CN112522445A - 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 application discloses a primer probe combination, a kit and a method for detecting novel coronavirus, wherein the primer probe combination is shown as SEQ ID NO.1-9, and the primer probe combination can be used for detecting ORF1ab gene fragment and N gene fragment of novel coronavirus nucleic acid, so that the novel coronavirus can be accurately detected; the kit comprises a nucleic acid rapid amplification reaction solution, a nucleic acid rapid amplification enzyme mixed solution, a sample releasing agent, a positive control and a negative control, and is suitable for conventional fluorescent quantitative PCR methods, digital PCR methods and the like.
Description
Technical Field
The application relates to the technical field of biological detection, in particular to a primer probe combination, a kit and a method for detecting novel coronavirus.
Background
The pathogenic pathogen of the novel coronary pneumonia (Coronavir Disease 2019, COVID-19) is named as a novel Coronavirus (SARS-CoV-2), patients often show fever, dry cough and hypodynamia, severe cases can cause acute respiratory syndrome, multi-organ failure and even death, the infection route is mainly through respiratory droplets and close contact transmission, and the crowd is generally susceptible, so that the establishment of a rapid and accurate detection method is particularly important.
The nucleic acid detection is used as the gold standard of the current novel coronavirus detection method, and plays a vital role in the processes of rapid diagnosis, curative effect evaluation and epidemic prevention and control. However, the detection kit used in the conventional nucleic acid detection method is not only required to be used in a laboratory with a certain grade, but also required to have a certain detection equipment foundation and related trained operators, so that sometimes the screening of large-scale population is difficult to be rapidly completed, or a small batch of samples are rapidly detected at any time, and detection reports can be obtained only half a day or even every other day, which increases the time cost for the confirmation of patients and has a barrier effect on the timely prevention and control of epidemic situations. In addition, the related technology also has related detection of antigen antibody of the novel coronavirus, the detection takes about half an hour, but the detectable antibody is usually detected after a patient is infected with the virus for several days, so the detection sensitivity and the detection accuracy are limited, the rapid detection of the antigen antibody is often used in a rapid screening stage, and the nucleic acid detection is still a recognized standard for determining the novel coronavirus.
The existing nucleic acid detection kit for the novel coronavirus usually takes a long time for single circulation in the using process, so that the detection usually needs to be started after a large quantity of samples are collected, namely, the centralized delayed detection is usually given to the chance of virus re-propagation; in addition, the detection method needs a special PCR (polymerase chain reaction) laboratory to complete detection, and cannot meet the requirement of rapid detection along with detection, so that the mobility of detected personnel becomes wider, the application range of the existing nucleic acid detection product is limited, and the universality of biomolecule diagnosis cannot be promoted.
Disclosure of Invention
The application aims to provide a primer probe combination, a kit and a method for detecting novel coronavirus so as to realize efficient and accurate detection of the novel coronavirus.
The application is realized by the following technical scheme:
in a first aspect, the present application provides a primer probe combination for the detection of a novel coronavirus, comprising:
the first primer probe group for detecting the novel coronavirus ORF1ab gene comprises a first forward primer shown as SEQ ID NO.1, a first reverse primer shown as SEQ ID NO.2 and a first probe shown as SEQ ID NO. 3;
a second primer probe group for detecting the N gene of the novel coronavirus, which comprises a second forward primer shown as SEQ ID NO.4, a second reverse primer shown as SEQ ID NO.5 and a second probe shown as SEQ ID NO. 6; and the number of the first and second groups,
the third primer probe group for detecting the internal standard gene comprises a third forward primer shown as SEQ ID NO.7, a third reverse primer shown as SEQ ID NO.8 and a third probe shown as SEQ ID NO. 9.
Further, the fluorescent group marked at the 5' end of the first probe shown as SEQ ID NO.3 is FAM; the fluorescent group marked at the 5' end of the second probe shown as SEQ ID NO.6 is HEX; the 5' -end labeled fluorophore of the third probe shown in SEQ ID NO.9 is CY 5.
In a second aspect, the present application provides a use of the primer probe combination in the preparation of a kit for detecting a novel coronavirus.
In a third aspect, the present application provides a kit for detecting a novel coronavirus, including a nucleic acid rapid amplification reaction solution, a nucleic acid rapid amplification enzyme mixture, a sample releasing agent, a positive control and a negative control, wherein the nucleic acid rapid amplification reaction solution includes the primer probe combination.
Further, the concentration of each primer in the primer probe combination in the nucleic acid rapid amplification reaction solution is 0.2-1 μ M, and the concentration of each probe in the primer probe combination in the nucleic acid rapid amplification reaction solution is 0.2-1 μ M.
Further, the reaction solution for rapid amplification of nucleic acid also comprises MgCl2, a buffer solution and a dNTP solution.
Further, the nucleic acid rapid amplification enzyme mixture comprises reverse transcriptase, a nuclease inhibitor and DNA rapid polymerase with hot start property; wherein the reverse transcriptase is a reverse transcriptase with RNaseH activity deletion, and the amplification speed of the DNA rapid polymerase with the hot start property is not less than 50 bp/s.
Further, the concentration of the reverse transcriptase in the nucleic acid rapid amplification enzyme mixture is 4-12U/muL, and the concentration of the DNA rapid polymerase in the nucleic acid rapid amplification enzyme mixture is 0.02-0.2U/muL.
Further, the positive control comprises an in vitro transcription RNA segment containing ORF1ab gene, N gene and internal standard gene RnaseP; the negative control was ribozyme-free water treated with diethylpyronate.
In a fourth aspect, the present application provides a method for detecting a novel coronavirus, wherein the kit is used for detecting a novel coronavirus nucleic acid.
The implementation of the technical scheme of the application has the following beneficial effects:
the primer probe combination is shown as SEQ ID NO.1-9, and the primer probe combination can be used for detecting ORF1ab gene segments and N gene segments of novel coronavirus nucleic acid, so that the novel coronavirus can be accurately detected; the kit is suitable for conventional fluorescent quantitative PCR methods, digital PCR methods and the like, has the characteristic of rapidness and accuracy when being used for detecting the novel coronavirus, and can greatly shorten the time of the whole nucleic acid amplification process, so that the novel coronavirus can be efficiently and accurately detected.
Drawings
In order to more clearly illustrate the technical solutions and advantages of the embodiments of the present application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic flow chart of the fluorescent quantitative method for detecting novel coronavirus in the present application example;
FIG. 2 is a schematic diagram showing the comparison of the results of fluorescence quantitative PCR detection based on the kit of the present embodiment and the prior art kit;
FIG. 3 shows the result of detection of the amplification curve of ORF1ab gene fragment;
FIG. 4 shows the result of detection of the amplification curve of the N gene fragment;
FIG. 5 shows melting curves of the amplification results of ORF1ab gene fragment;
FIG. 6 is a melting curve showing the result of amplification of an N gene fragment;
FIG. 7 is a schematic flow chart of the digital PCR method for detecting the novel coronavirus in the example of the present application;
FIG. 8 is a graph showing the results of the reproducibility and stability of the detection of the ORF1ab target using the kit of the present embodiment;
FIG. 9 is a graph showing the results of the reproducibility and stability of the detection of N target using the kit according to the embodiment of the present application;
FIG. 10 is a graph showing the results of the reproducibility and stability of RNase P detection using the kit according to the embodiment of the present application;
FIG. 11 is a graph comparing the results of a digital PCR assay based on the kits of the present application with a quantitative fluorescent assay using a prior art kit;
FIG. 12 shows the test results of clinical samples according to the example of the present application;
FIG. 13 is a graph of the concentration results of the clinical samples corresponding to FIG. 12;
FIG. 14 shows the results of triple targets in different fluorescence channels;
fig. 15 is a time-temperature curve corresponding to fig. 14.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more clear, the present application will be further described in detail with reference to the following embodiments. It is to be understood that the embodiments described are only a few embodiments of the present application and not all 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 application.
It should be noted that the terms "first," "second," "third," and the like in the description and claims of this application and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the application described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or server that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
Examples
Aiming at the defect that the novel coronavirus nucleic acid detection can not be carried out rapidly, accurately and efficiently in the prior art, a diagnostic reagent capable of rapidly and accurately detecting the novel coronavirus is developed so as to realize the efficient and accurate detection of a sample to be detected, and the urgent need of epidemic situation prevention and control is met.
To this end, the present application provides a primer probe combination for detecting a novel coronavirus, specifically SARS-CoV-2, wherein the sequences of the primers and probes in the present embodiment are as follows:
a first primer probe combination for detecting a novel coronavirus ORF1ab gene, comprising:
first forward primer sequence (5 '-3'): GTGARATGGTCATGTGTGGCGG (SEQ ID No.1),
first reverse primer sequence (5 '-3'): CARATGTTAAASACACTATTAGCATA (SEQ ID NO.2),
first probe (5 '-3'): CAGGTGGAACCTCATCAGGAGATGC (SEQ ID NO.3),
the first probe is a Taqman probe, the fluorescence reporter group at the 5 'end is FAM, and the quenching group at the 3' end is BHQ 1.
A second primer probe combination for detecting a novel coronavirus N gene, comprising:
second forward primer sequence (5 '-3'): GGGGAACTTCTCCTGCTAGAAT (SEQ ID No.4),
second reverse primer sequence (5 '-3'): CAGACATTTTGCTCTCAAGCTG (SEQ ID NO.5),
second probe (5 '-3'): TTGCTGCTGCTTGACAGATT (SEQ ID NO.6),
the second probe is a Taqman probe, the fluorescence reporter group at the 5 'end is HEX, and the quenching group at the 3' end is BHQ 1.
A third primer probe combination for detecting an internal standard gene, wherein the internal standard gene is specifically an Rnase P gene and comprises the following components:
third forward primer sequence (5 '-3'): AGATTTGGACCTGCGAGCG (SEQ ID No.7),
third reverse primer sequence (5 '-3'): GAGCGGCTGTCTCCACAAGT (SEQ ID NO.8),
third probe (5 '-3'): TTCTGACCTGAAGGCTCTGCGCG (SEQ ID NO.9),
the third probe is a Taqman probe, the fluorescent reporter group at the 5 'end is CY5, and the quenching group at the 3' end is BHQ 2.
In this example, the 5' end of the probe nucleotide sequence shown in SEQ ID NO.3/SEQ ID NO.6/SEQ ID NO.9 was labeled with a fluorophore, and sequentially labeled with FAM, HEX, and CY 5. The primer probe combination can be used for detecting the ORF1ab gene segment and the N gene segment of the novel coronavirus nucleic acid, so that the novel coronavirus can be accurately detected.
In another embodiment of the present application, an application of the primer probe combination in the above embodiments in preparing a kit for detecting a novel coronavirus is provided, which can realize accurate detection of the novel coronavirus.
Another embodiment of the present application provides a kit for detecting a novel coronavirus, which comprises a nucleic acid rapid amplification reaction solution, a nucleic acid rapid amplification enzyme mixture, a sample releasing agent, a positive control and a negative control, wherein the nucleic acid rapid amplification reaction solution comprises the primer probe combination in the above embodiments.
In a specific embodiment, the nucleic acid rapid amplification reaction solution comprises specific primers and probes of target ORF1ab and N to be detected and internal standard RNaseP. Specifically, the concentration of each primer in the primer probe combination in the nucleic acid rapid amplification reaction solution is 0.2-1. mu.M, and the concentration of each probe in the primer probe combination in the nucleic acid rapid amplification reaction solution is 0.2-1. mu.M.
In one embodiment, the reaction solution for rapid amplification of nucleic acid further comprises MgCl2Buffer and dNTP solution.
It will be understood by those skilled in the art that in other embodiments, other components may be added to the nucleic acid rapid amplification reaction solution according to specific situations and actual needs, and the examples of the present application are not particularly limited thereto.
In one embodiment, the nucleic acid rapid amplification enzyme mixture comprises a reverse transcriptase, a nuclease inhibitor, and a DNA rapid polymerase having hot start properties. The DNA fast polymerase is a DNA polymerase with the amplification speed of not less than 50bp/s, which is 4-5 times of the amplification speed of the conventional DNA polymerase, so that the time of the conventional annealing and extending steps can be shortened to one fifth of the conventional reaction time in the target nucleic acid amplification process, so that the conventional PCR single cycle reduction which takes about 1min or even longer can be shortened to tens of seconds or even seconds, and the total time consumed by 35 to 45 cycles in total can be shortened by nearly 30 min; if be equipped with quick temperature rise platform and continue to shorten the temperature rise time that rises among the PCR thermal cycle process, will reduce whole PCR greatly and detect consuming time, shorten the nucleic acid detection reaction process that traditional spends several hours (generally about 2 h) to minute order of magnitude to can in time detect novel coronavirus effectively.
Specifically, the reverse transcriptase is a reverse transcriptase with an RNaseH activity deficiency, and the concentration of the reverse transcriptase in the nucleic acid rapid amplification enzyme mixture is 4-12U/. mu.L.
Specifically, the amplification speed of the DNA fast polymerase with the hot start characteristic is not less than 50bp/s, and the concentration of the DNA fast polymerase in the nucleic acid fast amplification enzyme mixed solution is 0.02-0.2U/muL.
In a specific embodiment, the positive control is a positive standard including an in vitro transcription RNA fragment containing ORF1ab gene, N gene and internal standard gene RnaseP, and the negative control is a negative quality control which is ribozyme-free water treated by diethyl dicarbonate.
In a specific embodiment, the kit package specification may be 24 reactions/cartridge, wherein each cartridge comprises a nucleic acid rapid amplification reaction solution with a specification of 300 μ L/tube; the specification of the nucleic acid rapid amplification enzyme mixed solution is 60 mu L/tube; a sample releasing agent with the specification of 1 mL/tube; a positive control with a specification of 200 μ L/tube; negative control, 200. mu.L/tube. Of course, in other embodiments, the skilled person can make any suitable adjustment to the specification of the kit, and the examples of the present application do not limit this.
In another embodiment of the present application, a method for detecting a novel coronavirus is provided, and the kit in the above embodiment is used for detecting a novel coronavirus nucleic acid.
In a specific embodiment, referring to fig. 1, the detection method in this embodiment includes the following steps:
s1: providing a sample to be detected, and extracting nucleic acid from the sample to be detected by using a sample releasing agent to obtain a nucleic acid sample to be detected;
in a specific embodiment, the sample to be tested comprises serum, plasma sample, nasopharyngeal swab sample, sputum, alveolar lavage fluid, and the like, wherein the nasopharyngeal swab sample is stored in a TE buffer/saline matrix.
Specifically, the sample processing operation in this embodiment is performed in the sample processing area, and the sample releasing agent in the kit is first taken out, and after the equilibrium is reached to room temperature, the sample releasing agent is mixed uniformly for use.
For serum, plasma and nasopharyngeal swab samples, sucking 10 μ L of sample releasing agent, adding the sample releasing agent into a PCR reaction tube, adding 5 μ L-15 μ L, preferably 10 μ L of sample to be detected into each tube, if the sample is turbid or has obvious precipitation, centrifuging, for example, centrifuging at 2000rpm for 30 seconds, sucking 10 μ L of supernatant, sucking by using a pipette for 3-5 times, mixing uniformly, and standing at room temperature for 10 minutes to obtain the nucleic acid sample to be detected.
For samples such as sputum, alveolar lavage fluid and the like, 100 and 200 mu L of uniformly mixed samples to be detected are put into a 1.5ml centrifuge tube and centrifuged at 12000rpm for 10 minutes; and (3) removing the supernatant, adding 50 mu L of sample release agent into the precipitate, shaking and uniformly mixing, and standing for 10 minutes to serve as a nucleic acid sample to be detected for later use.
S2: and uniformly mixing a plurality of groups of nucleic acid rapid amplification reaction liquid with preset volume with the nucleic acid rapid amplification enzyme mixed liquid according to a preset proportion, respectively adding the positive control, the negative control and the nucleic acid sample to be detected, and uniformly mixing again to obtain a reaction liquid system.
Specifically, the required components in the kit are thawed, shaken, mixed uniformly and centrifuged for a short time for later use. And preparing a nucleic acid rapid amplification reaction mixed solution according to the number of the nucleic acid samples to be detected, the number of the positive controls and the number of the negative controls. Taking 10 μ L of the nucleic acid sample to be detected as an example, a nucleic acid rapid amplification reaction mixture with a total volume of 15 μ L is prepared, which mainly comprises 12.5 μ L of the nucleic acid rapid amplification reaction mixture and 2.5 μ L of the nucleic acid rapid amplification enzyme mixture, i.e., the preset ratio is 5: 1. Adding 15 mu L of nucleic acid rapid amplification reaction mixed liquor into a certain quantity of marked PCR amplification tubes for standby application, respectively adding 10 mu L of positive control (directly used without extraction), 10 mu L of negative control (directly used without extraction) and 10 mu L of nucleic acid sample to be detected corresponding to the tube marks, uniformly mixing to form a reaction liquid system with the final volume of 25 mu L/tube, tightly covering the reaction tube, and performing instantaneous low-speed centrifugation to wait for detection on a computer.
It should be noted that, taking 10 μ L of the nucleic acid sample to be detected and 25 μ L of the reaction liquid system as an example, the preset volume of the nucleic acid rapid amplification reaction liquid is determined to be 12.5 μ L, and in other embodiments, a person skilled in the art can adjust the preset volume of the nucleic acid rapid amplification reaction liquid appropriately according to the volume of the nucleic acid sample to be detected and the volume of the reaction liquid system, so as to achieve the same effect as in this embodiment.
S3: and carrying out fluorescent quantitative PCR reaction on the reaction solution system to obtain an amplification curve.
In a specific embodiment, the prepared reaction solution system is added into a PCR amplification tube, and the real-time fluorescent quantitative PCR detection is completed on a fluorescent PCR instrument; FAM, HEX and CY5 channels are respectively selected to detect specific targets ORF1ab and N and internal standard RNaseP in the new coronavirus nucleic acid.
In a specific embodiment, the fluorescent quantitative PCR reaction comprises reverse transcription, pre-denaturation, and denaturing extension in sequence. In view of the use of the preferred fast DNA polymerases with hot start properties, the time set during a PCR cycle can be significantly shortened compared to conventional approaches.
Specifically, reverse transcription is carried out at the temperature of 42-50 ℃, the time of 30s-1min and the cycle number of 1; the pre-denaturation temperature is 95 ℃, the time is 30s-1min, and the cycle number is 1; the two-step PCR program can be set to 95 ℃ for 2-5 s; the temperature is 58 ℃, the time is 4-10s, and the cycle number is 45; or setting the temperature of the three-step PCR program to be 95 ℃ and the time to be 2-5 s; the temperature is 58 ℃ and the time is 4-6 s; the temperature is 72 ℃, the time is 2-4s, and the cycle number is 45.
It should be noted that, regarding specific parameters involved in the above-mentioned fluorescent quantitative PCR reaction, such as temperature, time, etc., those skilled in the art can make any appropriate adjustments according to specific situations and actual needs, and the examples of the present application are not limited to this.
S4: and judging the detection result of the nucleic acid sample to be detected according to the amplification curve.
In a specific embodiment, on the premise that the result of the detected sample is valid, when the detected sample has an obvious S-type amplification curve in an FAM channel or an HEX channel and the Ct value is less than or equal to 40, the sample is positive; when the tested sample has an obvious S-type amplification curve in a CY5 channel and the Ct value is less than or equal to 40, and no S-type amplification curve or no Ct value exists under both the FAM channel and the HEX channel, the sample is negative.
Firstly, the following requirements need to be met in the same detection experiment, the detection result is valid, otherwise, the detection is invalid, and the detection needs to be carried out again: the negative control result shows that the FAM, the HEX channel and the CY5 channel have no Ct value or the Ct value is more than 40; the positive control result shows that the Ct values of FAM, HEX channel and CY5 channel are less than 35.
For the nucleic acid sample to be tested, the following detection results may occur:
(a) positive: detecting a typical S-shaped amplification curve of a nucleic acid sample to be detected in a FAM or HEX channel, wherein the Ct value is less than or equal to 40;
(b) negative: the nucleic acid sample to be detected does not detect a typical S-type amplification curve or Ct value is more than 40 in FAM or HEX channel, the CY5 channel has an amplification curve, and the Ct value is less than or equal to 40;
(c) and (4) invalidation: the typical S-shaped curve or Ct more than 40 is not detected in FAM, HEX and CY5 channels of the nucleic acid sample to be detected, and the reaction is possibly inhibited due to too low target content or other interferents in the nucleic acid sample to be detected, so that the result is judged to be invalid, and the sample is re-sampled to perform a detection experiment after the reason is checked.
In the case of interpreting the detection result of the nucleic acid sample to be detected according to the amplification curve, a person skilled in the art can arbitrarily and appropriately adjust the Ct value according to the specific situation, which is not limited in the embodiment of the present application.
In this embodiment, the kit in the embodiment of the present application is used, a conventional fluorescence quantitative PCR method is used for detection, a detection result is shown as a in fig. 2, b in fig. 2 is shown as a temperature curve in an amplification process of the novel coronavirus detection kit in the prior art, time consumed in an RT-PCR (Reverse Transcription-Polymerase Chain Reaction, Reverse Transcription PCR) process is about 2 hours, and as can be seen from fig. 2, the kit in the embodiment of the present application can shorten the conventional several-hour RT-PCR process to within 45min, and has the characteristics of rapidness and accuracy compared with the prior art.
In order to verify the accuracy and repeatability of the experiment, according to the steps, the standard of the national metrology institute with gradient dilution is tested, and the standard obtains the important characteristic genes of the novel coronavirus through in vitro transcription, wherein the important characteristic genes comprise ORF1ab gene fragments, N genes and the like, and the standard is purchased from the national metrology science research institute. Specifically, the model of the real-time fluorescent PCR instrument is Roche 480, and the parameters are set as the reverse transcription temperature of 42 ℃, the time of 1min and the cycle number of 1; the pre-denaturation temperature is 95 ℃, the time is 1min, and the cycle number is 1; the denaturation temperature is 95 ℃, the time is 2S, the annealing and extension temperature is 58 ℃, the time is 4S, the cycle number is 45, the detection results shown in FIGS. 3-4 are detection results, the detection result shown in FIG. 3 is an amplification curve detection result of an ORF1ab gene fragment, and the detection result shown in FIG. 4 is an amplification curve detection result of an N gene fragment, all the detection results show a standard S-shaped amplification curve, and the Ct values between the gradient curves are uniformly spaced, so that the efficiency and uniformity of an amplification system are proved to be good, and the accuracy and repeatability of the experiment are excellent. In addition, it is also shown that the rapid detection kit in the examples of the present application has excellent detection effect on a conventional fluorescence PCR instrument.
In addition, the designed primer combination and SYBR Green dye were used to detect the serial standards in gradient dilution, and the same instrument setting parameters as those used in the amplification curve obtained above were used to obtain the melting curve, and the results are shown in FIGS. 5-6. FIG. 5 shows the melting curve of the amplification result of ORF1ab gene fragment, and FIG. 6 shows the melting curve of the amplification result of N gene fragment, and it can be seen that both show a single peak of specificity, further demonstrating that the primer sequences in the kit of the present example have excellent specificity.
In an alternative embodiment, after the step S2, a Digital PCR (Digital PCR, dPCR for short) method for rapidly and quantitatively detecting the novel coronavirus may be further adopted for detection, and referring to fig. 7, specifically:
s5: the reaction liquid system was subjected to digital PCR reaction.
In a specific embodiment, the absolute quantitative detection of the novel coronavirus nucleic acid is carried out by a rapid absolute quantitative digital PCR system, namely a rapid PCR instrument, in the PCR thermal cycle process, the rapid PCR instrument can be called as the rapid PCR instrument when the heating rate and the cooling rate both reach more than 10 ℃/s, the system comprises a sample reaction chip, a micro heating disc array for heating the sample reaction chip and a temperature control system connected with the micro heating disc to realize temperature control, and the system can realize the heating rate of more than 100 ℃/s and the cooling rate of more than 50 ℃/s. Specifically, after the reaction solution system is prepared in step S2, a digital PCR instrument is used to perform sample injection to generate 20000-100000 micro-scale small reaction units, including but not limited to microdroplets, micro-cavities, and microarrays; setting PCR circulation process parameters, specifically, reverse transcription at 42-50 deg.C for 15s-1min, and circulating number 1; the pre-denaturation temperature is 95 ℃, the time is 15s-1min, and the cycle number is 1; the two-step PCR program can be set to 95 ℃ for 1-3 s; the temperature is 58 ℃, the time is 4-10s, and the cycle number is 45; alternatively, the three-step PCR program can be set to a temperature of 95 ℃ for 1-3 s; the temperature is 58 ℃ and the time is 4-6 s; the temperature is 72 ℃, the time is 2-4s, and the cycle number is 45.
S6: and (4) carrying out result interpretation on the digital PCR reaction to obtain the detection result of the nucleic acid sample to be detected.
On the premise that the result of the detected sample is effective, the result is judged according to the following mode, wherein P indicates that the number of positive droplets is more than or equal to 4, N indicates that the number of positive droplets is 0, ND indicates that the number of positive droplets is more than 0 and less than 4, and the result judgment specifically comprises the following conditions:
(a) the FAM (ORF1ab) of the nucleic acid sample to be detected is P, HEX (N) is P, CY5 (internal standard RNaseP) is P or ND; or, if the single channels of FAM (ORF1ab) and HEX (N) in the nucleic acid sample to be detected are P and the CY5 (internal standard RNaseP) is P or ND, the detection of the novel coronavirus is reported.
(b) If the single or double channels of the nucleic acid sample FAM (ORF1ab) and HEX (N) to be detected are ND and the CY5 (internal standard RNaseP) is P, the retest: reporting as detection of a new coronavirus if the results are consistent with the previous; if the inconsistency is reported as undetected.
(c) When FAM (ORF1ab) and HEX (N) in the nucleic acid sample to be detected are N and CY5 (internal standard RNaseP) is P, the detection result is reported as that no novel coronavirus is detected.
(d) And if the test nucleic acid sample CY5 (internal standard RNaseP) is N, reporting that the result is invalid, and re-testing.
It should be noted that, for the specific parameters involved in the above digital PCR reaction, such as temperature, time, etc., and for the specific values of "P" and "ND" during the interpretation of the above results, those skilled in the art can make any suitable adjustments according to specific situations and actual needs, and the examples of the present application are not limited to this.
The kit in the embodiment of the application is used for detecting the novel coronavirus, the digital PCR system in the embodiment can realize that the single cycle is less than 10s, and the absolute quantitative detection of the nucleic acid of the novel coronavirus is completed within 5-10min in total time consumption, so that the aim of quickly and accurately quantitatively detecting the novel coronavirus in a sample is fulfilled, and the detection efficiency of the 2019 novel coronavirus is greatly improved.
In order to verify the repeatability and stability of the kit in the embodiment of the application, a nucleic acid rapid absolute quantitative digital PCR system is matched to perform a gradient dilution standard repeatability test. Since only the dual targets of ORF1ab and N are within the national institute of metrology standards, to better mimic the detection of real case samples, the target RNase P extracted from human salivary nucleic acids was homogeneously mixed with the initial concentration of standard template, which was then subjected to gradient dilution. According to the initial template concentration provided by the instruction in the national institute of metrology standards, the concentration of the diluted ORF1ab target and N target is 10, 50, 100, 1000, 5000copies/μ L, and because the concentration of the RNase P target is not quantified, the dilution gradient is used as the abscissa for plotting the repeatability and stability test results, and the dilution gradient is 10-3、5×10-3、10-2、10-1、5×10-1Repeating the detection at least three times, and showing the detection results in FIGS. 8-10, wherein a is the fluorescence photograph corresponding to each dilution gradient of the sample, and b is the corresponding fitting result, fromAs can be seen in the figure, the consistency R of the detected concentration and the calculated concentration of the triple target2>0.99, the repeatability and stability of the nucleic acid rapid quantitative detection kit are proved to be good.
Further, the detection result of the digital PCR detection performed by the kit of the present application in the embodiment of the present application is compared with the detection result of a kit of the prior art based on a qPCR instrument (model number: roche 480), and the comparison result is shown in fig. 11, which shows that the detection result of the digital PCR detection performed by the kit of the present application in the embodiment of the present application and the detection result of the real-time fluorescence quantitative detection performed by the kit of the prior art have better consistency.
In addition, the kit in the embodiment of the application is matched with a digital PCR system based on rapid absolute quantification of nucleic acid to test clinical samples, so that not only can the positive and negative of the samples be judged, but also the virus load in the samples can be calculated and obtained. For example, 9 clinical samples to be detected are provided, and the detection process of the clinical samples is specifically as follows:
sample adding: 20000-30000 micron-sized droplet reaction units are formed using a microfluidic chip matched with a rapid digital PCR system.
Amplification: the program is set as follows: reverse transcription was performed at 42 ℃ for 15s, followed by pre-denaturation at 95 ℃ for 15s, and finally a rapid PCR reaction was performed for 40 cycles, about 5 min.
And (4) interpretation of results: FAM, HEX and CY5 channels are selected for detection, and the result is judged under the condition of system stability, wherein the system stability means that an internal standard gene RNase P can observe obvious positive droplets under the CY5 channel, and the results of a positive control and a negative control are normal. The following results may occur:
(1) the detection result is positive: obvious positive droplets are detected in FAM or HEX channels, and the virus copy number in the sample can be obtained through calculation according to a Poisson distribution equation.
(2) The detection result is negative: no significant positive droplets were detected in both FAM and HEX channels.
Fig. 12-13 show the detection results of the above 9 clinical samples to be detected, where a in fig. 12 is a negative control and B is a positive control, and all the nucleic acid samples 1-6 of 6 patients with new coronavirus that have been diagnosed are accurately detected to be positive, while all the nucleic acid samples 7-9 of 3 healthy people do not detect positive droplets under FAM and HEX channels, and table 1 shows the results of the virus copy number in each sample calculated according to the poisson distribution equation.
TABLE 1 nucleic acid test results of confirmed patients and healthy human samples
Further, based on the rapid detection kit system in the embodiment of the application, a rapid absolute quantitative digital PCR system is combined, time consumption of each step in RT-PCR is shortened, and ultra-rapid qualitative analysis detection is obtained, wherein the qualitative analysis is to find that an obvious positive droplet signal appears, and to prove that a target gene, namely a new crown specific gene fragment, exists in the sample at this time, so that the sample is a positive sample. In a specific embodiment, the total time consumed by a single cycle is shortened to be within 4s, the total cycle number is set to be 28, and the ultra-fast RT-PCR reaction within 2 minutes is realized, as shown in fig. 14, the results of the triple targets (the new coronavirus specific target gene ORF1ab gene fragment and the N gene fragment, and the internal standard gene RNase P gene fragment) under different fluorescence channels (FAM, HEX and Cy5) are respectively, the droplets have obvious negative and positive scores, and the gray scale of the positive droplets is obviously higher than that of the negative droplets. In addition, the time-temperature curve is shown in fig. 15, the time consumption of the whole detection is less than 2 minutes, which indicates that the kit of the present application also has the potential of faster detection efficiency when the novel coronavirus is detected by rapid qualitative analysis, and the RT-PCR reaction within 2 minutes can be rapidly realized by combining with a digital PCR system, so as to complete the differentiation of positive and negative samples. Therefore, the kit in the embodiment of the application has the capability of rapidly screening and qualitatively analyzing the sample by the novel coronavirus nucleic acid, and the qualitative analysis can be shortened to less than 5 minutes by matching with a rapid PCR instrument.
The above embodiment of the present application has the following beneficial effects:
the primer probe combination in the embodiment of the application is shown as SEQ ID NO.1-9, and the primer probe combination can be used for detecting ORF1ab gene segment and N gene segment of novel coronavirus nucleic acid, so that the novel coronavirus can be accurately detected.
The kit in the embodiment of the application is suitable for conventional fluorescent quantitative PCR methods, digital PCR methods and the like, the kit has the characteristic of rapidness and accuracy in detection of the novel coronavirus, and the time of the whole nucleic acid amplification process can be greatly shortened, so that the novel coronavirus can be efficiently and accurately detected.
The application can shorten the conventional PCR process for hours to within 45min, and even shorten the whole nucleic acid amplification process to about 5min by matching with a rapid PCR instrument, so that the purpose of rapidly and accurately quantitatively detecting the novel coronavirus in a sample is achieved, and the detection efficiency of the 2019 novel coronavirus is greatly improved.
It should be noted that: the sequence of the embodiments of the present application is only for description, and does not represent the advantages and disadvantages of the embodiments. And specific embodiments thereof have been described above. Other embodiments are within the scope of the following claims. In some cases, the actions or steps recited in the claims may be performed in a different order than in the embodiments and still achieve desirable results.
The foregoing is a preferred embodiment of the present application, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present application, and these modifications and decorations are also regarded as the protection scope of the present application.
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Claims (10)
1. A primer probe combination for the detection of a novel coronavirus, comprising:
the first primer probe group for detecting the novel coronavirus ORF1ab gene comprises a first forward primer shown as SEQ ID NO.1, a first reverse primer shown as SEQ ID NO.2 and a first probe shown as SEQ ID NO. 3;
a second primer probe group for detecting the N gene of the novel coronavirus, which comprises a second forward primer shown as SEQ ID NO.4, a second reverse primer shown as SEQ ID NO.5 and a second probe shown as SEQ ID NO. 6; and the number of the first and second groups,
the third primer probe group for detecting the internal standard gene comprises a third forward primer shown as SEQ ID NO.7, a third reverse primer shown as SEQ ID NO.8 and a third probe shown as SEQ ID NO. 9.
2. The primer probe combination of claim 1, wherein the 5' -end labeled fluorophore of the first probe shown in SEQ ID No.3 is FAM; the fluorescent group marked at the 5' end of the second probe shown as SEQ ID NO.6 is HEX; the 5' -end labeled fluorophore of the third probe shown in SEQ ID NO.9 is CY 5.
3. Use of a primer probe combination according to claim 1 or 2 for the preparation of a kit for the detection of a novel coronavirus.
4. A kit for detecting a novel coronavirus, which comprises a nucleic acid rapid amplification reaction solution, a nucleic acid rapid amplification enzyme mixed solution, a sample releasing agent, a positive control and a negative control, wherein the nucleic acid rapid amplification reaction solution comprises the primer probe combination of claim 1 or 2.
5. The kit according to claim 4, wherein the concentration of each primer in the primer probe combination in the nucleic acid rapid amplification reaction solution is 0.2 to 1 μ M, and the concentration of each probe in the primer probe combination in the nucleic acid rapid amplification reaction solution is 0.2 to 1 μ M.
6. The kit according to claim 4, wherein the reaction solution for rapid amplification of nucleic acid further comprises MgCl2Buffer and dNTP solution.
7. The kit according to claim 4, wherein the nucleic acid rapid amplification enzyme mixture comprises reverse transcriptase, a nuclease inhibitor and a DNA rapid polymerase having a hot start property; wherein the reverse transcriptase is a reverse transcriptase with RNaseH activity deletion, and the amplification speed of the DNA rapid polymerase with the hot start property is not less than 50 bp/s.
8. The kit according to claim 7, wherein the concentration of the reverse transcriptase in the nucleic acid rapid amplification enzyme mixture is 4 to 12U/μ L, and the concentration of the DNA rapid polymerase in the nucleic acid rapid amplification enzyme mixture is 0.02 to 0.2U/μ L.
9. The kit of claim 4, wherein the positive control comprises an in vitro transcribed RNA fragment comprising ORF1ab gene, N gene, and internal standard gene RnaseP; the negative control was ribozyme-free water treated with diethylpyronate.
10. A method for detecting a novel coronavirus, which comprises detecting a novel coronavirus nucleic acid using the kit according to any one of claims 4 to 9.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113337642A (en) * | 2021-06-22 | 2021-09-03 | 中国人民解放军陆军军医大学 | RT-PCR kit for detecting novel coronavirus |
| CN113684287A (en) * | 2021-08-31 | 2021-11-23 | 中国科学院上海微系统与信息技术研究所 | Primer probe group, PCR reagent and kit for detecting mycoplasma pneumoniae |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180127836A1 (en) * | 2015-05-07 | 2018-05-10 | Emerging Viral Diagnostic Limited | Improved compositions and methods for detection of viruses |
| CN111057797A (en) * | 2020-01-19 | 2020-04-24 | 华中科技大学同济医学院附属同济医院 | Novel coronavirus 2019-nCoV real-time fluorescent quantitative PCR detection primer, probe, kit and method |
| CN111304368A (en) * | 2020-03-19 | 2020-06-19 | 艾康生物技术(杭州)有限公司 | Method, oligonucleotide and kit for detecting novel coronavirus |
| CN111304369A (en) * | 2020-02-06 | 2020-06-19 | 中山大学达安基因股份有限公司 | Novel dual detection kit for coronavirus |
| CN111334614A (en) * | 2020-04-21 | 2020-06-26 | 尹秀山 | Method for detecting novel coronavirus by RT-qPCR technology |
| CN111471717A (en) * | 2020-04-17 | 2020-07-31 | 复百澳（苏州）生物科技有限公司 | Preparation method of pseudovirus for 2019 new coronavirus nucleic acid detection |
| CN111549183A (en) * | 2020-06-08 | 2020-08-18 | 国药集团武汉血液制品有限公司 | Method for simultaneously detecting SARS-CoV-2 virus and other pathogens in plasma of convalescent coronary pneumonia convalescent patient |
| CN111690626A (en) * | 2020-07-02 | 2020-09-22 | 南京诺唯赞生物科技股份有限公司 | Fusion type Taq DNA polymerase and preparation method and application thereof |
| CN111733290A (en) * | 2020-07-16 | 2020-10-02 | 北京贝尔生物工程股份有限公司 | Kit for detecting novel coronavirus and near-field coronavirus and preparation method thereof |
| CN111733291A (en) * | 2020-07-20 | 2020-10-02 | 中山大学达安基因股份有限公司 | Method and kit for detecting novel coronavirus nucleic acid by digital PCR (polymerase chain reaction) |
| CN112111607A (en) * | 2020-09-30 | 2020-12-22 | 菁良基因科技(深圳)有限公司 | COVID-19 virus nucleic acid quality control product and application thereof |
-
2020
- 2020-12-23 CN CN202011541548.9A patent/CN112522445A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180127836A1 (en) * | 2015-05-07 | 2018-05-10 | Emerging Viral Diagnostic Limited | Improved compositions and methods for detection of viruses |
| CN111057797A (en) * | 2020-01-19 | 2020-04-24 | 华中科技大学同济医学院附属同济医院 | Novel coronavirus 2019-nCoV real-time fluorescent quantitative PCR detection primer, probe, kit and method |
| CN111304369A (en) * | 2020-02-06 | 2020-06-19 | 中山大学达安基因股份有限公司 | Novel dual detection kit for coronavirus |
| CN111304368A (en) * | 2020-03-19 | 2020-06-19 | 艾康生物技术(杭州)有限公司 | Method, oligonucleotide and kit for detecting novel coronavirus |
| CN111471717A (en) * | 2020-04-17 | 2020-07-31 | 复百澳（苏州）生物科技有限公司 | Preparation method of pseudovirus for 2019 new coronavirus nucleic acid detection |
| CN111334614A (en) * | 2020-04-21 | 2020-06-26 | 尹秀山 | Method for detecting novel coronavirus by RT-qPCR technology |
| CN111549183A (en) * | 2020-06-08 | 2020-08-18 | 国药集团武汉血液制品有限公司 | Method for simultaneously detecting SARS-CoV-2 virus and other pathogens in plasma of convalescent coronary pneumonia convalescent patient |
| CN111690626A (en) * | 2020-07-02 | 2020-09-22 | 南京诺唯赞生物科技股份有限公司 | Fusion type Taq DNA polymerase and preparation method and application thereof |
| CN111733290A (en) * | 2020-07-16 | 2020-10-02 | 北京贝尔生物工程股份有限公司 | Kit for detecting novel coronavirus and near-field coronavirus and preparation method thereof |
| CN111733291A (en) * | 2020-07-20 | 2020-10-02 | 中山大学达安基因股份有限公司 | Method and kit for detecting novel coronavirus nucleic acid by digital PCR (polymerase chain reaction) |
| CN112111607A (en) * | 2020-09-30 | 2020-12-22 | 菁良基因科技(深圳)有限公司 | COVID-19 virus nucleic acid quality control product and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| E. K. WHEELER等: ""Under-three minute PCR: Probing the limits of fast amplification"", 《ANALYST》 * |
| ELIZABETH C. STAHL等: ""IGI-LuNER: single-well multiplexed RT-qPCR test for SARS-CoV-2"", 《MEDRXIV》 * |
| HAO YIN等: ""Ultrafast multiplexed detection of SARS-CoV-2 RNA using a rapid droplet digital PCR system"", 《BIOSENSORS AND BIOELECTRONICS》 * |
| XINJIN LIU等: ""Analytical comparisons of SARS-COV-2 detection by qRT-PCR and ddPCR with multiple primer/probe sets"", 《EMERGING MICROBES & INFECTIONS》 * |
| 何吕芬等: ""新型冠状病毒实时荧光RT-PCR检测的性能验证"", 《分子诊断与治疗杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113337642A (en) * | 2021-06-22 | 2021-09-03 | 中国人民解放军陆军军医大学 | RT-PCR kit for detecting novel coronavirus |
| CN113684287A (en) * | 2021-08-31 | 2021-11-23 | 中国科学院上海微系统与信息技术研究所 | Primer probe group, PCR reagent and kit for detecting mycoplasma pneumoniae |
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