CN111455098A - Coronavirus nucleic acid detection kit and detection method thereof - Google Patents
Coronavirus nucleic acid detection kit and detection method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biological detection, and discloses a coronavirus nucleic acid detection kit, which comprises a kit body, wherein a containing block is embedded in the kit body, a first test tube, a second test tube, a third test tube and a fourth test tube are placed in the containing block, SARS-CoV-2_ PCR reaction solution is contained in the first test tube, SARS-CoV-2_ PCR enzyme mixed solution is contained in the second test tube, SARS-CoV-2 positive quality control products are contained in the third test tube, SARS-CoV-2 negative quality control products are contained in the fourth test tube, SARS-CoV-2_ PCR reaction solution, SARS-CoV-2_ PCR enzyme mixed solution, SARS-CoV-2 positive quality control products and SARS-CoV-2 negative quality control products are matched with an RNA extraction kit, a fluorescence quantitative PCR instrument, a vortex mixing instrument, a centrifuge and a PCR reaction tube for use, thereby detecting the negativity and the positivity of the RNA sample; the invention has the characteristic of being capable of detecting coronavirus rapidly, accurately and quantitatively.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a coronavirus nucleic acid detection kit and a detection method thereof.
Background
Coronaviruses (CoV) belong to the family Coronaviridae (Coronaviridae) and the subfamily Coronaviridae (Coronaviridae), are enveloped single-stranded positive-strand RNA viruses having the largest genomic sequence length among known RNA viruses.
Disclosure of Invention
In view of this, the present invention provides a coronavirus nucleic acid detection kit and a detection method thereof.
In order to solve the technical problems, the technical scheme of the invention is as follows: a coronavirus nucleic acid detection kit comprises a kit body, wherein a containing block is embedded in the kit body, a first test tube, a second test tube, a third test tube and a fourth test tube are placed in the containing block, SARS-CoV-2_ PCR reaction liquid is contained in the first test tube, SARS-CoV-2_ PCR enzyme mixed liquid is contained in the second test tube, SARS-CoV-2 positive quality control products are contained in the third test tube, and SARS-CoV-2 negative quality control products are contained in the fourth test tube.
Preferably, the SARS-CoV-2_ PCR reaction solution comprises dNTPs, MgC L2, Tris-HC L, a primer probe and UNG enzyme.
Preferably, the SARS-CoV-2_ PCR enzyme mixture comprises a Tap DNA polymerase and a reverse transcriptase.
Preferably, the SARS-CoV-2 positive quality control substance comprises a positive template.
Preferably, the SARS-CoV-2 negative quality control material comprises a negative template.
On the other hand, the detection method adopting the coronavirus nucleic acid detection kit is provided, which comprises the steps of preparing an RNA extraction kit, a fluorescent quantitative PCR instrument, a vortex mixing instrument, a centrifugal machine and a PCR reaction tube;
the method comprises the following specific steps:
firstly, extracting RNA in a freshly collected serum, plasma or throat swab sample by using an RNA extraction kit, ensuring that the quality and quantity of the extracted RNA meet the experimental requirements, and immediately detecting or storing the extracted RNA sample at the temperature of below 70 ℃;
secondly, after the first test tube, the second test tube, the third test tube and the fourth test tube are subjected to short-time oscillation, the first test tube, the second test tube, the third test tube and the fourth test tube are placed into a centrifuge for centrifugation for several seconds, if the first test tube, the second test tube, the third test tube and the fourth test tube are stored in an environment below 70 ℃ below zero, an RNA sample needs to be taken out in advance for 30min, and after room temperature fusion, oscillation centrifugation can be performed; after the oscillation centrifugation is finished, determining the reaction number and the amount of SARS-CoV-2_ PCR reaction solution and SARS-CoV-2_ PCR enzyme mixed solution to be added;
thirdly, taking 1.5ml of a sterile centrifuge tube, adding the SARS-CoV-2_ PCR reaction solution and the SARS-CoV-2_ PCR enzyme mixed solution into the sterile centrifuge tube, uniformly mixing by using a vortex mixer, and centrifuging for several seconds by using a centrifuge to prepare a mixed solution;
fourthly, subpackaging the prepared mixed solution into PCR reaction tubes by 20 ul/tube;
fifthly, respectively adding 5ul of SARS-CoV-2 positive quality control product, 5ul of SARS-CoV-2 negative quality control product and 5ul of RNA sample into different PCR reaction tubes, covering the tube cover tightly, then using a centrifuge to centrifuge for a short time, throwing all liquid on the tube wall to the tube bottom, and immediately putting the PCR reaction tube into a fluorescence quantitative PCR instrument to perform PCR reaction;
sixthly, selecting a FAM/TEXAS RED/Cy5 channel on a fluorescent quantitative PCR instrument to detect coronavirus, selecting a VIC/HEX channel to detect an internal reference gene, selecting a reaction volume of 25ul, and selecting None with reference fluorescence;
and seventhly, after the fluorescent quantitative PCR instrument finishes detection, analyzing data to know whether the RNA sample to be detected is negative or positive so as to judge whether the patient is the patient.
Preferably, the fluorescent quantitative PCR instrument comprises 5 stages which are respectively RNA reverse transcription stages, the set temperature is 50 ℃, the time is 30 minutes, and the cycle time is 1 time; in the pre-denaturation stage, the temperature is set to be 95 ℃, the time is set to be 3 minutes, and the cycle time is set to be 1 time; in the denaturation stage, the temperature is set to be 95 ℃, the time is set to be 15 seconds, and the cycle time is set to be 40 times; annealing, extending and collecting fluorescence signals, setting the temperature at 60 ℃, the time at 30 seconds and the cycle number at 40 times; and in the instrument cooling stage, the temperature is set to be 25 ℃, the time is set to be 1 minute, and the cycle number is set to be 1 time.
Preferably, the ratio of the cycle number of the RNA reverse transcription stage, the pre-denaturation stage, the annealing, the extension, the fluorescence signal collection stage and the instrument cooling stage is 1:1:40:40: 1.
Preferably, the RNA extraction Kit is a Viral RNA extraction Kit (DP315-R) named QIAamp Viral RNA Mini Kit (52904) or Tiangen Biochemical technology (Beijing) Ltd.
Compared with the prior art, the invention has the advantages that:
the invention uses SARS-CoV-2_ PCR reaction liquid, SARS-CoV-2_ PCR enzyme mixed liquid, SARS-CoV-2 positive quality control material and SARS-CoV-2 negative quality control material, and matches with RNA extraction kit, fluorescent quantitative PCR instrument, vortex mixing instrument, centrifuge and PCR reaction tube to detect RNA sample collected by human body, and can quickly, accurately and quantitatively detect coronavirus so as to judge the negative or positive presented in RNA sample, and can provide reference basis for determining whether human body is infected with coronavirus, so that it is convenient for management department to better control coronavirus, and can prevent the infection range of coronavirus from continuously expanding.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic structural diagram of a nucleic acid detection kit according to the present invention.
Reference numerals: 1. a kit body; 2. receiving the block; 3. a first test tube; 4. a second test tube; 5. A third test tube; 6. and a fourth test tube.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1, the present invention provides a coronavirus nucleic acid detection kit, which comprises a kit body 1, wherein a containing block 2 is designed to be embedded in the kit body, a first test tube 3, a second test tube 4, a third test tube 5 and a fourth test tube 6 are placed in the containing block 2, a SARS-CoV-2_ PCR reaction solution is contained in the first test tube 3, the components of the ARS-CoV-2_ PCR reaction solution comprise dNTPs, MgC L2, Tris-HC L, a primer probe and UNG enzyme, a SARS-CoV-2_ PCR enzyme mixed solution is contained in the second test tube 4, the components of the SARS-CoV-2_ PCR enzyme mixed solution comprise Tap DNA polymerase and reverse transcriptase, a SARS-CoV-2 positive quality control product is contained in the third test tube 5, the SARS-CoV-2 positive quality control product comprises a positive template, and a SARS-CoV-2 negative quality control product comprises a negative template in the fourth test tube 6.
Example (b): the embodiment provides a detection method adopting a coronavirus nucleic acid detection Kit, which comprises the steps of preparing an RNA extraction Kit, a fluorescent quantitative PCR instrument, a vortex mixing instrument, a centrifugal machine and a PCR reaction tube, wherein the RNA extraction Kit is a virus RNA extraction Kit (DP315-R) named as QIAamp Viral RNA Mini Kit (52904) or Tiangen Biochemical technology (Beijing) Co., Ltd.;
the method comprises the following specific steps:
the method comprises the following steps of firstly, collecting a sample, namely collecting a fresh human serum, plasma or throat swab sample, wherein the collection mode of the serum sample is that the collected blood sample is placed in a test tube without anticoagulant, then natural agglutination is carried out for 20-30min at room temperature, then a kapok stick is used for separating blood clots along the wall of the test tube, then the test tube is sealed, the test tube is placed in a centrifuge for centrifugation for 10min at the speed of 2000-3000rpm, finally a capillary pipette is used for removing the serum, and the name (or number) and the type of the sample are marked; the plasma sample collection method comprises the steps of collecting a blood sample, placing the blood sample in an EDTA anticoagulation tube, inverting and uniformly mixing the blood sample, placing the blood sample in a centrifuge, centrifuging the blood sample for 5min at the speed of 2000rpm, then sucking supernatant by using a pipette, injecting the supernatant into a new EP tube, then placing the new EP tube in the centrifuge, centrifuging the blood sample for 3min at the speed of 12000rpm, then sucking the supernatant by using the pipette, injecting the supernatant into the new EP tube to obtain separated plasma, and marking the name (or number) and the type of the sample; the collection mode of the throat swab sample collection is that a polyester fiber head swab is used for wiping the posterior wall of the pharynx and the tonsils on the two sides of the posterior part of the uvula (uvula or uvula) for multiple times by moderate force, the tongue is prevented from being touched, the uvula is taken out after wiping and placed in a sampling tube, then a plastic handle of a hand contact part is broken off, the swab is soaked in a sampling liquid, a tube cover is screwed, and the name (or the number) and the type of the sample are marked; it should be noted that the sample to be tested, i.e. the collected fresh human serum, plasma or throat swab sample, must be preserved in a frozen state at-20 + -5 deg.C for no more than 1 week, and preserved in a frozen state at-70 deg.C for no more than 6 months.
And secondly, treating a sample, namely extracting the obtained fresh human serum, plasma or throat swab sample by using an RNA extraction kit, wherein the quality and quantity of the extracted RNA meet the experimental requirements, and the extracted RNA sample is immediately detected or stored at the temperature of below 70 ℃.
Thirdly, preparing a reagent, namely placing the first test tube 3, the second test tube 4, the third test tube 5 and the fourth test tube 6 into a centrifuge for centrifugation for several seconds after short-term oscillation, taking out an RNA sample 30min in advance if the first test tube 3, the second test tube 4, the third test tube 5 and the fourth test tube 6 are stored in an environment below 70 ℃, and performing oscillation centrifugation after room temperature fusion; after the oscillation centrifugation is finished, determining the reaction number and the amount of SARS-CoV-2_ PCR reaction liquid and SARS-CoV-2_ PCR enzyme mixed liquid which need to be added, wherein the amount of the reaction number is the amount of the RNA sample which needs to be detected plus 1 tube of SARS-CoV-2 positive quality control product and 1 tube of SARS-CoV-2 negative quality control product, the amount of SARS-CoV-2_ PCR reaction liquid is the amount of the reaction number multiplied by 19, the amount of SARS-CoV-2_ PCR enzyme mixed liquid is the amount of the reaction number multiplied by 1, if the RNA sample which needs to be detected is 9 tubes, the amount of the reaction number is 11 tubes, the amount of SARS-CoV-2_ PCR reaction liquid is 209 tubes, and the amount of SARS-CoV-2_ PCR enzyme mixed liquid is 11 tubes; after the quantity is determined, taking 1.5ml of sterile centrifuge tube, and mixing the SARS-CoV-2_ PCR reaction solution and the SARS-CoV-2_ PCR enzyme mixed solution according to the proportion of 19: 1, mixing uniformly by using a vortex mixer, centrifuging for several seconds by using a centrifuge to prepare a mixed solution, and subpackaging the mixed solution into PCR reaction tubes by 20 ul/tube.
And fourthly, adding sample, namely respectively adding 5ul of SARS-CoV-2 positive quality control product, 5ul of SARS-CoV-2 negative quality control product and 5ul of RNA sample into different PCR reaction tubes each time according to the selected amount, then tightly covering the tube covers of the PCR reaction tubes, then using a centrifugal machine to carry out short-time centrifugation, throwing all liquid on the tube walls to the tube bottoms, and immediately putting the PCR reaction tubes into a fluorescence quantitative PCR instrument to carry out PCR reaction.
Fifthly, setting a fluorescent quantitative PCR instrument, selecting a FAM/TEXAS RED/Cy5 channel on the fluorescent quantitative PCR instrument to detect coronavirus, selecting a VIC/HEX channel to detect an internal reference gene, selecting a reaction volume of 25ul, selecting None as reference fluorescence, wherein the fluorescent quantitative PCR instrument comprises 5 stages when in use, namely an RNA reverse transcription stage, and setting the temperature to be 50 ℃, the time to be 30 minutes and the cycle number to be 1 time; in the pre-denaturation stage, the temperature is set to be 95 ℃, the time is set to be 3 minutes, and the cycle time is set to be 1 time; in the denaturation stage, the temperature is set to be 95 ℃, the time is set to be 15 seconds, and the cycle number is set to be 40 times; annealing, extending and collecting fluorescence signals, setting the temperature at 60 ℃, the time at 30 seconds and the cycle number at 40 times; and in the instrument cooling stage, the set temperature is 25 ℃, the time is 1 minute, the cycle number is 1, the ratio of the cycle number of the RNA reverse transcription stage, the pre-denaturation stage, the annealing, the extension, the fluorescence signal acquisition stage and the instrument cooling stage is 1:1:40:40:1, and the cycle number of the RNA reverse transcription stage, the pre-denaturation stage, the annealing, the extension, the fluorescence signal acquisition stage and the instrument cooling stage is selected according to the actual situation.
Sixthly, setting result analysis conditions, wherein the ABI7500 fluorescence quantitative instrument or the S L AN96S fluorescence quantitative instrument is commonly used for the fluorescence quantitative PCR instrument, the setting method of the base line and the threshold value of the ABI7500 fluorescence quantitative instrument is that the starting point of the base line is generally set to be 3, the end point is set to be 15, the threshold value of each channel is respectively set, when a certain channel threshold value line is set, a detected negative reference substance is firstly selected, the hooked automatic threshold value line is removed, the option ' Auto ' is changed to ' □ Auto ', then the threshold value line is manually adjusted, the highest point of a FAM/TEXAS RED/Cy5 channel amplification curve of the threshold value line just exceeds the normal blank reference substance is taken as the reference, the base line and the threshold value setting method of the S L AN96 fluorescence quantitative PCR instrument are that the starting point of the base line is set to be 6, the end point is set to be 12, the threshold value of each channel is respectively set, when a certain channel threshold value line is set, firstly, the base line optimization ' in the basic parameter ' is manually optimized ', then the highest point of the FAM blank reference substance of the.
Seventhly, controlling quality, wherein the requirement of SARS-CoV-2 negative quality control products is as follows: the channel detection of FAM/TEXAS RED/Cy5 has no Ct value or the Ct value is 0 and the channel detection of VIC/HEX is a typical S-shaped curve with the Ct value less than or equal to 32; the positive quality control requirements of SARS-CoV-2 are as follows: the detection of the FAM/TEXAS RED/Cy5 channel and the VIC/HEX channel is in a typical S-shaped curve, and the Ct value is less than or equal to 32; the RNA samples required: the amplification curve of the VIC/HEX channel is S-shaped, and the Ct value detected is less than or equal to 32; and the requirements need to be met in the same experiment, otherwise, the experiment is invalid and needs to be carried out again.
And eighthly, analyzing positive and negative Tm values, analyzing a critical value detected by the coronavirus nucleic acid detection kit by adopting an ROC curve analysis method, and determining that the Ct value of the positive judgment value of the kit for detecting the novel coronavirus nucleic acid is less than or equal to 40.
Ninth, analyzing the test result, if the amplification curve of any channel of the tested RNA sample FAM/CY5/TEXAS RED is S-shaped curve and Ct value is less than or equal to 40, the coronavirus nucleic acid detection is positive; if the amplification curve of the FAM/CY5/TEXASRED channel detection of the tested RNA sample is not in an S-shaped curve, the Ct value of the sample is 0 or no Ct value, and the Ct value of the VIC/HEX channel detection is less than or equal to 32, the sample is negative in the nucleic acid detection of the novel coronavirus; if the detected amplification curve of any channel of the tested RNA sample FAM/CY5/TEXAS RED is not in an S-shaped curve, the Ct value of the sample is 0 or no Ct value, and the Ct value detected by the VIC/HEX channel is greater than 32 or countless values, the experiment is invalid, and the detection needs to be carried out again.
In the description of the present invention, it is to be understood that the terms "coaxial", "bottom", "one end", "top", "middle", "other end", "upper", "one side", "top", "inner", "front", "center", "both ends", and the like, indicate orientations or positional relationships based on those shown in the drawings, and are only for convenience of description and simplicity of description, and do not indicate or imply that the referenced device or element must have a particular orientation, be constructed and operated in a particular orientation, and thus, are not to be construed as limiting the present invention.
In the present invention, unless otherwise expressly specified or limited, the terms "mounted," "disposed," "connected," "secured," "screwed" and the like are to be construed broadly, e.g., as meaning fixedly connected, detachably connected, or integrally formed; can be mechanically or electrically connected; the terms may be directly connected or indirectly connected through an intermediate, and may be communication between two elements or interaction relationship between two elements, unless otherwise specifically limited, and the specific meaning of the terms in the present invention will be understood by those skilled in the art according to specific situations.
The above are only typical examples of the present invention, and besides, the present invention may have other embodiments, and all technical solutions formed by equivalent substitutions or equivalent transformations fall within the scope of the present invention as claimed.
Claims (9)
1. A coronavirus nucleic acid detection kit comprises a kit body (1), and is characterized in that: the kit body (1) is internally embedded with a containing block (2), a first test tube (3), a second test tube (4), a third test tube (5) and a fourth test tube (6) are placed in the containing block (2), SARS-CoV-2_ PCR reaction liquid is placed in the first test tube (3), SARS-CoV-2_ PCR enzyme mixed liquid is placed in the second test tube (4), SARS-CoV-2 positive quality control products are placed in the third test tube (5), and SARS-CoV-2 negative quality control products are placed in the fourth test tube (6).
2. The kit of claim 1, wherein the SARS-CoV-2_ PCR reaction solution comprises dNTPs, MgC L2, Tris-HC L, primer probe and UNG enzyme.
3. The coronavirus nucleic acid detection kit of claim 1, wherein: the SARS-CoV-2_ PCR enzyme mixed liquor comprises components of Tap DNA polymerase and reverse transcriptase.
4. The coronavirus nucleic acid detection kit of claim 1, wherein: the SARS-CoV-2 positive quality control material comprises a positive template.
5. The coronavirus nucleic acid detection kit of claim 1, wherein: the SARS-CoV-2 negative quality control material comprises negative template.
6. The method of any one of claims 1 to 5, which comprises the steps of: the method comprises the steps of preparing an RNA extraction kit, a fluorescent quantitative PCR instrument, a vortex mixing instrument, a centrifuge and a PCR reaction tube;
the method comprises the following specific steps:
firstly, extracting RNA in a freshly collected serum, plasma or throat swab sample by using an RNA extraction kit, ensuring that the quality and quantity of the extracted RNA meet the experimental requirements, and immediately detecting or storing the extracted RNA sample at the temperature of below 70 ℃;
secondly, after the first test tube (3), the second test tube (4), the third test tube (5) and the fourth test tube (6) are subjected to short-term oscillation, the first test tube (3), the second test tube (4), the third test tube (5) and the fourth test tube (6) are placed into a centrifuge for centrifugation for several seconds, if the first test tube (3), the second test tube (4), the third test tube (5) and the fourth test tube (6) are stored in an environment below 70 ℃, an RNA sample needs to be taken out 30min in advance, and after room-temperature fusion, oscillation centrifugation can be performed; after the oscillation centrifugation is finished, determining the reaction number and the amount of SARS-CoV-2_ PCR reaction solution and SARS-CoV-2_ PCR enzyme mixed solution to be added;
thirdly, taking 1.5ml of a sterile centrifuge tube, adding the SARS-CoV-2_ PCR reaction solution and the SARS-CoV-2_ PCR enzyme mixed solution into the sterile centrifuge tube, uniformly mixing by using a vortex mixer, and centrifuging for several seconds by using a centrifuge to prepare a mixed solution;
fourthly, subpackaging the prepared mixed solution into PCR reaction tubes by 20 ul/tube;
fifthly, respectively adding 5ul of SARS-CoV-2 positive quality control product, 5ul of SARS-CoV-2 negative quality control product and 5ul of RNA sample into different PCR reaction tubes, covering the tube cover tightly, then using a centrifuge to centrifuge for a short time, throwing all liquid on the tube wall to the tube bottom, and immediately putting the PCR reaction tube into a fluorescence quantitative PCR instrument to perform PCR reaction;
sixthly, selecting a FAM/TEXAS RED/Cy5 channel on a fluorescent quantitative PCR instrument to detect coronavirus, selecting a VIC/HEX channel to detect an internal reference gene, selecting a reaction volume of 25ul, and selecting None with reference fluorescence;
and seventhly, after the fluorescent quantitative PCR instrument finishes detection, analyzing data to know whether the RNA sample to be detected is negative or positive so as to judge whether the patient is the patient.
7. The method according to claim 6, wherein the detection method comprises the steps of: the fluorescent quantitative PCR instrument comprises 5 stages which are respectively RNA reverse transcription stages, the temperature is set to be 50 ℃, the time is set to be 30 minutes, and the cycle time is set to be 1 time; in the pre-denaturation stage, the temperature is set to be 95 ℃, the time is set to be 3 minutes, and the cycle time is set to be 1 time; in the denaturation stage, the temperature is set to be 95 ℃, the time is set to be 15 seconds, and the cycle number is set to be 40 times; annealing, extending and collecting fluorescence signals, setting the temperature at 60 ℃, the time at 30 seconds and the cycle number at 40 times; and in the instrument cooling stage, the temperature is set to be 25 ℃, the time is set to be 1 minute, and the cycle number is set to be 1 time.
8. The method of claim 7, wherein the detection method comprises the steps of: the ratio of the cycle times of the RNA reverse transcription stage, the pre-denaturation stage, the annealing stage, the extension stage, the fluorescence signal acquisition stage and the instrument cooling stage is 1:1:40:40: 1.
9. The method according to claim 6, wherein the detection method comprises the steps of: the RNA extraction Kit used was a Viral RNA extraction Kit (DP315-R) named QIAamp Viral RNA Mini Kit (52904) or Tiangen Biochemical technology (Beijing) Ltd.
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