CN113046483A - Novel real-time fluorescent RT-RAA primer, probe and detection kit for coronavirus - Google Patents
Novel real-time fluorescent RT-RAA primer, probe and detection kit for coronavirus Download PDFInfo
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Abstract
The invention discloses a novel real-time fluorescent RT-RAA primer, a probe and a detection kit for coronavirus, belonging to the technical field of virus nucleic acid detection, wherein the real-time fluorescent RT-RAA primer and the probe are designed according to a novel coronavirus gene sequence, the primer sequences are SEQ ID NO 1 and SEQ ID NO 2, and the probe sequence is SEQ ID NO 1; the kit provided by the invention comprises the primer and the probe; the final concentration of the primers of the kit is respectively 420 nmol/muL, and the final concentration of the probe is 120 nmol/muL; the kit also comprises A Buffer, B Buffer, a recombinase, a polymerase and a freeze-dried enzyme preparation of single-stranded DNA binding protein, a positive control and a negative control. The invention adopts a real-time fluorescence RT-RAA method to detect the primer, the probe and the kit of the novel coronavirus, the detection is rapid, and the operation is simple and convenient; the specificity is good, and the sensitivity is high; is suitable for field detection and large-scale screening.
Description
The technical field is as follows:
the invention belongs to the technical field of virus nucleic acid detection, and particularly relates to a primer, a probe and a kit for rapidly detecting novel coronavirus through a real-time fluorescence isothermal recombinase amplification method (RT-RAA).
Background art:
the novel coronavirus pneumonia is caused by infection of novel coronavirus (SARS-CoV-2), and has the characteristics of strong infectivity and general susceptibility of people. Clinical symptoms mainly include fever, dry cough, asthenia, and dyspnea, and serious patients are complicated with Acute Respiratory Distress Syndrome (ARDS), septic shock, systemic inflammatory response syndrome, metabolic acidosis difficult to correct, and hemorrhagic coagulation dysfunction. However, some patients do not develop SARS-CoV-2 and become asymptomatic SARS-CoV-2 infected persons (asymptomatic infected persons). Due to the strong infectivity and concealment of the virus, asymptomatic infected persons are very likely to become the source of infection and cause the occurrence of an aggregated epidemic.
The detection method aiming at SARS-CoV-2 mainly uses nucleic acid detection and antibody detection, and uses the negative result of nucleic acid detection as therapeutic recovery standard, so that the sample and selective detection method are important. The nucleic acid amplification technology based on molecular biology is the only method capable of detecting infection in the first hours after symptom onset at present, and particularly, the logarithm of the human body begins to increase from 3 rd to 5 th after the human body is infected with virus, and the logarithm can reach 10%4～106Copies/. mu.L. The detection threshold of the nucleic acid amplification technology is 1-10 copies/. mu.L, so that the virus can be found in the period, and the virus can be found in clinical samples earlier.
Commonly used nucleic acid detection methods are known to mainly include two technologies, fluorescence quantitative PCR (RT-PCR) and isothermal amplification. The RT-PCR method has strict requirements on laboratory equipment and laboratory personnel, the detection consumes long time, and the recombinase constant-temperature amplification technology has the advantages of specific and simple primer design, convenient operation, rapid amplification and constant reaction temperature. The Recombinase constant temperature amplification technology comprises a Recombinase polymerase constant temperature amplification technology (RPA) and a Recombinase mediated constant temperature amplification technology (RAA), and is characterized in that the RAA is a Recombinase obtained from bacteria or fungi to replace a phage Recombinase which is difficult to obtain in the RPA technology, the whole amplification processes of the two are consistent, and in-vitro DNA amplification is realized through combination, strand displacement and extension. RAA is a novel isothermal amplification technology which does not need a thermostable enzyme and a complex thermal cycler, and can complete nucleic acid amplification by using single strand DNA binding protein (SSB), recombinase and DNA polymerase at 37-42 ℃ for 5-30 minutes. The principle of the technology is that recombinase is extracted from bacteria or fungi, the recombinase is tightly combined with a primer under an isothermal environment to form a primer-enzyme aggregate, when the primer with the recombinase searches a DNA sequence which is completely complementary with the primer in a reaction system, a DNA chain is uncoiled and opened under the help of SSB, and a new DNA complementary chain is formed by amplification under the action of DNA polymerase. Under the condition of constant temperature, the process is continuously repeated and efficiently amplified, and the amplification product of the target gene can reach the detection level when reacting for 5 minutes at the fastest speed. The amplification product can be visualized in conjunction with various detection methods, such as real-time fluorescent quantitative PCR.
At present, along with the trend that novel coronavirus normalizes and detects, prior art need break through to the restriction in personnel, place, need shorten when detecting, and check out test set's portable and convenient degree need promote simultaneously to promote the diagnosis and move down, realize suspected patient's quick diagnosis and the quick screening in scene of the crowd of intimate contact, and provide effectual accurate quick detection scheme to different demands. Therefore, the novel coronavirus nucleic acid recombinase isothermal amplification detection kit is simple, convenient, rapid and effective, and can be used for field diagnosis and large-scale screening, and has great significance.
The invention content is as follows:
the invention aims to carry out rapid and accurate identification and amplification on the specific nucleic acid of the novel coronavirus, and by optimizing the proportion of a primer probe and each component in a constant-temperature amplification system, an amplification reagent can sensitively, rapidly and accurately detect the novel coronavirus, can keep stability and reliability in the storage and transportation processes, and realizes rapid detection and analysis of the nucleic acid of the novel coronavirus.
The technical scheme adopted by the invention for solving the technical problems is as follows:
the invention provides a primer and probe combination for detecting a novel coronavirus through a real-time fluorescent RT-RAA method, which comprises a specific primer pair and a probe aiming at an S gene segment of the novel coronavirus, wherein an upstream primer sequence is SEQ ID NO. 1, a downstream primer sequence is SEQ ID NO. 2, and a probe sequence is SEQ ID NO. 3.
The invention also provides application of the primer and probe combination in a novel coronavirus detection kit or a detection reagent.
The invention also provides a kit comprising the primer and probe combination.
Furthermore, the kit is a novel real-time fluorescence RT-RAA method detection kit for the coronavirus.
Further, the final concentrations of the primers and the probe were 420 nmol/. mu.L and 120 nmol/. mu.L, respectively.
Furthermore, the kit also comprises A Buffer, B Buffer, recombinase, polymerase and freeze-dried enzyme preparation of single-stranded DNA binding protein, a positive control and a negative control.
Further, the A Buffer is 20% PEG, and the B Buffer is 280mM MgAc.
Compared with the prior art, the invention provides the primer, the probe and the kit for detecting the novel coronavirus by the real-time fluorescence RT-RAA method, and the detection is rapid, sensitive and simple and convenient to operate. The method adopts RAA technology, and can analyze the result only by reacting for 5-20 min at 37-42 ℃ (preferably 42 ℃), without complex reaction procedures; and can be used for portable gene amplification equipment, and is suitable for field and field detection and large-scale screening. The amplification primers and the probe provided by the invention have strong specificity and high sensitivity, can accurately detect the novel coronavirus, and have stable detection result and good repeatability; the method can realize the on-site rapid detection of the novel coronavirus through a single tube, realize the totally-enclosed reaction, monitor the fluorescence data in real time, avoid the subsequent treatment, avoid the pollution and ensure the reliability of the detection result.
Description of the drawings:
FIG. 1 is a graph showing the results of RAA reaction tests at different temperatures, in which 1 is an amplification curve at 42 ℃, 2 is an amplification curve at 41 ℃, 3 is an amplification curve at 40 ℃, 4 is an amplification curve at 39 ℃, 5 is an amplification curve at 38 ℃ and 6 is an amplification curve at 37 ℃.
FIG. 2 is a diagram showing the result of specificity test of SARS-CoV-2RT-RAA method.
FIG. 3 is a graph showing the results of sensitivity test of SARS-CoV-2RT-RAA method, in which 1 is 1.0X 107Copy/. mu.L amplification curve, 2 is 1.0X 106Amplification Curve at 1.0X 10 for copy/. mu.L, 35Copy/. mu.L amplification curve, 4 is 1.0X 104Copy/. mu.L amplification Curve, 5 is 1.0X 103Amplification Curve at 1.0X 10 copies/. mu.L, 62Copy/. mu.L amplification Curve, 7 is 1.0X 101Copy/. mu.L amplification curve.
FIG. 4 is a diagram showing the result of a repetitive test of the SARS-CoV-2RT-RAA method.
The specific implementation mode is as follows:
the invention provides a novel recombinase amplification rapid detection kit for coronavirus, which aims to overcome the defect of detecting new coronavirus in the prior art, break through the limitation of the prior art on personnel and places, shorten the detection time, simultaneously improve the portability and the convenience of detection equipment, promote the forward movement and the downward movement of diagnosis, and realize the rapid diagnosis of suspected patients and the on-site rapid screening of close contact people.
The recombinase constant-temperature amplification technology is used as a novel nucleic acid in-vitro amplification technology for reaction under the condition of constant temperature, and has the advantages of easy transportation, high sensitivity, strong specificity, quick reaction time, simple operation and the like in the form of freeze-dried powder. The recombinase constant-temperature amplification technology fills the gap outside the traditional culture and temperature-dependent equipment technology, and can realize instant detection in field and non-laboratory environments. Compared with RPA, the RAA recombinase has relatively wide sources, is a novel isothermal nucleic acid amplification technology independently developed in China, and meets the requirement of rapid detection in China. The RAA technology can realize the high-efficiency and rapid amplification of trace nucleic acid in vitro in a constant or wide temperature range through a dynamic environment of automatic circulation for about 30min, and then an end point detection method is used for detecting an amplification product, so that an ideal detection result can be obtained.
In order to clearly illustrate the technical features of the present invention, the following further describes a specific embodiment of the present invention with reference to the examples. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby. Unless otherwise indicated, the technical means used in the examples are conventional technical means well known to those skilled in the art.
Example one
This example is a specificity and sensitivity test of the SARS-CoV-2RT-RAA method.
1. Experimental materials and instruments
1.1 Main instruments and consumables
The Tianlong NP968 nucleic acid extractor was purchased from Suzhou Tianlong Biotech limited; the portable fluorescence detector bio-cener H1600 Isothermal was purchased from Olympic instruments, Inc., Hangzhou;
96 real-time fluorescent quantitative PCR instrument purchased from Roche; pipettes were purchased from Eppendorf corp; pipette tips were purchased from Thermo Fisher Scientific; roche
480Multiwell Plate 96, white available from Roche corporation; refrigerators were purchased from Haier corporation; sterile 0.2mL, 0.5mL, 1mL, and 1.5mL centrifuge tubes from Guangzhou Jiete BiotechFilter stocks, inc; the biological safety cabinet is purchased from Likang biological medical science and technology control GmbH; the mini eight-tube centrifuge is purchased from Tosheng Innovation Biotech limited, Beijing; desktop refrigerated high speed centrifuges were purchased from Thermo Fisher Scientific.
1.2 Primary reagents
RT-RAA nucleic acid amplification reagent (fluorescent type) kit is purchased from Hangzhou mass inspection, and nucleic acid extraction kit (EX-RNA/DNA virus, T009) is purchased from Suzhou Tianlong biotechnology limited; TranscriptAId T7 High Yield transformation kit from Thermo Fisher; RNase Inhibitor was purchased from TAKARA; ddH2O was purchased from TAKARA; other reagents required were provided by the laboratory.
2. Sequence analysis and primer Probe design
Primers and probes specific to the recombinase were designed based on the SARS-CoV-2 gene sequence (NC-045512.2) registered in genebank, with reference to the RAA guide. The RAA nucleic acid amplification technology has certain difference between the design of a primer and the design of a conventional PCR primer, and two oligonucleotides form a pair of primers which respectively and specifically recognize the upstream and downstream nucleotide sequences of a nucleic acid target; the length is between 30 and 35 nucleotides (nt), and the sequence has no palindromic sequence, continuous single-base repeated sequence and internal secondary structure region; the Tm value of the primer is not considered as a main factor in designing; the optimal primer pair needs to be screened out through experimental optimization. The designed sequence of the probe is not overlapped with the recognition site of the specific primer, the length is 46-52nt, and the sequence avoids palindromic sequence, internal secondary structure and continuous repeated basic groups; the total number of the four modification sites is four, and the middle position which is more than or equal to 35nt away from the 5' end is marked with Tetrahydrofuran (THF) which is used as a recognition site of exonuclease; the upstream of the THF site is marked with a fluorescent group, the downstream is marked with a quenching group, and the distance between the two groups is 2-4 nt; THF is more than or equal to l5nt from the 3 'end, and a modifying group is marked at the 3' end.
And designing and synthesizing the RAA primer and the RAA-exo probe according to the design principle of the RAA primer probe, further performing sequence comparison analysis on the primer and the probe through BLAST, and comparing the sequence matching degrees of the designed probe and the primer with the sequences of H1N1, H5N1 and SRAS. The specificity of the primers and probes was determined, and all primers and probes were synthesized by Shanghai Bioengineering technology services, Inc. (Table 1). The plasmids of the target region sequences for SARS-CoV-2, SARS, H1N1 and H5N1 described below were constructed by Shanghai's biosynthesization.
TABLE 1 primer and Probe sequence information
3. Real-time fluorescent RAA reaction system and conditions
The S gene DNA construction plasmid of SARS-CoV-2 is used as template, and RAA reaction is carried out according to the kit instruction. The total reaction system was 50. mu.L, and first 32. mu.L of A Buffer, ddH was added to a reaction tube to which exo RT enzyme had been previously added2O9.5 mu L, upstream primer (F)1 mu L, downstream primer (R)1 mu L, probe (P)1 mu L and RNA template 3 mu L, B Buffer 2.5 mu L is added on the tube cover, after fully mixing, the reaction EP tube is put into a portable fluorescence detector, the reaction temperature and time are set, and the constant temperature amplification reaction is carried out at a specific temperature. The portable fluorescence detector measures and records the intensity of a fluorescence signal emitted by FAM every 20s, records the intensity as a fluorescence value, and judges that the sample is positive if the fluorescence value emitted by the sample is three standard deviations of the fluorescence value of the sample detected within a threshold time after the amplification is carried out for 19-20 min. The threshold time used in this experiment was 0-1min from the start of the amplification reaction.
According to the reaction conditions recommended in the kit instructions, the primers are used for optimizing the reaction conditions of the SARS-CoV-2RT-RAA detection method to be established in the test, and each reaction is repeated three times. First, the RT-RAA reaction was carried out for 20min at a reaction temperature of 40 ℃ and a probe concentration of 120 nmol/. mu.L and primer concentrations of 300 nmol/. mu.L, 360 nmol/. mu.L, 420 nmol/. mu.L, 480 nmol/. mu.L and 540 nmol/. mu.L, respectively, and the optimal primer concentration was selected. The results show that the fluorescence detection values of the primer concentrations of 300 nmol/. mu.L, 360 nmol/. mu.L, 420 nmol/. mu.L, 480 nmol/. mu.L and 540 nmol/. mu.L respectively reach three standard deviations of the fluorescence intensity at the threshold time, and the primer concentrations are shortest when used under the condition of 420 nmol/. mu.L concentration, and the detection efficiency is highest.
Then, the determined primer concentration of 420 nmol/. mu.L was used to perform RT-RAA reaction at 40 ℃ for 20min under the conditions of probe concentrations of 100 nmol/. mu.L, 120 nmol/. mu.L, and 140 nmol/. mu.L, respectively, and the optimal probe concentration was selected. The results showed that the fluorescence detection values at probe concentrations of 100 nmol/. mu.L, 120 nmol/. mu.L, and 140 nmol/. mu.L, respectively, all reached three standard deviations of the fluorescence intensity at the threshold time, with the shortest time of use at a concentration of 120 nmol/. mu.L.
Finally, RT-RAA reaction is carried out for 20min under the conditions of optimal primer concentration of 420 nmol/. mu.L, probe concentration of 120 nmol/. mu.L and temperature of 37, 38, 39, 40, 41 and 42 ℃ respectively, and the optimal reaction temperature is screened. The results show that the fluorescence intensity of the reaction at 37, 38, 39, 40, 41 and 42 ℃ can reach three standard deviations of the fluorescence intensity at the threshold time with the increase of the temperature, but in contrast, the detection efficiency is the highest and the time is the shortest at 42 ℃, see fig. 1.
Therefore, the optimal reaction conditions of the SARS-CoV-2Real-time RT-RAA detection method established by the test are that the concentration of the primer is 420 nmol/muL, the concentration of the probe is 120 nmol/muL, the reaction temperature is 42 ℃, and the reaction time is 20 min.
4. Specificity test
In order to determine the specificity of the method, an optimal reaction system of 3 screens is applied, SARS-CoV-2, H1N1, H5N1 and cDNA of SARS are selected to react, the concentration of the cDNA is 10 ng/mu L, the dosage is 2 mu L, the optimal reaction condition is utilized to carry out real-time fluorescence RAA detection, the specificity of the method is evaluated, and the test result is shown in figure 2.
As can be seen from FIG. 2, with the lapse of reaction time, after 10min of reaction, the fluorescence intensity of SARS-CoV-2 positive cDNA is obviously increased, compared with that of other virus positive cDNA, the fluorescence intensity measured has no obvious increase change, which indicates that the method can specifically amplify new corona nucleic acid and can be used for detecting new corona.
5. Sensitivity test
In order to determine the minimum detection amount of the real-time fluorescence isothermal amplification method, the optimal reaction system screened in 3 is applied, S gene DNA construction plasmid of SARS-CoV-2 virus is used as a template, and the template is dividedRespectively diluted to 7 concentrations according to 10 times of the ratio, namely 10-107Copying/mu L of seven templates with concentration gradients, detecting by using the optimal reaction conditions, taking the real-time fluorescence intensity of 1000mV as a positive detection limit, and carrying out sensitivity comparison with a fluorescence quantitative PCR method, wherein the test result is shown in figure 3.
The results show that the RAA method can detect a concentration of 1X 10 within 6min7The Ct value of the corresponding fluorescent quantitative PCR of the copied/mu L sample is 17.47 +/-0.03, the sample with the lowest concentration of 1 multiplied by 10 copies/mu L can be detected within 20min, the Ct value of the corresponding fluorescent quantitative PCR is 34.41 +/-0.46, the real-time fluorescent RAA sensitivity is equivalent to that of the fluorescent quantitative PCR method, but the time required by detection is shortened to 20min from 2h of the fluorescent quantitative PCR, and is far faster than that of the fluorescent quantitative PCR method.
6. Repeatability test
To verify the repeatability of the fluorescent quantitative PCR detection result, 10 was selected7The diluted plasmid samples were amplified 3 times in a 50. mu.L system and the results are shown in FIG. 4. The result shows that the experimental amplification curve has good repeatability and good stability.
Example two
This example is the testing of clinical mock samples.
60 throat swab or nose swab samples are simultaneously subjected to recombinase rapid detection, conventional PCR and real-time fluorescent quantitative PCR detection, the detection effects of the three detection methods are compared, and 60 samples are from different areas of Shandong. As can be seen from Table 2, the positive rate of the recombinase rapid detection is 91.67%, which is consistent with the gold standard fluorescent quantitative PCR detection rate of the new crown nucleic acid detection, while the positive rate of the conventional PCR detection is 80%. The result shows that the method for rapidly detecting the recombinase has reliable detection result, shortest time and simplest operation.
Results of recombinase rapid detection, conventional PCR and real-time fluorescent quantitative PCR detection of 260 clinical samples in table
EXAMPLE III
This example is the preparation of a test kit.
The preparation and operation process of the kit is based on the RAA detection technology. The kit contains specific amplification primers and probes, wherein the primer sequences are SEQ ID No. 1 and SEQ ID No. 2, and the probe sequence is SEQ ID No. 3. The final concentrations of the primers and the probes are respectively 420 nmol/. mu.L and 120 nmol/. mu.L, and the kit also comprises corresponding RAA-based fluorescence universal reaction reagent and reaction buffer solution, such as 20% PEG, 280mM MgAc, recombinase, polymerase and lyophilized enzyme preparation of single-stranded DNA binding protein, ddH2O, etc., which are well known to those skilled in the art. In addition, there may be a positive control and a negative control, the positive control should be able to completely amplify the targeted gene segment corresponding to the primer during the amplification process, for example, the positive control may be a plasmid solution containing the targeted gene segment (S gene segment) corresponding to the viral primer probe; negative control is ddH2And O. The value of this kit lies in that the sample includes pharynx swab, nose swab, nasopharynx extract or respiratory tract extract, the sputum that deeply coughs, and do not need other tissue samples of human body, through retrenching with specific primer and probe detection, adopt recombinase amplification technique to carry out quick accurate amplification and discernment to novel coronavirus specificity nucleic acid, and is not only stable, and the detection is convenient, and is accurate, and the sensitivity and the specificity of viral diagnosis are greatly improved, consequently put this kit into practice, can simplify the operating procedure, shorten check-out time, reduce detectioner's operating time, reduce the infection risk, and the detection efficiency is improved, and is suitable for large-scale screening. The relevant primer and probe information is shown in the table 1 part of the specification.
Sequence listing
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Claims (7)
1. A primer and probe combination for detecting novel coronavirus by real-time fluorescent RT-RAA method is characterized in that: the primer and probe combination comprises a specific primer pair and a probe aiming at an S gene segment of the novel coronavirus, wherein the sequence of an upstream primer is SEQ ID NO. 1, the sequence of a downstream primer is SEQ ID NO. 2, and the sequence of the probe is SEQ ID NO. 3.
2. Use of the primer and probe combination of claim 1 for the preparation of a kit or a reagent for the detection of a novel coronavirus.
3. A kit comprising the primer and probe combination of claim 1.
4. The kit of claim 3, wherein: the kit is a novel real-time fluorescent RT-RAA detection kit for coronavirus.
5. The kit of claim 4, wherein: the final concentrations of the primers and the probe were respectively 420 nmol/. mu.L and 120 nmol/. mu.L, respectively.
6. The kit of claim 5, wherein: the kit also comprises A Buffer, B Buffer, a recombinase, a polymerase and a freeze-dried enzyme preparation of single-stranded DNA binding protein, a positive control and a negative control.
7. The kit of claim 6, wherein: the A Buffer is 20% PEG, and the B Buffer is 280mM MgAc.
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