CN111910023A - Primer-probe combination, kit and method for detecting novel coronavirus - Google Patents
Primer-probe combination, kit and method for detecting novel coronavirus Download PDFInfo
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Abstract
The invention relates to a primer-probe combination, a kit and a method for detecting novel coronavirus, which comprise two groups of primers and probes for detecting novel coronavirus nucleic acid and a group of internal control primers and probes, wherein the detection channel of the probe I and the primer pair I is FAM, the detection channel of the probe II and the primer pair II is VIC, and the detection channel of the internal control primers and the probes is CY 5. The kit provided by the invention introduces a plurality of marker loci into a detection system to carry out multiple fluorescence labeling, and a multiple digital PCR system simultaneously detects a plurality of novel coronavirus loci, so that the detection accuracy is improved. The kit has high sensitivity and specificity, can detect the positivity as low as 5 copies/reaction, and avoids the false negativity possibly appearing in fluorescence quantification; two detection channels and an internal control channel are arranged simultaneously, so that possible pollution caused in the sample adding process is avoided.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a primer probe combination, a kit, a method and application for detecting novel coronavirus.
Background
2019 novel coronavirus (COVID-19) was named by the world health organization on 12/1/2020. After people are infected with coronavirus, the common signs of the person are respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. The molecular diagnostic technology required for developing a kit for detecting the new coronavirus is relatively mature, but the high sensitivity and high specificity of detection of the kit on the market are required to be improved and promoted.
The existing nucleic acid detection method usually adopts a one-step method fluorescence quantitative PCR technology to detect a single gene locus after extracting RNA, so that the sensitivity of platform detection is low, detection omission and false negative are easily caused, and the existing fluorescence quantitative detection usually can only realize the simultaneous detection of two fluorescence channels and cannot give consideration to internal control channels; or detecting two parts, namely, respectively adopting two genes and one internal control gene to form a detection system.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to develop a primer probe combination, a kit, a method and application for detecting novel coronavirus, wherein the kit can ensure high sensitivity and specificity of detection, can detect the positive of 5 copies/reaction, and avoids the false negative possibly appearing in fluorescence quantification; and the detection of the two detection channels and the internal control channel is carried out simultaneously, so that the pollution possibly caused in the sample adding process is avoided.
The digital PCR technology is an absolute quantitative technology of nucleic acid molecules. The digital PCR process at least comprises 3 main links, namely sample dispersion, PCR amplification, fluorescent signal acquisition and data analysis. Namely, 1 sample is diluted and divided into hundreds of even millions of independent reaction units, each reaction unit contains or does not contain 1 or more copies of target molecules, all the independent reaction units are amplified in parallel, after the amplification is finished, negative or positive fluorescence signals of each reaction unit are read, and the copy number of the original sample is calculated according to the poisson distribution principle. According to the invention, multiple fluorescence labeling is carried out by introducing multiple labeled sites into the system, and the multiple digital PCR system simultaneously detects multiple novel coronavirus sites, so that the accuracy of the detection result is improved.
The technical scheme adopted by the invention is as follows:
a primer probe combination for detecting novel coronavirus, which comprises two groups of primers and probes for detecting novel coronavirus nucleic acid: the nucleotide sequence of the probe I is shown as SEQ ID NO. 1, and the sequences of the primer pair I are shown as SEQ ID NO. 2 and SEQ ID NO. 3; the nucleotide sequence of the probe II is shown as SEQ ID NO. 4, and the sequences of the primer pair II are shown as SEQ ID NO. 5 and SEQ ID NO. 6;
also comprises a group of internal control primers and probes: the nucleotide sequence of the internal control probe is shown as SEQ ID NO. 7, and the sequences of the internal control primer pair are shown as SEQ ID NO. 8 and SEQ ID NO. 9.
Furthermore, the detection channel of the probe I and the primer pair I is FAM, the detection channel of the probe II and the primer pair II is VIC, and the detection channel of the internal control primer and the probe is CY 5.
Further, the working concentration of the primer is 800-1200nM, and the working concentration of the probe is 150-250 nM.
Further, two sets of primers and probes for detecting the novel coronavirus nucleic acid were used to detect the 2 conserved region M gene fragments and Rdrp gene fragments of the novel coronavirus nucleic acid.
A kit for detecting a novel coronavirus, comprising the primer-probe combination for detecting a novel coronavirus: a probe I, a primer pair I, a probe II, a primer pair II, an internal control primer and a probe.
Further, the kit also comprises reaction buffer, reverse transcriptase, Taq DNA polymerase, ROX dye, internal control primer probe group, internal control DNA and nuclease-free water.
Further, the reaction Buffer is 2 × RT Buffer; the internal control DNA is the genome DNA of human oral exfoliative cells extracted by a blood/cell/tissue genome DNA extraction kit, and is diluted to 10 ng/mu L for later use.
Further, the working concentration of the reaction Buffer is 1 × RT Buffer, the working concentration of the reverse transcriptase is 0.1U/reaction, the working concentration of the Taq DNA polymerase is 0.1U/reaction, the working concentration of the ROX dye is 1 ×, and the working concentration of the internal control DNA is 0.67ng/μ L.
A method for detecting novel coronavirus nucleic acid, comprising the following steps:
extracting and purifying nucleic acid of the collected clinical sample to obtain a sample to be detected; detecting a sample to be detected by using the novel coronavirus detection kit of claim 6 to obtain detection data; and reading the detection data to obtain a detection result.
Further, the clinical sample is a nasal swab, a pharyngeal swab, sputum or alveolar lavage fluid of a human body; the detection is carried out by a multiple digital PCR method; the detection limit of the sample to be detected is more than or equal to 5 copies/reaction.
Further, the method of the multiplex digital PCR sequentially comprises three steps of reverse transcription, pre-denaturation, denaturation and extension, wherein the reverse transcription temperature in the reverse transcription step is 45-55 ℃, the reverse transcription time is 30-60min, and the cycle number is 1; the pre-denaturation temperature in the pre-denaturation step is 93-97 ℃, the pre-denaturation time is 3-7min, and the number of cycles is 1; the number of cycles of denaturation and extension is 42-48, wherein the denaturation temperature is 92-98 ℃, the denaturation time is 10-20s, the extension temperature is 55-65 ℃, and the extension time is 40-80 s.
The primer probe combination for detecting the novel coronavirus is applied to detecting the novel coronavirus.
The kit is applied to the detection of the novel coronavirus.
The application is an application for non-diagnostic purposes.
The gene sequences of SEQ ID NO 1-SEQ ID NO 9 are as follows:
the technical scheme of the invention has the beneficial effects that:
the invention provides two groups of primers and probes for detecting novel coronavirus, wherein the nucleotide sequence of the probe I is shown as SEQ ID NO. 1, and the sequences of the primer pair I are shown as SEQ ID NO. 2 and SEQ ID NO. 3; the nucleotide sequence of the probe II is shown as SEQ ID NO. 4, the sequence of the primer pair II is shown as SEQ ID NO. 5 and SEQ ID NO. 6, 2 conserved region M gene segments and Rdrp gene segments of the novel coronavirus nucleic acid can be detected by utilizing the primers and the probe, and the method can be used for accurately detecting the novel coronavirus; also comprises an internal control primer probe group, wherein the nucleotide sequence of the internal control probe in the internal control primer probe group is shown as SEQ ID NO. 7, and the sequence of the internal control primer pair is shown as SEQ ID NO. 8 and SEQ ID NO. 9. The detection channel of the probe I and the primer pair I is FAM, and the detection channel of the probe II and the primer pair II is VIC. The invention also provides a kit for detecting the novel coronavirus, which comprises the primer and the probe, and the kit also comprises reaction buffer solution, reverse transcriptase, Taq DNA polymerase, ROX dye, internal control DNA and nuclease-free water.
The invention also provides a method for detecting the novel coronavirus nucleic acid, which comprises the following steps: extracting and purifying nucleic acid of the collected clinical sample to obtain a sample to be detected; detecting a sample to be detected by adopting the novel coronavirus detection kit to obtain detection data; and reading the detection data to obtain a detection result. The kit provided by the invention introduces a plurality of marker loci into a detection system to carry out multiple fluorescence labeling, and a multiple digital PCR system simultaneously detects a plurality of novel coronavirus loci, so that the detection accuracy is improved.
The kit has high sensitivity and specificity, can detect the positivity as low as 5 copies/reaction, and avoids the false negativity possibly appearing in fluorescence quantification; two detection channels and an internal control channel are arranged simultaneously, so that possible pollution caused in the sample adding process is avoided. The detection kit has high detection speed, and the whole detection process can be finished in only 4 hours, so that the market demand can be met.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
Example 1
This example provides a primer probe combination for detecting novel coronavirus by designing primer express software based on novel coronavirus conserved domain sequence, comprising two sets of primers and probes for detecting novel coronavirus nucleic acid: the nucleotide sequence of the probe I is shown as SEQ ID NO. 1, and the sequences of the primer pair I are shown as SEQ ID NO. 2 and SEQ ID NO. 3; the nucleotide sequence of the probe II is shown as SEQ ID NO. 4, and the sequences of the primer pair II are shown as SEQ ID NO. 5 and SEQ ID NO. 6; also comprises a group of internal control primers and probes: the nucleotide sequence of the internal control probe is shown as SEQ ID NO. 7, the sequences of the internal control primer pair are shown as SEQ ID NO. 8 and SEQ ID NO. 9, the internal control probe pair is stored at the temperature of minus 20 ℃ for later use, and the specific information is shown as table 1.
TABLE 1 primer and Probe sequences
Example 2
This example provides a kit for detecting a novel coronavirus, comprising the primer probe set for detecting a novel coronavirus described in example 1, 2 × RT Buffer, reverse transcriptase, Taq DNA polymerase, ROX dye, internal control DNA, and nuclease-free water.
Wherein the internal control DNA is: extracting the genome DNA of the human oral exfoliative cells by adopting a blood/cell/tissue genome DNA extraction kit, and diluting to 10 ng/. mu.L for later use.
This example provides a method for detecting a nasal swab using the kit described above:
1. extracting nucleic acid of a clinical sample nasal swab, and purifying to obtain a sample to be detected;
2. preparing a reaction system
Thawing the required components in the kit, reversing, uniformly mixing and centrifuging for a short time for later use; the primers and probes were mixed with double distilled water without nuclease, and detection reaction solutions were prepared according to the number of reaction tubes to be detected (number of samples + 2). times.1.1, as shown in Table 2.
TABLE 2 configuration table of detection reaction solution
| Component name | Take volume (. mu.L)/sample |
| Water (W) | 0.91 |
| 2×RT Buffer | 7.5 |
| Reverse transcriptase | 0.15 |
| Taq DNA polymerase | 0.15 |
| ROX dyes | 0.3 |
| M-F(100μM) | 0.15 |
| M-R | 0.15 |
| M-P | 0.03 |
| R-F | 0.15 |
| R-R | 0.15 |
| R-P | 0.03 |
| IC-F | 0.15 |
| IC-R | 0.15 |
| IC-P | 0.03 |
| Internal control DNA | 1 |
| Sample to be tested | 4 |
| Total volume | 15 |
The working concentration of the primers in the primer probe combination is 1000nM, and the working concentration of the probes is 200 nM; the working concentration of the reaction Buffer solution 2 × RT Buffer is 1 × RT Buffer, the working concentration of the reverse transcriptase is 0.1U/reaction, the working concentration of the Taq DNA polymerase is 0.1U/reaction, the working concentration of the ROX dye is 1 × and the working concentration of the internal control DNA is 0.67ng/μ L.
3. Reaction of
Uniformly mixing the prepared reaction system, subpackaging the mixture into an optical flat cover PCR reaction tube, transferring the reaction system into a sample processing chamber, centrifuging the reaction system at a low speed instantaneously, transferring the reaction system to a detection area, and detecting the reaction system by using a multiple digital PCR method, wherein a detection channel of a probe I and a primer pair I is FAM, a detection channel of a probe II and a primer pair II is VIC, and a detection channel of an internal control primer and a probe is CY 5; the amplification reaction was carried out according to the reaction conditions shown in Table 3:
TABLE 3 reaction temperature and time Table
4. Analysis of results
The digital PCR instrument software automatically performs threshold partitioning and gives the detected copy number.
And (3) judging standard:
4.1 negative control CY5 channel copy number is greater than or equal to 75copies and FAM and VIC channel copy number is less than 5copies or no detection;
4.2 the copy number of CY5 channel as positive control is more than or equal to 75copies and the copy number of FAM and VIC channels is more than or equal to 5 copies;
4.3 copy number of CY5 channel in clinical sample equal to or greater than 75 copies:
4.3.1 copy number of FAM channel and VIC channel < 5copies or no detection, negative;
4.3.2 the copy number of FAM channel and VIC channel is more than or equal to 5copies, and the FAM channel and the VIC channel are positive;
4.3.3FAM and VIC channels, one channel with copy number greater than or equal to 5copies, the other channel with copy number less than 5copies or no detection, is suspected.
The sample to be detected in this embodiment is a suspected sample obtained by detecting a sample to be detected of clinical sample nasal swab nucleic acid, and needs to be further confirmed in combination with clinical tests.
Example 3
This example differs from example 2 only in that the clinical sample is a pharyngeal swab, the working concentration of the primers in the primer probe combination is 800nM, the working concentration of the probe is 150nM, and the amplification reaction conditions are: the reverse transcription temperature in the reverse transcription step is 45 ℃, the reverse transcription time is 60min, and the cycle number is 1; the pre-denaturation temperature in the pre-denaturation step is 93 ℃, the pre-denaturation time is 7min, and the number of cycles is 1; the number of cycles of denaturation and extension was 42, wherein the denaturation temperature was 92 ℃, the denaturation time was 20s, the extension temperature was 55 ℃, and the extension time was 80 s.
Example 4
This example differs from example 2 only in that the working concentration of primers in the primer probe combination was 1200nM, the working concentration of probes was 250nM, and the amplification reaction conditions were: the reverse transcription temperature in the reverse transcription step is 55 ℃, the reverse transcription time is 30min, and the cycle number is 1; the pre-denaturation temperature in the pre-denaturation step is 97 ℃, the pre-denaturation time is 3min, and the number of cycles is 1; the number of cycles of denaturation and extension was 48, wherein the denaturation temperature was 98 ℃, the denaturation time was 10s, the extension temperature was 65 ℃, and the extension time was 40 s.
Examples of the experiments
1. Reverse transcription program optimization
After the same amount of pseudoviral RNA is adopted and the digital PCR liquid drops are generated, the three reverse transcription programs in the table 4 are respectively adopted to carry out comparative tests, and the obtained amplification copy numbers of the M gene and the Rdrp gene are shown in the table 5.
TABLE 4 reverse transcription procedure
TABLE 5 reverse transcription program optimization
As can be seen from tables 4 and 5, the copy numbers of the M gene and Rdrp gene detected in scheme 2 were higher than those in scheme 1 and scheme 3, indicating that the reverse transcription efficiency was the highest at 50 ℃.
2. Example 2 kit minimum detection Limit test
Reaction systems were prepared as described in example 2, and then new coronavirus RNA reaction templates were set at different concentrations in each reaction system, in the following order: 5 copies/reaction system, 10^2 copies/reaction system, 10^3 copies/reaction system, set up the reaction parameter and carry out the result analysis according to corresponding reaction condition, specific result is shown in Table 6.
TABLE 6 lowest detection Limit test results
As can be seen from Table 6, the minimum detection limit of the kit in the embodiment 2 of the invention is more than or equal to 5 copies/reaction, so that false negative possibly occurring in fluorescence quantification is avoided, and the detection sensitivity of the kit for detecting the novel coronavirus is improved.
3. Example 2 kit specificity test
30 normal human pharyngeal swabs were examined, and the results are shown in Table 7.
TABLE 7-results of the kit specificity test
As can be seen from Table 7, the kit of example 2 of the present invention has detection specificity.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
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Claims (10)
1. A primer probe combination for detecting novel coronavirus, which is characterized by comprising two groups of primers and probes for detecting novel coronavirus nucleic acid: the nucleotide sequence of the probe I is shown as SEQ ID NO. 1, and the sequences of the primer pair I are shown as SEQ ID NO. 2 and SEQ ID NO. 3; the nucleotide sequence of the probe II is shown as SEQ ID NO. 4, and the sequences of the primer pair II are shown as SEQ ID NO. 5 and SEQ ID NO. 6;
also comprises a group of internal control primers and probes: the nucleotide sequence of the internal control probe is shown as SEQ ID NO. 7, and the sequences of the internal control primer pair are shown as SEQ ID NO. 8 and SEQ ID NO. 9.
2. The primer probe combination of claim 1, wherein the detection channel of probe I and primer pair I is FAM, the detection channel of probe II and primer pair II is VIC, and the detection channel of the internal control primer and probe is CY 5.
3. The primer probe combination of claim 1, wherein two sets of primers and probes for detecting novel coronavirus nucleic acid are used for detecting 2 conserved M gene segments and Rdrp gene segments of novel coronavirus nucleic acid.
4. A kit for detecting a novel coronavirus, comprising the primer-probe set for detecting a novel coronavirus according to claim 1.
5. The kit as claimed in claim 4, wherein the working concentration of the primer in the primer probe combination is 800-1200nM and the working concentration of the probe in the primer probe combination is 150-250 nM.
6. The kit of claim 4, further comprising a reaction buffer, reverse transcriptase, Taq DNA polymerase, ROX dye, internal control DNA, and nuclease-free water.
7. A method for detecting a novel coronavirus nucleic acid, comprising the steps of:
extracting and purifying nucleic acid of the collected clinical sample to obtain a sample to be detected; detecting a sample to be detected by using the novel coronavirus detection kit of claim 5 to obtain detection data; and reading the detection data to obtain a detection result.
8. The method of detecting according to claim 7, wherein the clinical sample is a nasal swab, a pharyngeal swab, sputum, or alveolar lavage of a human body; the detection is carried out by a multiple digital PCR method; the detection limit of the sample to be detected is more than or equal to 5 copies/reaction.
9. The detection method according to claim 8, wherein the multiplex digital PCR method comprises three steps of reverse transcription, pre-denaturation, denaturation and extension in sequence, wherein the reverse transcription temperature in the reverse transcription step is 45-55 ℃, the reverse transcription time is 30-60min, and the number of cycles is 1; the pre-denaturation temperature in the pre-denaturation step is 93-97 ℃, the pre-denaturation time is 3-7min, and the number of cycles is 1; the number of cycles of denaturation and extension is 42-48, wherein the denaturation temperature is 92-98 ℃, the denaturation time is 10-20s, the extension temperature is 55-65 ℃, and the extension time is 40-80 s.
10. Use of the primer probe combination of any one of claims 1 to 4 for the detection of novel coronaviruses.
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