阅读说明：本技术 癌症相关的eb病毒核酸含量检测试剂盒及方法 (Kit and method for detecting nucleic acid content of EB (Epstein-Barr) virus related to cancer ) 是由 陈琦 梁昊原 谷东风 于 2018-05-31 设计创作，主要内容包括：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。(The invention provides a cancer-related EB virus nucleic acid content detection kit and a method. The inner support is movably arranged in the inner cavity through a pulley and two sliding rails, the inner support is provided with eight detection concave holes which are arranged in a 2x 4 array, reagent tubes are inserted into seven detection concave holes, and the other detection concave hole is a standby hole. The reagent tubes comprise a reaction reagent tube filled with RT-PCR reaction liquid, an enzyme reagent tube filled with enzyme mixed liquid, a negative control reagent tube filled with EB virus negative quality control substances, a positive control reagent tube filled with EB virus positive quality control substances, a double-distilled water reagent tube filled with sterilized double-distilled water, an ROX reagent tube filled with ROX correction liquid and a primer probe liquid reagent tube. And a semiconductor temperature controller is arranged in the inner cavity and below the inner support and used for adjusting the environmental temperature of the inner cavity to the temperature required by EB virus detection. The invention can quickly and accurately detect the EB virus content, and has strong specificity, high sensitivity, stability and convenience.)

1. The utility model provides a cancer-related EB virus nucleic acid content detection kit, includes shell, inner chamber and interior support, its characterized in that, be provided with two slide rails in the inner chamber, four angle bottoms of interior support set up a pulley respectively, interior support is passed through pulley and two slide rail activities set up in the inner chamber, interior support is last to be provided with eight detection shrinkage pools of arranging with 2x 4 array, and wherein seven detection shrinkage pools intussuseption have the reagent pipe, and another one detects the shrinkage pool and is equipped with the hole, wherein:

The reagent tubes comprise a reaction reagent tube filled with RT-PCR reaction liquid, an enzyme reagent tube filled with enzyme mixed liquid, a negative control reagent tube filled with an EB virus negative quality control product, a positive control reagent tube filled with an EB virus positive quality control product, a double-distilled water reagent tube filled with sterilized double-distilled water, an ROX reagent tube filled with ROX correction liquid and a primer probe liquid reagent tube;

The temperature-adjustable semiconductor temperature controller is arranged in the inner cavity and below the inner support, the power line input end of the semiconductor temperature controller is electrically connected with the storage battery, and the semiconductor temperature controller is used for adjusting the environmental temperature in the inner cavity to the temperature required by EB virus detection so as to realize reagent heat preservation.

2. The kit for detecting the nucleic acid content of the EB virus related to the cancer according to claim 1, wherein the aperture of the detection concave hole of the reagent tube of the EB-PCR reaction solution is larger than the apertures of seven other detection concave holes, the apertures of the seven other detection concave holes are equal in size, and each reagent tube is a sterilized screw glass tube.

3. The kit for detecting the nucleic acid content of cancer-associated epstein barr virus according to claim 1, wherein the inner cavity is further provided with a driver, an input end of a power line of the driver is electrically connected to a storage battery, and the storage battery provides driving electric energy for the driver.

4. The kit for detecting the nucleic acid content of cancer-associated epstein-barr virus according to claim 3, wherein the driver comprises 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 holder, the other end of the free telescopic rod is electrically connected to the driving motor, and the driving motor drives the inner holder to freely slide in the inner cavity along two sliding rails when the driving motor drives the free telescopic rod to extend and retract up and down.

5. The kit for detecting the nucleic acid content of EB virus related to cancer according to claim 3, wherein the housing on the side of the driver opposite to the inner holder is provided with a sliding slot, the two ends of the sliding slot are matched with the two ends of the two sliding rails, the height of the sliding slot is greater than the thickness of the inner holder, and the length of the sliding slot is greater than the width of the inner holder.

6. The cancer-associated EB virus nucleic acid content detection kit according to claim 3, wherein a warehouse-out button and a warehouse-in button are arranged on the surface of the shell, and both the warehouse-out button and the warehouse-in button are electrically connected to the control end of the driver; when a detector presses the delivery button, the driver drives the inner support to pass through the sliding groove and slide out of the inner cavity to the outside of the reagent box body; when the inspector presses the warehouse entering button, the driver drives the inner support to penetrate through the sliding groove along the two sliding rails to automatically push the inner cavity.

7. the kit for detecting the nucleic acid content of cancer-associated epstein-barr virus according to claim 1, wherein the enzyme mixture contained in the enzyme reagent tube comprises reverse transcriptase and an amplification enzyme.

8. the kit for detecting the nucleic acid content of cancer-associated epstein-barr virus according to claim 1, wherein an inner wall of the casing is coated with a heat insulation layer, and the heat insulation layer is made of a heat insulation material.

9. a method for detecting the nucleic acid content of cancer-associated epstein barr virus using the kit of any one of claims 1 to 8, comprising the steps of:

Absorbing a sample to be detected by adopting a primer probe liquid reagent tube;

Extracting virus RNA from a sample to be detected through an enzyme reagent tube filled with enzyme mixed liquor, and performing virus RNA amplification;

Carrying out PCR reaction on a reaction reagent tube filled with EB virus RT-PCR reaction solution to prepare EB virus reaction solution;

Putting the prepared EB virus reaction solution into a fluorescent PCR detector for fluorescent quantitative RT-PCR detection;

After the fluorescent quantitative RT-PCR detection is finished, the fluorescent PCR detector automatically records and stores the fluorescent signal of each cycle;

And selecting a fluorescence channel to adjust the threshold value of the fluorescence signal, and carrying out result judgment by observing the amplification curve and Ct value of the EB virus to obtain the nucleic acid content of the EB virus.

10. The method for detecting the nucleic acid content of epstein-barr virus associated with cancer according to claim 9, wherein the result of the detection of the nucleic acid content of epstein-barr virus is determined as follows:

When the Ct value is more than or equal to 30.0 or no Ct value, the result shows that the sample to be detected has no EB virus nucleic acid or the nucleic acid content thereof is lower than the detection limit value of the detection kit and is negative;

when the Ct value is less than 30, the result shows that the sample to be detected contains EB virus nucleic acid and is positive.

Technical Field

The invention relates to the technical field of EB virus detection, in particular to a kit and a method for detecting the nucleic acid content of EB virus related to cancer.

Background

The EB virus belongs to the DNA virus of human herpesvirus gammalidae lymphotropic virus, the infection rate of people in China reaches more than 90 percent, and each organ and system of the whole body can be affected; EB virus has the biological characteristic of specifically infecting human and some primate B cells in vivo and in vitro; humans are hosts for EB virus infection, primarily by salivary transmission; EB virus is proliferated in oropharyngeal epithelial cells and then infects B lymphocytes, and the cells largely enter blood circulation to cause systemic infection and can be latent in human lymph tissues for a long time; EBV infection can be manifested as proliferative and latent infection; different antigens are expressed in different infection states, the antigens expressed in the proliferative infection stage comprise EBV early antigens, EBV capsid proteins and EBV membrane antigens, and the antigens expressed in the latent infection stage comprise EBV nuclear antigens and latent membrane proteins; the virus infection has various clinical manifestations and is easy to miss diagnosis and misdiagnose.

the Polymerase Chain Reaction (PCR) technology is widely applied to animal epidemic disease detection, and the technology is mainly based on the specificity of pathogenic microorganism nucleic acid and designs a specific primer aiming at a target to carry out exponential amplification on a DNA or RNA segment; therefore, the establishment of the EB virus detection method by using the fluorescence PCR technology has important significance for quickly and accurately detecting the EB virus. At present, a plurality of kits for detecting EBV-DNA are marketed abroad, the detection method mainly comprises fluorescence immunoassay (HIGH), the domestic EBV detection mainly adopts an ELISA method, and no approved kit for detecting EBV-DNA is available on the market. Based on the serious EBV infection, it is important to develop a simple, feasible and low-cost detection kit. Most of the existing reagent boxes have the problems of complicated steps, mutual pollution of detection reagents, difficulty in distinguishing between reagent tubes and the like, and the reagent preservation temperature in the reagent box cannot be in a constant-temperature preservation state, so that the detection precision of the EB virus content is influenced, and the sensitivity is low.

disclosure of Invention

The invention mainly aims to provide a cancer-related EB virus nucleic acid content detection kit and a method, and aims to solve the problem that the detection precision of EB virus content is influenced because the storage temperature of reagents in the kit cannot be in a constant-temperature storage state in the prior art.

in order to achieve the above object, the present invention provides a cancer-related EB virus nucleic acid content detection kit, comprising a housing, an inner cavity, and an inner holder, wherein two slide rails are disposed in the inner cavity, a pulley is disposed at each of the bottoms of four corners of the inner holder, the inner holder is movably disposed in the inner cavity via the pulley and the two slide rails, eight detection concave holes arranged in a 2 × 4 array are disposed on the inner holder, wherein a reagent tube is inserted into seven detection concave holes, and the other detection concave hole is a spare hole, wherein:

the reagent tubes comprise a reaction reagent tube filled with RT-PCR reaction liquid, an enzyme reagent tube filled with enzyme mixed liquid, a negative control reagent tube filled with an EB virus negative quality control product, a positive control reagent tube filled with an EB virus positive quality control product, a double-distilled water reagent tube filled with sterilized double-distilled water, an ROX reagent tube filled with ROX correction liquid and a primer probe liquid reagent tube;

The temperature-adjustable semiconductor temperature controller is arranged in the inner cavity and below the inner support, the power line input end of the semiconductor temperature controller is electrically connected with the storage battery, and the semiconductor temperature controller is used for adjusting the environmental temperature in the inner cavity to the temperature required by EB virus detection so as to realize reagent heat preservation.

Preferably, the aperture of the detection concave hole of the EB-PCR reaction solution reagent tube is larger than the apertures of the other seven detection concave holes, the apertures of the other seven detection concave holes are equal in size, and each reagent tube is a sterilized screw glass tube.

Preferably, the inner cavity is further provided with a driver, the power line input end of the driver is electrically connected to a storage battery, and the storage battery provides driving electric energy for the driver.

Preferably, the driver comprises 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, the other end of the free telescopic rod is electrically connected to the driving motor, and when the driving motor drives the free telescopic rod to extend and retract up and down, the inner support is driven to freely slide in the inner cavity along the two sliding rails.

Preferably, the shell on one side of the opposite surface of the inner support connected by the driver is provided with a sliding chute, the positions of the two ends of the sliding chute are matched with the positions of the two ends of the two sliding rails, the height of the sliding chute is greater than the thickness of the inner support, and the length of the sliding chute is greater than the width of the inner support.

Preferably, a delivery button and an entry button are arranged on the surface of the shell, and both the delivery button and the entry button are electrically connected to the control end of the driver; when a detector presses the delivery button, the driver drives the inner support to pass through the sliding groove and slide out of the inner cavity to the outside of the reagent box body; when the inspector presses the warehouse entering button, the driver drives the inner support to penetrate through the sliding groove along the two sliding rails to automatically push the inner cavity.

Preferably, the enzyme mixture contained in the enzyme reagent tube is reverse transcriptase and an amplification enzyme.

Preferably, the inner wall of the outer shell is coated with a heat insulation layer, and the heat insulation layer is made of a heat insulation material.

In another aspect, the present invention also provides a method for detecting the nucleic acid content of cancer-associated epstein-barr virus, comprising the steps of: absorbing a sample to be detected by adopting a primer probe liquid reagent tube; extracting virus RNA from a sample to be detected through an enzyme reagent tube filled with enzyme mixed liquor, and performing virus RNA amplification; carrying out PCR reaction on a reaction reagent tube filled with EB virus RT-PCR reaction solution to prepare EB virus reaction solution; putting the prepared EB virus reaction solution into a fluorescent PCR detector for fluorescent quantitative RT-PCR detection; after the fluorescent quantitative RT-PCR detection is finished, the fluorescent PCR detector automatically records and stores the fluorescent signal of each cycle; and selecting a fluorescence channel to adjust the threshold value of the fluorescence signal, and carrying out result judgment by observing the amplification curve and Ct value of the EB virus to obtain the nucleic acid content of the EB virus.

Preferably, the result of detecting the nucleic acid content of the EB virus is judged as follows: when the Ct value is more than or equal to 30.0 or no Ct value, the result shows that the sample to be detected has no EB virus nucleic acid content or the nucleic acid content is lower than the detection limit value of the detection kit, and the sample to be detected is negative; when the Ct value is less than 30, the result shows that the sample to be detected contains EB virus nucleic acid and is positive.

Compared with the prior art, the kit and the method for detecting the nucleic acid content of the EB virus related to the cancer adopt the technical scheme, and the following technical effects are achieved: the kit can quickly and accurately detect cancer-related EB virus, has strong specificity, high sensitivity, stability and convenience, does not pollute detection reagents, and can accurately detect the content of the EB virus; coating a heat insulation material on the inner wall of the reagent box body, wherein the inner support is made of hard foam; in addition, through placing the semiconductor temperature controller in the kit body, when being convenient for the kit body to be taken outside, can guarantee that the reagent temperature in the kit body is in the constant temperature save state, avoid influencing the detection precision because of the temperature surpasses save temperature.

Drawings

FIG. 1 is a schematic structural diagram of the kit for detecting the nucleic acid content of cancer-associated EB virus according to the present invention;

FIG. 2 is a flow chart of the preferred embodiment of the method for detecting the nucleic acid content of cancer-associated EB virus according to the invention.

The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.

Detailed Description

To further explain the technical means and effects of the present invention adopted to achieve the predetermined objects, the following detailed description of the embodiments, structures, features and effects of the present invention will be given with reference to the accompanying drawings and preferred embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

referring to fig. 1, fig. 1 is a schematic structural diagram of the kit for detecting the nucleic acid content of the cancer-associated epstein barr virus according to the present invention. In this embodiment, the kit for detecting the content of EB virus associated with cancer includes a reagent kit body 10, the reagent kit body 10 is in a rectangular parallelepiped shape with a pull-out structure, and the reagent kit body 10 includes an outer shell 1, an inner cavity 2, and an inner support 3. The inner wall of the shell 1 is coated with a heat insulation layer 11, the heat insulation layer 11 is made of heat insulation materials, heat insulation of reagents is achieved, EB virus detection precision is improved, and detection accuracy is prevented from being affected by temperature factors.

As a preferred embodiment, two sliding rails 12 are arranged in the inner cavity 2, the inner support 3 is of a rectangular parallelepiped structure, the bottom of each of the four corners of the inner support 3 is provided with a pulley 13, and each pulley 13 can freely slide on the sliding rail 12. The inner support 3 is movably arranged in the inner cavity 2 through the pulley 13 and the two slide rails 12. The distance between two slide rails 12 arranged in the inner cavity 2 is equal to the length of the inner support 3, so that the inner support 3 can freely slide in the inner cavity 2 along the two slide rails 12 through pulleys 13 arranged at the bottoms of four corners. 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 electric energy 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, and when the driving motor 140 drives the free telescopic rod 141 to extend and retract up and down, the inner support 3 is driven to freely slide in the inner cavity 2 along the two slide rails 12.

as a preferred embodiment, the inner holder 3 is made of rigid foam, eight detection concave holes arranged in a 2 × 4 array are arranged on the inner holder 3, and reagent tubes are inserted into seven of the eight detection concave holes; the reagent tubes comprise a reaction reagent tube 31 filled with EB virus RT-PCR reaction liquid, an enzyme reagent tube 32 filled with enzyme mixed liquid, a negative control reagent tube 33 filled with EB virus negative quality control substances, a positive control reagent tube 34 filled with EB virus positive quality control substances, a double-distilled water reagent tube 35 filled with sterilized double-distilled water, a ROX reagent tube 36 filled with ROX correction liquid and a primer probe liquid reagent tube 37. The other detection concave hole is a spare hole 38, the aperture of the detection concave hole inserted with the EB-PCR reaction solution reagent tube 31 is larger than the apertures of the other seven detection concave holes, the apertures of the other seven detection concave holes are equal in size, and an electronic tag is attached to the outside of each reagent tube and used for distinguishing EB virus detection reagents in each reagent tube.

In this embodiment, the EB virus RT-PCR reaction solution contained in the reaction reagent tube 31 is used for performing a PCR reaction on a sample to be tested to prepare an EB virus reaction solution, and the enzyme mixture contained in the enzyme reagent tube 32 is reverse transcriptase and an amplification enzyme, and is used for extracting viral nucleic acid (RNA) from the EB virus reaction solution and performing viral RNA amplification. The EB virus negative quality control substance contained in the negative control reagent tube 33 and the EB virus positive quality control substance contained in the positive control reagent tube 34 can detect the nucleic acid of the EB virus or the nucleic acid content thereof from the sample to be detected. The double distilled water reagent tube 35 is filled with sterilized double distilled water for configuring the concentration of the EB virus reaction solution, the ROX correction fluid filled in the ROX reagent tube 36 is used for correcting the concentration of the EB virus reaction solution, and the primer probe fluid reagent tube 37 is used for sucking a sample to be detected.

As the preferred embodiment, each reagent pipe is a sterilized screw glass pipe, a temperature-adjustable semiconductor temperature controller 4 is arranged in the inner cavity 2 and below the inner support 3, the power line input end of the semiconductor temperature controller 4 is electrically connected with the storage battery 15, the semiconductor temperature controller 4 can adjust the environmental temperature in the inner cavity 2 to the temperature required by EB virus detection, the heat preservation of the reagent is realized, the EB virus detection precision is improved, and the detection accuracy is prevented from being influenced by temperature factors.

As a preferred embodiment, the shell 1 on one side of the opposite surface of the driver 14 connected with the inner holder 3 is provided with a sliding slot 16, the two end positions of the sliding slot 16 are matched with the two end positions of the two sliding rails 12, the height of the sliding slot 16 is greater than the thickness of the inner holder 3, and the length of the sliding slot 16 is greater than the width of the inner holder 3, so that the inner holder 3 can slide out of the reagent box body 10 from the inner cavity 2 through the sliding slot 16. In the present embodiment, the surface of the housing 1 of the reagent cartridge body 10 is provided with a bin outlet button 17 and a bin inlet button 18, and both the bin outlet button 17 and the bin inlet button 18 are electrically connected to the control terminal of the driver 14. When the inspector presses the delivery button 17, the driver 14 can drive the inner holder 3 to slide out of the reagent box body 10 from the inner cavity 2 through the sliding chute 16, so that the inspector can add a solution to be tested (such as saliva or sweat) into the reagent tube; when the examiner finishes adding the solution to be tested in the reagent tube and presses the warehousing button 18, the driver 14 can automatically push the inner support 3 into the inner cavity 2 through the sliding chute 16 along the two sliding rails 12.

The kit for detecting the nucleic acid content of the cancer-related EB virus can quickly and accurately detect the cancer-related EB virus, has strong specificity, high sensitivity, stability and convenience, does not pollute detection reagents, and can accurately detect the content of the EB virus; coating a heat insulation material on the inner wall of the reagent box body, wherein the inner support is made of hard foam; in addition, through placing the semiconductor temperature controller in the kit body, when being convenient for the kit body to be taken outside, can guarantee that the reagent temperature in the kit body is in the constant temperature save state, avoid influencing the detection precision because of the temperature surpasses save temperature.

as shown in FIG. 2, FIG. 2 is a flow chart of the preferred embodiment of the method for detecting the nucleic acid content of EB virus related to cancer according to the present invention. In this embodiment, the method for detecting the nucleic acid content of cancer-associated epstein-barr virus comprises the following steps: step S21: a primer probe solution reagent tube 37 is adopted to absorb a sample to be detected; step S22: extracting virus RNA from a sample to be detected through an enzyme reagent tube 32 filled with enzyme mixed liquor, and performing virus RNA amplification; step S23: the reaction reagent tube 31 filled with EB virus RT-PCR reaction liquid carries out PCR reaction to prepare EB virus reaction solution, wherein the total reaction system is 25 mu L, and comprises 12 mu L of 2XRT-PCR buffer solution, 7.5 mu L of primer probe suction amount, 0.5 mu L of amplification enzyme and 5 mu L of sample nucleic acid.

Step S24: and putting the prepared EB virus reaction solution into a fluorescent PCR detector for fluorescent quantitative RT-PCR detection. The PCR (polymerase chain reaction) instrument is a pathogen quantitative nucleic acid molecular diagnostic instrument, and is widely applied to the rapid quantitative detection and early diagnosis of infectious diseases such as hepatitis B HBV, hepatitis C HCV, HIV, STD and the like and tumor cancers. As a preferred embodiment, the reaction cycle parameters: 2min at 50 ℃ for 1 cycle; 2min at 95 ℃ for 1 cycle; collecting fluorescence at 58 ℃ in 45 cycles of 1min at 91 ℃ and 15s → 58 ℃; wherein, the upstream primer has a sequence shown as Seq ID No.1, the downstream primer has a sequence shown as Seq ID No.2, and the EB virus TaqMan fluorescent probe has a sequence shown as 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 EB virus TaqMan fluorescent probe has FAM as the 5 'end fluorescent reporter group and BHQ1 as the 3' end marker.

step S25: after the fluorescent quantitative RT-PCR detection is finished, the fluorescent PCR detector automatically records and stores the fluorescent signal of each cycle; step S26: selecting a fluorescence channel (FAM) to adjust the threshold value of the fluorescence signal, and judging the result by observing the amplification curve and Ct value of the EB virus to obtain the nucleic acid content of the EB virus; when the negative control has no fluorescence logarithmic increase, no Ct value is detected; the positive control has the logarithmic increase of fluorescence, and the fluorescence channel FAM presents a typical amplification curve; in this example, the results of the detection of nucleic acid content of EB virus were judged as follows: when the Ct value is more than or equal to 30.0 or no Ct value, the result shows that the sample to be detected has no EB virus nucleic acid or the nucleic acid content thereof is lower than the detection limit value of the detection kit and is negative; when the Ct value is less than 30, the result shows that the sample to be detected contains EB virus nucleic acid and is positive.

The cancer-related EB virus nucleic acid content detection method can quickly and accurately distinguish EB viruses and accurately detect the EB virus content; the disease condition of the EB virus can be known more comprehensively, the diagnosis efficiency of the EB virus is improved, corresponding prevention and control measures are provided, and unnecessary economic loss is reduced; coating a heat insulation material on the inner wall of the reagent box body, wherein the inner support is made of hard foam; in addition, through placing the semiconductor temperature controller in the kit body, when being convenient for kit integument is taken outward, can guarantee that the reagent temperature in the kit is in the constant temperature save state, avoid influencing the detection precision because of the temperature surpasses save temperature.

The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by using the contents of the present specification and the accompanying drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.