内托的宽度。
  6. 如权利要求3所述的癌症相关的EB病毒核酸含量检测试剂盒，其特征在于，所述外壳的表面设置有出仓按钮和进仓按钮，所述出仓按钮和进仓按钮均电连接至所述驱动器的控制端；当检测者按下出仓按钮时，所述驱动器驱动内托穿过滑槽从内腔滑出至试剂盒体外；当检测者按下进仓按钮时，所述驱动器驱动内托沿着两条滑轨穿过滑槽自动推进内腔内。
  7. 如权利要求1所述的癌症相关的EB病毒核酸含量检测试剂盒，其特征在于，所述酶试剂管内装有的酶混合液为逆转录酶和扩增酶。
  8. 如权利要求1所述的癌症相关的EB病毒核酸含量检测试剂盒，其特征在于，所述外壳的内壁涂覆有隔热层，所述隔热层由保温材料制成。
  9. 一种利用如权利要求1至8任一项所述的试剂盒检测癌症相关的EB病毒核酸含量检测方法，其特征在于，该方法包括步骤：
    采用引物探针液试剂管吸取待测样品；
    通过装有酶混合液的酶试剂管从待测样品提取病毒RNA，并进行病毒RNA扩增；
    装有EB病毒RT-PCR反应液的反应试剂管进行PCR反应配置EB病毒反应溶液；
    将配置完成的EB病毒反应溶液放入荧光PCR检测仪中进行荧光定量RT-PCR检测；
    荧光定量RT-PCR检测结束后，荧光PCR检测仪将自动记录保存每个循环的荧光信号；
    选定荧光通道将荧光信号进行阈值调整，并通过观察EB病毒的扩增曲线及Ct值进行结果判断得到EB病毒核酸含量。
  10. 如权利要求9所述的癌症相关的EB病毒核酸含量检测方法，其特征在于，所述EB病毒核酸含量检测结果按照如下方式判断：
    当Ct值≥30.0或无Ct值时，结果表明待检测样品中无EB病毒核酸或其核酸含量低于检测试剂盒的检测限值，呈阴性；
    当Ct值<30时，结果表明待检测样品中含有EB病毒核酸，呈阳性。
PCT/CN2019/089246 2018-05-31 2019-05-30 癌症相关的eb病毒核酸含量检测试剂盒及方法 WO2019228449A1 (zh)

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