CN111471803A - Novel coronavirus COVID-19 infection detection kit - Google Patents
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Abstract
The invention discloses a novel coronavirus COVID-19 infection detection kit, and belongs to the field of in-vitro diagnostic reagents. The kit comprises a primer and a probe for detecting a specific gene ORF1ab of the novel coronavirus COVID-19, a primer and a probe for detecting a positive internal standard gene GAPDH, and a primer and a probe for detecting an exogenous monitoring gene EGFP, wherein the sequences of the primers and the probes are respectively shown as SEQ ID NO. 1-9; the kit improves the detection flux on the premise of ensuring the accuracy, stability and stability of the detection result. The invention uses Trizol reagent as sample preserving fluid, which ensures the biological safety, nucleic acid integrity and virus load. The RNA of the exogenous monitoring gene is introduced during the extraction of the RNA, so that the RNA loss condition in the whole extraction and detection process can be monitored, the normalization of the whole operation process is ensured, and false negative caused by the defects of any step in the operation process is eliminated.
Description
Technical Field
The invention belongs to the field of in-vitro diagnostic reagents, and particularly relates to a novel coronavirus COVID-19 infection detection kit.
Background
Since the new coronavirus pneumonia epidemic situation occurs, nucleic acid detection is always the 'gold standard' for clinical diagnosis, rehabilitation discharge and isolation release. The fluorescent quantitative PCR method commonly used at present comprises the following steps: sample collection, nucleic acid extraction, fluorescent quantitative PCR detection, and further judging whether the sample contains the novel coronavirus or not by detecting whether the sample to be detected contains the specific gene of the novel coronavirus.
However, the current nucleic acid detection has the common problems of false negative and false positive, namely the problems of accuracy and sensitivity. The new coronavirus is an RNA virus, and the RNA enzyme has certain stability and wide sources, and the RNA enzyme which is ubiquitous in a human body and the environment can degrade RNA in each link of sample collection, transportation, storage, extraction and detection; in addition, the sampling volume is large, and the virus load is diluted; the test results do not allow quantification of viral load in different patients; lack of monitoring of the entire process, etc. For the detection result, on one hand, it is impossible to judge whether the sampling fails or the viral load is insufficient due to sample dilution, on the other hand, it is impossible to confirm the nucleic acid loss condition in the experimental operation, and it is also impossible to accurately describe the viral loads carried by different patients. Severely hampers the confirmation of suspected cases and the screening of potential carriers, as well as monitoring the course of the patient.
Regarding sample collection, two methods which are commonly used at present mainly comprise non-inactivated virus preservation solution and inactivated virus preservation solution, the sampling volume is generally 3-4m L, the existing method usually takes 0.2-1m L sample to extract nucleic acid, so that the sample is diluted, the potential virus load is reduced, and further the detection sensitivity is greatly reduced.
Regarding sample extraction, the existing methods usually use direct column extraction and magnetic bead extraction, but sample loss may be caused by operation procedures or human factors during extraction, so that sample extraction failure is caused, how to define sampling failure or false negative caused by sample extraction failure, and how to define extraction efficiency difference between different samples is to be solved.
Regarding RT-QPCR detection, determination of the detection result is not determined whether the detection result is a negative detection result of a virus-specific gene or a false negative due to sampling transport failure or nucleic acid extraction failure, and a positive internal standard gene needs to be set. Meanwhile, compared with RT and QPCR step-by-step methods and dye method detection, the one-step probe method detection is a more accurate and efficient detection method, but because the method does not have a step of digesting genome pollution, genome detection interference caused by positive internal standard genes can mislead the judgment of sample qualification. The increase of the detection of the internal standard gene improves the reading of the result accuracy, but also limits the detection flux and increases the detection cost.
Regarding the problem of false positive caused by aerosol contamination, a commonly used method is to strictly perform laboratory partition management and set a negative control. In addition, the use of UDG enzyme in the reaction is also a very effective means for preventing aerosol contamination.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provide a novel coronavirus COVID-19 infection detection kit; in addition, a novel coronavirus COVID-19 sample preservation method and an RNA extraction method are also provided.
The purpose of the invention is realized by the following technical scheme:
a novel coronavirus covi-19 infection detection kit, comprising: the primers and the probes for detecting the specific genes of the novel coronavirus COVID-19, the primers and the probes for detecting the positive internal standard genes and the primers and the probes for detecting the exogenous monitoring genes. Wherein, the specific gene of the novel coronavirus COVID-19 and the positive internal standard gene are subjected to double PCR.
Furthermore, the specific gene of the novel coronavirus COVID-19 is ORF1ab, the positive internal standard gene is GAPDH, and the exogenous monitoring gene is EGFP.
Furthermore, the primers and probes for detecting the specific gene ORF1ab of the novel coronavirus COVID-19 are as follows:
ORF1ab F primer: CCCTGTGGGTTTTACACTTAA (SEQ ID NO.1),
ORF1ab R primer: ACGATTGTGCATCAGCTGA (SEQ ID NO.2),
ORF1ab probe: 5 'FAM-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ 1-3' (SEQ ID NO. 3);
the primers and probes for detecting the positive internal standard gene GAPDH are as follows:
GAPDH F primer: ATCAGCAATGCCTCCTGCAC (SEQ ID NO.4),
GAPDH R primers: ACAGTCTTCTGGGTGGCAGT (SEQ ID NO.5),
GAPDH probe: 5 'VIC-ACCAACTGCTTAGCACCCCTG-BHQ 1-3' (SEQ ID NO. 6);
the primers and the probes for detecting the exogenous monitoring gene EGFP are as follows:
EGFP primer: ATGTTGTGGCGGATCTTGAAGT (SEQ ID NO.7),
EGFP R primers: GTCTATATCATGGCCGACAAGCA (SEQ ID NO.8),
EGFP probe: 5 'FAM-CACCTTGATGCCGTTCT-BHQ 1-3' (SEQ ID NO. 9).
Furthermore, the novel coronavirus COVID-19 infection detection kit comprises dUTP/dNTP Mix, UDG enzyme, a fluorescent quantitative PCR reagent and the like.
Furthermore, the novel coronavirus COVID-19 infection detection kit comprises RNA standard products of the novel coronavirus COVID-19 specific gene, the positive internal standard gene and the exogenous monitoring gene.
A method for preserving a novel coronavirus COVID-19 sample is to use Trizol reagent to preserve, furthermore, the sample comprises a nasal swab, an oropharyngeal swab, cerebrospinal fluid, blood and the like, and the preservation volume of the sample is 1m L.
A method for extracting novel coronavirus COVID-19RNA is characterized in that the RNA is extracted by a Trizol method, and RNA of an exogenous monitoring gene with known molecular number is added into a sample before the RNA is extracted. Further, the exogenous monitoring gene is EGFP.
The invention has the following beneficial effects:
(1) in order to solve the problems of RNA degradation prevention and biological safety in the process of sample collection and transportation, Trizol reagents are selected as cracking, extracting and storing reagents, the Trizol reagents are mainly prepared by guanidine salt matched with reagents such as Tween, phenol and the like, the guanidine salt components can effectively inhibit the activity of RNA enzyme, the integrity of RNA is ensured, meanwhile, the reagent components can fully inactivate viruses possibly existing, the biological safety is ensured, the sampling volume is only 1m L, all collected samples can be subjected to nucleic acid extraction, the virus load is further improved, the detection sensitivity is increased, and the sampling cost is reduced.
(2) Setting an exogenous monitoring gene EGFP, and comparing primers and probes for EGFP gene detection, wherein the primers and the probes are not homologously matched with the new coronavirus and the human genome. Adding EGFP RNA with known molecular number before RNA extraction, determining the number of the residual EGFP molecules in the sample according to an EGFP standard curve in QPCR detection, further obtaining the EGFP yield, and indirectly reflecting the RNA extraction and detection yield in the sample. The RNA loss condition of the whole extraction and detection process can be monitored through the exogenous monitoring gene, the normalization of the whole operation process is ensured, and false negative caused by the defects of any step in the operation process is eliminated.
(3) Setting positive internal standard gene GAPDH, comparing the primer and probe detected by GAPDH gene, and not matching with new coronavirus; and the primers are designed by spanning introns, so that the genome pollution is avoided. The positive internal standard gene can monitor the false negative problem caused by sample collection and transportation loss and evaluate whether the sample is qualified.
(4) Setting the standard, the pseudoviral RNA standard is 10^5 molecules, 10^4 molecules, 10^3 molecules, 10^2 molecules, 10 molecules and 1 molecules. And (3) detecting the standard substance and the sample in the same batch, drawing a standard curve, carrying out absolute quantification on the detection result according to the standard curve, and carrying out quantitative analysis on the virus load of the patient sample. For infected samples, the number of new coronaviruses infected can be given, and the possible infection degree can be presumed according to the existing knowledge and algorithm, and the detection sensitivity of each reaction is as low as 10 molecules.
(5) The virus specific gene ORF1ab and human GAPDH are selected as detection targets, and double PCR is performed, so that the accuracy and stability of results are ensured, and the detection flux is improved. The virus specific gene ORF1ab is compared and has sequence conservation in the downloaded 118 new coronavirus genomes; no match exists among coronavirus hku1, coronavirus nl63, respiratory syncytial virus, human metapneumovirus, rhinovirus, human bocavirus, MERS virus and SARS virus, and the conservation and specificity of ORF1ab detection primers and probes are good.
(6) In the reaction system, a dUTP/UDG anti-pollution system is used, and the dUTP/dNTP Mix with optimized proportion is added with Heat-simple Uracil-DNA glycylase from psychrophilic marine bacteria. The Heat-labileUracil-DNA Glycosylase has high activity at room temperature, and can fully degrade double-stranded DNA pollution containing U in the mixing process of a reaction system. When the reaction system is heated to 50 ℃, the Heat-laboratory Uracil-DNA Glycosylase is quickly and thoroughly inactivated, the integrity of cDNA is maintained, the detection sensitivity is not influenced, and false positive brought by aerosol pollution is eliminated.
Drawings
FIG. 1 is a flow chart of a complete set of technical schemes for detection of infection by the novel coronavirus COVID-19.
FIG. 2 is a graph showing the amplification of ORF1ab and GAPDH, and the amplification of sample Nos. 2 and 5 on the left and right, respectively.
FIG. 3 is a standard graph of ORF1ab and GAPDH duplex PCR detection, single gene detection.
Detailed Description
The flow chart of the complete technical scheme for detecting the infection of the novel coronavirus COVID-19 is shown in figure 1, and specifically comprises the following steps:
firstly, collecting samples
1) Nasal swab and oropharyngeal swab sampling
Adding 1m L Trizol reagent (ambion, 15596-.
2) Cerebrospinal fluid sample sampling
The cerebrospinal fluid sample is mixed with equal volumes of Trizol reagent, e.g., 4.5m L cerebrospinal fluid +4.5m L Trizol reagent.
3) Blood sample sampling
The anticoagulated blood sample is mixed with Trizol reagent in a volume ratio of 1:2, for example 2m L anticoagulated blood +4m L Trizol reagent.
Second, nucleic acid extraction
In a biosafety secondary laboratory, mainly a biosafety cabinet, the following operations are carried out:
(1) thawing the sample tube on ice, transferring the sample in the sample tube to a 1.5m L EP tube, slightly pressing a swab head by using a pipette head, fully extruding the sample, and completely transferring the sample to the EP tube, approximately taking out the sample preserved by 700 mu L Trizol, adding 2.5 x 10^4 molecules of EGFP standard RNA into the sample, fully mixing the EGFP standard RNA and the EGFP standard RNA, and then carrying out subsequent nucleic acid extraction.
(2) For each sample, 140. mu. L chloroform (1/5 sample volumes) was added and mixed by vortexing for 5 seconds.
(3) The samples were sequentially placed in a refrigerated centrifuge and centrifuged at 13200rpm at 4 ℃ for 20 minutes.
(4) While centrifuging, another set of EP tubes was prepared, each tube being charged with 1 μ L glycogen (20mg/m L), 35 μ L sodium acetate at 3M pH5.2 and 350 μ L isopropanol in that order.
(5) After centrifugation is finished, the sample tubes in the centrifuge are carefully taken out and are sequentially placed in an EP tube frame, liquid in the EP tube is divided into 3 layers, a medium-sized gun head is sleeved with a small-sized gun head to carefully absorb an upper-layer aqueous phase without touching an intermediate layer or a lower organic layer, about 350 mu L aqueous phase can be absorbed (if the intermediate layer or the lower-layer liquid is inadvertently absorbed, the non-aqueous-phase liquid in the gun head is gently knocked off, whether the residual liquid keeps pure aqueous phase or not is judged, the residual liquid is retained and not discarded), and the liquid is sequentially added into the EP tube prepared in the previous step.
(6) Shaking with vortex shaker for 5s, mixing well the liquid in EP tube, and precipitating at-20 deg.C for 30 min.
(7) After precipitation, the mixture was centrifuged at 13200rpm at 4 ℃ for 20 minutes, and the RNA white precipitate was visually observed at the bottom of the centrifuge tube.
(8) The large gun head is sleeved with the small gun head, the supernatant is gently sucked and discarded along the opposite tube wall of the sediment, and the supernatant is not completely discarded because ethanol washing is carried out later.
(9) Adding 75% ethanol of 1m L into each tube, performing vortex oscillation for 5s to make visible white precipitate more clearly visible, centrifuging at 13200rpm at 4 ℃ for 5 minutes, sleeving a small gun head on a large gun head, slightly sucking and discarding supernatant along the opposite tube wall of the precipitate, centrifuging at 13200rpm at 4 ℃ for 30s to allow a small amount of residual ethanol to be remained on the tube wall, and carefully sucking residual liquid by using a small gun head (if batch samples are processed simultaneously, when ethanol supernatant is removed from the former sample, the tube cover needs to be closed, and the problem that drying time of the same batch of samples is inconsistent during subsequent drying is avoided)
(10) The cover is opened and the mixture is dried at room temperature for 3min, the optimal time for complete drying is just when the white precipitate becomes transparent, and the RNA quality is influenced by too long or too short drying time.
(11) And adding 15 mu L DEPC water for redissolving, wherein the RNA can be directly subjected to RT-QPCR experiments or transferred to a refrigerator for storage at-70 ℃.
Third, fluorescent quantitative PCR detection
(1) Unfreezing the RNA sample at 4 ℃, and unfreezing the related reagent; according to the number of samples to be detected, configuring related Mix, and setting a negative control sample (DEPC water used in the experiment), positive control standard ORF1ab RNA, GAPDHRNA and EGFP RNA (the number of molecules is respectively 10^5 molecules, 10^4 molecules, 10^3 molecules, 10^2 molecules, 10 molecules and 1 molecule).
(2) QPCR system with Taqman probe method
Primer probe information:
TABLE 1
ORF1ab and GAODH duplex PCR assay:
TABLE 2
EGFP detection:
TABLE 3
(3) Reaction procedure
TABLE 4
(4) Analysis of results
The fluorescence collecting time can be properly adjusted according to different models, and the program is set according to an ABI Quant Studio6Flex fluorescence quantitative PCR instrument.
Setting method of ABI Quant Studio6Flex fluorescence quantitative PCR instrument baseline and threshold:
baseline (baseline) settings: the automatic setting was removed, the manual setting was selected, the baseline starting point was set to 3, and the end point was set to 15.
Threshold (threshold) setting: removing the automatic setting, selecting the manual setting, respectively setting the threshold values of all channels, when setting a certain channel threshold value line, firstly selecting the detected negative reference substance, removing the hooked automatic threshold value line, and then manually adjusting the threshold value line, wherein the highest point of the channel amplification curve (irregular noise line) of the normal blank reference substance which is just exceeded by the threshold value line is taken as the standard.
(5) Determination of results
a) If the ORF1ab amplification curve of the sample to be detected is a typical S-shaped curve and the Ct value of ORF1ab is less than or equal to 38, the sample can be judged to be positive;
b) if the amplification curve of the sample ORF1ab to be detected does not show an S-shaped curve, or ORF1ab has no Ct value, and the Ct value of the internal reference is less than or equal to 32, the amplification curve of the internal reference is a typical S-shaped curve, and the sample can be judged to be negative;
c) if the amplification curve of the sample ORF1ab to be detected is an S-shaped curve and the Ct value of the detected sample is more than 38, retesting is required, and the determination is carried out according to the condition;
d) if the amplification curve of the sample ORF1ab to be detected does not show an S-shaped curve, or ORF1ab has no Ct value, and the Ct value of the internal reference is greater than 32 or has no numerical value, the amplification curve of the internal reference does not show a typical S-shaped curve, the sample is determined to be unqualified, and the re-detection is needed.
The advantages and effects of the present invention are further shown below in conjunction with specific test examples.
Example 1
The above protocol was used to collect cerebrospinal fluid and blood samples, RNA extraction and fluorescent quantitative PCR assays for 11 human nasal and oropharyngeal swabs and 8 patients, with the results shown in table 1 and fig. 2.
The Ct values detected by positive reference genes GAPDH of the nasal swab and the oropharyngeal swab are between 12 and 25 (table 5), the Ct values detected by the positive reference genes GAPDH of the cerebrospinal fluid sample are between 23 and 31, and the Ct values detected by the positive reference genes GAPDH of the blood sample are between 16 and 18 (table 6), so that the requirements of qualified sample storage and extraction are met; in addition, the RNA of the cerebrospinal fluid sample preserved and extracted by the Trizol reagent is found to have new coronavirus in the subsequent RNA-seq detection, which is not found in the preservation and extraction of the existing method, so that the reliability, the stability and the sensitivity of the preservation of the sample by the Trizol reagent and the nucleic acid extraction are fully proved.
TABLE 5
TABLE 6
Accurate detection of copy numbers of GAPDH and ORF1ab in samples of oropharyngeal swabs from COVID-19 patients, as well as the copy number of EGFP added exogenously (Table 5): in 11 detected nasal swabs and oropharyngeal swabs, the molecular number of the detected positive internal reference gene GAPDH is calculated according to a standard curve and is between 7500 and 3 x 10^8, which indicates that the number of the cells taken by different samples is different; the number of EGFP molecules detected is between 401.1 and 4300.2 molecules, and the recovery rate of EGFP is between 8 and 86 percent according to the initial input quantity of EGFP and 5000 molecules (Table 5); in addition, 2 positive samples were detected at this time, the number of detected ORF1ab molecules was obtained by the standard curve of ORF1ab, and the number of detected viral copies of the patient was indirectly obtained, and the samples were found to be equally positive, but the viral loads were very different (FIG. 2), for example, the viral load of sample No.2 was 4 x 10^5, and the viral load of patient No.5 was only 0.1. It is concluded that patient 2 may be in a high infectious stage, and because of the high virus load, isolation is highly important and the associated medical personnel are also particularly concerned about protection; while patient 5 may be in a stage of immediate infection, or a stage of immediate cure, not very infectious, still requiring isolation and protection, but may be downgraded from the former. Therefore, the detection result of the invention can accurately give the result of the RNA extraction and detection efficiency of the whole process according to the recovery rate of EGFP; according to the GAPDH result, a sample qualification result is given; and the accurate virus infection copy number of the positive sample is given, which is beneficial to clinical judgment.
Example 3
The results of the single-gene detection and the double-PCR detection of the GAPDH and ORF1ab standards using the above-described fluorescent quantitative PCR detection method are shown in Table 7 and FIG. 3. As can be seen from the results, the Ct value detected by the single gene and the Ct value detected by the duplex PCR are almost the same for the standard with the same number of molecules, which indicates that the specificity of the primers is good, and the 2 groups of primers and the probes have no obvious influence and the linear relationship is also good.
TABLE 7
The above examples are some of the embodiments of the present invention, but the embodiments of the present invention are not limited by the above examples, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be regarded as equivalent substitutions, and are included in the scope of the present invention.
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Claims (10)
1. A novel coronavirus COVID-19 infection detection kit is characterized in that: comprises a primer and a probe for detecting a specific gene of the novel coronavirus COVID-19, a primer and a probe for detecting a positive internal standard gene, and a primer and a probe for detecting an exogenous monitoring gene.
2. The novel coronavirus COVID-19 infection detection kit according to claim 1, wherein: the specific gene of the novel coronavirus COVID-19 and the positive internal standard gene are subjected to double PCR.
3. The novel coronavirus COVID-19 infection detection kit according to claim 1, wherein: the specific gene of the novel coronavirus COVID-19 is ORF1ab, the positive internal standard gene is GAPDH, and the exogenous monitoring gene is EGFP.
4. The novel coronavirus COVID-19 infection detection kit according to claim 3, wherein:
the primers and the probes for detecting the specific gene ORF1ab of the novel coronavirus COVID-19 are as follows:
ORF1ab F primer: CCCTGTGGGTTTTACACTTAA the flow of the air in the air conditioner,
ORF1ab R primer: ACGATTGTGCATCAGCTGA the flow of the air in the air conditioner,
ORF1ab probe: 5 'FAM-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ 1-3';
the primers and probes for detecting the positive internal standard gene GAPDH are as follows:
GAPDH F primer: ATCAGCAATGCCTCCTGCAC the flow of the air in the air conditioner,
GAPDH R primers: ACAGTCTTCTGGGTGGCAGT the flow of the air in the air conditioner,
GAPDH probe: 5 'VIC-ACCAACTGCTTAGCACCCCTG-BHQ 1-3';
the primers and the probes for detecting the exogenous monitoring gene EGFP are as follows:
EGFP primer: ATGTTGTGGCGGATCTTGAAGT the flow of the air in the air conditioner,
EGFP R primers: GTCTATATCATGGCCGACAAGCA the flow of the air in the air conditioner,
EGFP probe: 5 'FAM-CACCTTGATGCCGTTCT-BHQ 1-3'.
5. The novel coronavirus COVID-19 infection detection kit according to any one of claims 1 to 4, wherein: comprises dUTP/dNTP Mix, UDG enzyme and fluorescent quantitative PCR reagent.
6. The novel coronavirus COVID-19 infection detection kit according to any one of claims 1 to 4, wherein: an RNA standard product containing a novel coronavirus COVID-19 specific gene, a positive internal standard gene and an exogenous monitoring gene.
7. A method for preserving a novel coronavirus COVID-19 sample is characterized by comprising the following steps: preservation for Trizol reagent; the sample comprises a nasal swab, an oropharyngeal swab, cerebrospinal fluid and blood.
8. The method for preserving a sample of the novel coronavirus COVID-19 according to claim 7, wherein the sample preservation volume is 1m L.
9. A method for extracting novel coronavirus COVID-19RNA is characterized by comprising the following steps: for the Trizol method to extract RNA, RNA of exogenous monitoring genes with known molecular number is added into a sample before the RNA is extracted.
10. The method for extracting the novel coronavirus COVID-19RNA according to claim 9, wherein the method comprises the following steps: the exogenous monitoring gene is EGFP.
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