• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
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    • 专利摘要:
    The present disclosure provides an RT—LAMP primer set and kit for detecting novel coronavirus SARS-CoV—Z. The primer set for detecting SARS-CoV—2 includes 6 primers including outer primers F3 and B3, inner primers FIP and BIP, and loop primers LF and LB, the sequences of which are set forth in SEQ ID N01 to SEQ ID NO:6. The primer set mentioned above can accurately detect the novel coronavirus SARS-CoV—Z, without cross-reacting with other pneumonia-causing viruses. The primer set, RT—LAMP reaction solution, positive control, negative control, and DEPC water constitute the RT-LAMP kit for detecting SARS-CoV—Z. SARS-CoV—2 may be detected by isothermal amplification at 63°C in 20 minutes at the earliest, with the detection sensitivity reaching 80 copies/HL. The disclosure has the advantages of simplicity, rapidity, sensitivity, and specificity, which can directly determine the result by observing the colour change of the reaction tube with naked eyes without using expensive instruments, can realize rapid and accurate detection of SARS-CoV—Z. It is suitable for on-site testing and clinical testing in most domestic primary hospitals and other institutions that lack real-time fluorescent PCR machines. It is of great significance for the diagnosis, monitoring, prevention and control of the novel coronavirus SARS-CoV—Z. 1
    公开号:NL2027315A
    申请号:NL2027315
    申请日:2021-01-14
    公开日:2021-10-19
    发明作者:Yan Shigan；Zhu Liping
    申请人:Univ Qilu Technology；
    IPC主号:
    专利说明:

    [01] [01] This application claims the priority of Chinese Patent Application NO.202010150527.8, entitled “RT-LAMP primer set for detecting novel coronavirus SARS-CoV-2 and kit thereot™ filed with China National Intellectual Property Administration on March 06, 2020, which is incorporated herein by reference in its entirety.TECHNICAL FIELD
    [02] [02] The disclosure belongs to the technical field of viral nucleic acid detection, and relates to an RT-LAMP primer set, a kit and a detection method for detecting novel coronavirus SARS-CoV-2 by rapid one-step color development method.BACKGROUND ART
    [03] [03] The pathogen of novel coronavirus pneumonia is the novel coronavirus SARS-CoV-2. Genome sequence alignment analysis shows that SARS-CoV-2 and two other coronaviruses (i.e. severe acute respiratory syndrome coronavirus (SARS-Cov), Middle East respiratory syndrome coronavirus (MERS-Cov)) that can cause severe pneumonia belong to the B virus genus of the coronavirus family, but the homology of SARS-CoV-2 with SARS-Cov and MERS-Cov is only 79.5% and 40% respectively. The above three coronaviruses may cause severe pneumonia in humans. In addition, there are endemic human coronaviruses that cause mild influenza in human beings, the representative strains are HKU1, OC43, NL63 and 229E.
    [04] [04] Human viral pneumonia accounts for 70-80% of respiratory tract infections. In addition to the three coronaviruses of SARS-CoV-2, MERS-Cov and SARS-Cov, the viruses that can cause severe pneumonia also include highly pathogenic avian influenza viruses (H7N9, H5NI1, etc), seasonal influenza viruses (such as type A HIN], type B Victoria, etc.), respiratory syncytial virus, rhinovirus, adenovirus, parainfluenza virus, enterovirus, herpes simplex virus, human metapneumovirus, reovirus, measles virus, cytomegalovirus, etc,
    [05] [05] Only by achieving differential diagnosis of SARS-CoV-2 and other important pneumonia viruses can the early diagnosis, isolation, treatment, and control of novel coronavirus pneumonia be achieved, and the novel coronavirus pneumonia epidemic be defeated as soon as possible.
    [06] [06] The diagnosis of novel coronavirus pneumonia is based on epidemiological investigation, clinical symptoms (fever, cough, dyspnea, etc.) and CT examination results to identify a suspected case. The final diagnosis is based on the viral nucleic acid detection.
    [07] [07] At present, the commonly used methods for detecting SARS-CoV-2 include virus isolation and culture, electron microscopy observation method, immunological detection method, and molecular biology detection method, etc. Viral isolation is the "gold standard” for viral detection, but due to limitation of conditions, it is impossible to isolate and culture viruses from each patient's specimen. Electron microscopy observation method requires the acquisition of viruses, which takes a long time, most institutions have no such conditions, and most viruses have no indicative form. Immunoassay has low specificity and sensitivity for those viruses that are prone to antigen variation. However, the molecular biological detection method has the advantages in specificity, sensitivity and accuracy. The molecular biological methods commonly used to identity viruses include PCR, multiplex PCR, fluorescence PCR, genome sequencing and the like. PCR or multiplex PCR needs to combine electrophoresis and imaging observation to judge the results, which takes a long time. Fluorescent PCR, genome sequencing and other methods need expensive professional instruments, which have high requirements for operators and operation technology, which are not conducive to clinical application. Therefore, there is an urgent need to develop more efficient, simple and practical molecular biology detection technology.
    [08] [08] Loop-mediated isothermal amplification(LAMP) is a new nucleic acid amplification method, which requires 4 specific primers and 2 loop primers to specifically identify 6 different regions of the target gene, and relies on strand displacement of Bst DNA polymerase with high activity and large fragments by automatic circulation to produce loop-mediated isothermal amplification, thus forming a large number of amplified products with multiple subtype structures. Reverse transcription LAMP (RT-LAMP) is a combination of reverse transcription and LAMP technology, which can realize the amplification and detection of RNA. The loop-mediated isothermal amplification has higher amplification efficiency (DNA can be amplified by 10°-10' times in 15-60min), less time (15min as the fastest), and higher sensitivity(up to several copies) than fluorescent PCR amplification, which has been used for nucleic acid detection of pathogenic microorganisms. At present, there is no report of RT-LAMP detection kit and RT-LAMP method used for detecting novel coronavirus SARS-CoV-2 at home and abroad.
    [09] [09] Compared with the existing nucleic acid detection methods of novel coronavirus:
    [10] [10] Currently, real-time fluorescent RT-PCR detection is the recommended standard for the diagnosis of novel coronavirus pneumonia. WHO and CDC in China have designed specific fluorescent RT-PCR primers and probes for specific regions of ORF lab, N, and E genes, and have established a fluorescent RT-PCR method for detecting SARS-CoV-2. The detection time of the fluorescent RT-PCR method is about 2 hours, with strong specificity and high sensitivity. However, the fluorescent RT-PCR method requires an expensive fluorescent PCR instrument, which requires high technical requirements for operators and is not conducive to clinical application, resulting in low detection efficiency, a large number of suspected cases can not be diagnosed in time, delaying isolation and treatment, and affecting epidemic control. In contrast, the RT-LAMP amplification method does not need expensive instruments, ordinary water bath or metal bath will do. The result can be directly determined by observing the turbidity or colour of the product by naked eyes. The RT-LAMP has higher detection sensitivity, shorter detection time, and low technical requirements for operators. It has the advantages of intuitive results, rapidity, simplicity, specificity and sensitivity, and can be used for rapid and accurate identification of SARS-CoV-2.SUMMARY OF THE INVENTION
    [11] [11] In order to solve the problems mentioned in the above background art such as long time-consumption, expensive specialized instruments needing, high technical requirements for operators, and unfavorable clinical applications in the fluorescent RT-PCR detection of the novel coronavirus SARS-CoV-2, the present disclosure provides an RT-LAMP detection primer set, kit and detection method for loop-mediated isothermal amplification of
    [12] [12] The present disclosure is realized in the following solutions:
    [13] [13] The RT-LAMP primer set and kit for detecting the novel coronavirus SARS-CoV-2, wherein the primer set includes 6 primers, and the sequences of the primers are set forth in as follows:
    [14] [14] primer F3: 5'-TGGTATATTAGAGTAGGAGCTAGA-3' (SEQ ID NO: 1),
    [15] [15] primer B3: 5-AGTCTTCATAGAACGAACAAC-3' (SEQ ID NO: 2),
    [16] [16] primer FIP: S'-TCGATGTACTGAATGGGTGATTTAGTCAGCACCTTTAATTGAATTGTG-3' (SEQ ID NO: 3),
    [17] [17] primer BIP: 5-TACAGTTTCCTGTTCACCTTTTACAGCACTACAAGACTACCCAATT-3' (SEQ ID NO: 4),
    [18] [18] primer LF: 5'-ATTAATTGCCAGGAACCTA-3' (SEQ ID NO: 5),
    [19] [19] primer LB: 5'-AACCAGCCTCATCCACG-3' (SEQ ID NO: 6).
    [20] [20] In some embodiments, the molar ratio of outer primers (F3, B3): inner primers (FIP, BIP): loop primers (LF, LB) is 1:8:4, and each 25u4L of RT-LAMP reaction solution contains Spmol of primer F3, 5pmol of primer B3, 40pmol of primer FIP, 40pmol of primer BIP, 20pmol of primer LF and 20pmol of primer LB.
    [21] [21] The RT-LAMP kit for detecting novel coronavirus SARS-CoV-2 comprises 2xRT-LAMP reaction solution, positive control, negative control, DEPC water and the above primer set.
    [22] [22] In some embodiments, the 2xRT-LAMP reaction solution contains 40mM Tris-HCI (pH 8.8), 20mM KCl, 16mM MgSO4, 20mM (NH4)2S04, 0.2% tween 20, 1.6M betaine, 28mM dNTPs, 1.28U/uL Bst DNA polymerase, 16U/uL AMV reverse transcriptase,
    [23] [23] In some embodiments, the positive control is obtained by connecting the full-length nucleotide sequence of ORF8 gene of novel coronavirus SARS-CoV-2 to the positive recombinant plasmid screened and identified by pUCS7 vector, transcribing in vitro to obtain RNA fragment, and finally purifying by HPLC. The copy number of lug plasmid is
    [24] [24] The detection steps of RT-LAMP primer set and kit for detecting novel coronavirus SARS-CoV-2 are as follows: adding 7uL of primer, 12.5uL of 2xRT-LAMP reaction solution, luL of positive template or 1-3uL of sample RNA to be tested to the reaction tube in the ice 5 box, adding DEPC water to make up the volume to 25uL, closing the lid of the reaction tube, mixing the reaction solution evenly, centrifuging instantaneously to make the reaction solution located at the bottom of the tube, placing the reaction tube in a water bath, a metal bath, a thermostat or a PCR machine, and isothermal amplifying at a fixed temperature in the range of 58-65'C for 40-60 minutes, preferably 63°C for 5Ominutes, then reacting at 85°C for 5 minutes to terminate the reaction, and observing the colour of the reaction solution under natural light or ultraviolet lamp. If the colour of the reaction solution changed to green, it is determined that the sample to be tested contains the novel coronavirus SARS-CoV-2; if the colour of the reaction solution remains the original brown, it is determined that the sample to be tested does not contain the novel coronavirus SARS-CoV-2.
    [25] [25] The RT-LAMP primer set and kit for detecting novel coronavirus SARS-CoV-2, wherein the primer set and kit for use in detecting the presence of the novel coronavirus SARS-CoV-2 in clinical samples. By using the RT-LAMP primer set, kit and detection method provided by the disclosure, whether the samples contain novel coronavirus can be directly judged visually without relying on special instruments.
    [26] [26] The beneficial effects of the present disclosure are:
    [27] [27] (1) The established RT-LAMP detection method for rapid one-step color development detection of SARS-CoV-2 has the advantages of simple operation, good practicability and low cost. There is no need for expensive specialized instruments, with simply use of a common water bath, and the experiment results can be intuitively judged by observing the colour change by the naked eyes. The operation is simple and fast, and the technical requirements for operators are low. The kit of the present disclosure can be applied to on-site testing, which is convenient for the detection of SARS-CoV-2 and epidemic prevention and control in primary hospitals, CDC's and other institutions.
    [28] [28] (2) The RT-LAMP detection method for novel coronavirus SARS-CoV-2 of the present disclosure is fast and efficient. Due to the use of isothermal amplification, there 1s no need for the temperature change reaction in PCR amplification. At the same time, the highly active strand displacement Bst DNA polymerase is used, which shortens the detection time and simplifies the operation process.
    [29] [29] (3) For the RT-LAMP detection method and kit for detecting SARS-CoV-2 of the present disclosure, the reaction solution is added with calcein dye and manganese ions in advance, so that the result can be judged directly according to the color change of the reaction tube, thus avoiding the needs for second addition of dyes, since the observation of results after the reaction will cause the released products to form aerosols to pollute the environment. Because the RNase inhibitor is added to the reaction solution, the concentration of the dye and manganese ions are optimized, and the inhibition of RT-LAMP reaction caused by adding dye beforehand is avoided, and the rapid one-step color development RT-LAMP detection of SARS-CoV-2 is realized.
    [30] [30] (4) The RT-LAMP detection method for detecting SARS-CoV-2 of the present disclosure has strong specificity, and the design of primer set is the key technology. The RT-LAMP primer set is designed by using orf8 gene sequence in novel coronavirus SARS-CoV-2, which includes six specific primers, namely outer primers F3 and B3, inner primers FIP and BIP, and loop primers LF and LB. Primer set can only specifically amplify the nucleic acid of novel coronavirus SARS-CoV-2, but can not amplify other coronaviruses, nor can amplify the nucleic acids of influenza virus, respiratory syncytial virus, rhinovirus, adenovirus, parainfluenza virus, enterovirus, herpes simplex virus, human metapneumovirus, reovirus, measles virus, cytomegalovirus and other viruses causing pneumonia.
    [31] [31] (5) The RT-LAMP detection method and kit for detecting SARS-CoV-2 of the present disclosure have high amplification efficiency, which is related to the addition of loop primers and RNAase inhibitors. Adding a pair of loop primers and RNase inhibitors can significantly improve amplification efficiency, so that the direct addition of fluorescent dyes in the reaction tube can directly develop colours, thus solving the problem that directly adding dyes to the reaction tube will inhibit the amplification efficiency of Bst DNA polymerase, and avoiding the reduction of sensitivity and specificity.BRIEF DESCRIPTION OF THE DRAWINGS
    [32] [32] FIG 1 shows the judgement of the RT-LAMP detection results of SARS-CoV-2 (the left tube shows green, which 1s positive; the right tube remains the original brown, which is negative).
    [33] [33] FIG 2 shows the LAMP sensitivity test results of SARS-CoV-2 (The nucleic acid concentrations of positive templates corresponding to tubes 1#-9# are 8x103, 8x107, 8x106, 8<10%, 8x10% 8x10°, 8x10% 8x10! 0.8 copies/uL in sequence, tubes 10# and 11# are negative control and blank control respectively. 1#-8# tubes show positive results, and 9#-11# tubes show negative results. FIG. 2A shows the visual results under natural light, FIG 2B shows the visual results under ultraviolet lamp).DETAILED DESCRIPTION OF THE EMBODIMENTS
    [34] [34] The technical scheme of the disclosure will be described clearly and completely in combination with the embodiments, but the protection scope of the present disclosure is not limited thereto.
    [35] [35] (1) Design and synthesis of RT-LAMP primers and preparation of primer set solutions
    [36] [36] Log on to 2019 Novel Coronavirus Resource (2019nCoVR) in China National Center for Bioinformation (https://bigd.big.ac.cn/ncov). The genome sequences of novel coronaviruses, other coronaviruses and other pneumonia-causing viruses (influenza virus, respiratory syncytial virus, rhinovirus, adenovirus, parainfluenza virus, enterovirus, herpes simplex virus, human metapneumovirus, reovirus, measles virus, cytomegalovirus, etc.) were retrieved and downloaded, the genome sequences were comparatively analyzed by using CLUSTAL, DNASTAR, BIOEDIT and other bioinformatics software. The analysis results showed that there was no significant variation in the genome sequence among the isolates of the novel coronavirus SARS-CoV-2, which was recently epidemic. Therefore, the genome sequence NC 045512 of BetaCoV/Wuhan/WH-01/2019, which was a representative strain of the novel coronavirus SARS-CoV-2, was selected as template to design primers. Then the genome sequences of SARS-CoV-2 and coronaviruses such as SARS-Cov, MERS-Cov,
    [37] [37] primer F3: 5'-TGGTATATTAGAGTAGGAGCTAGA-3' (SEQ ID NO: 1),
    [38] [38] primer B3: S'-AGTCTTCATAGAACGAACAAC-3' (SEQ ID NO: 2),
    [39] [39] primer FIP: 5'-TCGATGTACTGAATGGGTGATTTAGTCAGCACCTTTAATTGAATTGTG-3' (SEQ ID NO: 3),
    [40] [40] primer BIP: 5-TACAGTTTCCTGTTCACCTTTTACAGCACTACAAGACTACCCAATT-3' (SEQ ID NO: 4),
    [41] [41] primer LF: 5'-ATTAATTGCCAGGAACCTA-3' (SEQ ID NO: 5),
    [42] [42] primer LB: 5'-AACCAGCCTCATCCACG-3' (SEQ ID NO: 6).
    [43] [43] The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and purified by HPLC.
    [44] [44] Each primer was prepared into an aqueous solution of a certain molar concentration with DEPC water, wherein the primers F3 and B3 were prepared into an aqueous solution with a molar concentration of 10uM respectively, and the primers FIP, BIP, LF, and LB were prepared into an aqueous solution with a molar concentration of 20uM. The primer aqueous solution was measured according to the volume ratio of 1 (F3): 1 (B3): 4 (FIP): 4 (BIP): 2 (LF): 2 (LB), the mixture was mixed evenly to prepare a primer set solution.
    [45] [45] During detection, 7uL of primer set solution was added to each 25uL reaction system, so that the moles of each primer in each reaction system were: primer F3, Spmol; primer B3, 5pmol; primer FIP, 40pmol; primer BIP, 40pmol; primer LF, 20pmol; primer LB, 20pmol.
    [46] [46] (2) Preparation of 2xRT-LAMP Reaction Solution
    [47] [47] The components and concentrations thereof in the 2xRT-LAMP reaction solution were: 40mM Tris-HCI (pH 8.8), 20mM KCl, 16mM MgSO,, 20mM (NH4)2804, 0.2% Tween 20,
    [48] [48] During detection, 12.5uL of 2:=RT-LAMP reaction solution was added to each reaction system (25uL), so that each component and the final concentration thereof were 20mM Tris-HCI (pH 8.8), 10mM KCI, 8mM MgSO, 10mM (NH4)2S04, 0.1% Tween 20, 0.8M betaine, 1.4mM dNTPs, 0.64U/uL Bst DNA polymerase, 8U/uL AMV reverse transcriptase,
    [49] [49] (3) Assembly of the kit
    [50] [50] The detection kit was consisted of 0.5mL of primer set aqueous solution (step (1)), ImL of 2xRT-LAMP reaction solution (step (2)), 0.1mL of positive control, 0.5mL of negative control and 1mL of DEPC water.
    [51] [51] Positive control of SARS-CoV-2: full-length DNA of synthetic orf8 gene was prepared by conventional methods in the field, the full-length DNA was inserted into pUCS7 vector by ligation and transformation, the RNA fragments were obtained by in vitro transcription, and the fragments were purified by HPLC to obtain positive control. The positive control was identified by gene sequencing after purification.
    [52] [52] 12.5uL of 2xLAMP reaction solution, 7uL of primer set aqueous solution, luL of positive template were added to each RT-LAMP reaction system (25uL), and sterilized
    [53] [53] The reaction temperatures of RT-LAMP were set to 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, and the reaction time was 50 minutes, then the reaction was terminated after reacting at 85°C for Sminutes, and the detection results was intuitively determined by visual inspection of the colour under natural light or visual inspection of the fluorescence under ultraviolet lamp, and the effects of different reaction temperatures on RT-LAMP detection were compared.
    [54] [54] In the present example, a water bath was used as a heating device for RT-LAMP reaction. In other examples, a PCR machine or a thermostatic metal bath can also be practically used as a heating device.
    [55] [55] If the reaction tube showed bright green visible fluorescence, the test result is determined to be positive; if the reaction tube still remained the original orange-brown, the test result is determined to be negative. See attached FIG. 1 for details.
    [56] [56] The RT-LAMP detection kit of the present disclosure was used to detect the positive control of SARS-CoV-2, bright green fluorescence could be detected in all the solution at 59-65°C for 50 minutes, but the fluorescence brightness of the products amplified at 63-65°C was slightly stronger than that of the products amplified at 59-62°C, so the subsequent detection temperature was fixed at 63°C.
    [57] [57] The positive template was diluted 10-folds with DEPC water. The concentrations of positive templates in the 1# to 9# tubes were 8x105, 8x107, 8x109, 8x105, 8x10%, 8-10’, 8x10% 8x101, 0.8 copies/uL, respectively. The 10# and 11# tubes were blank control and negative control respectively.
    [58] [58] The RT-LAMP reaction system was the same as the reaction system in Example 2.
    [59] [59] In the present example, a water bath was used as a heating device for RT-LAMP reaction. In other examples, a PCR machine or a thermostatic metal bath can also be practically used as a heating device.
    [60] [60] The reaction temperature of RT-LAMP was set to 63°C, and the colour of the liquid in each reaction tube was observed every 10minutes, the relationship between different nucleic acid concentrations and the occurrence time when the detection results appeared were compared, so as to find out the reaction time when RT-LAMP detects the lowest nucleic acid content, avoiding the occurrence of false negative results.
    [61] [61] If the reaction tube visually showed bright green visible fluorescence under natural light or ultraviolet lamp, it was determined that the detection result was positive; if the reaction tube remained the original orange-brown, it was determined that the detection result was negative.
    [62] [62] Table 1 Correspondence between the nucleic acid concentration of SARS-CoV-2 and the occurrence time of RT-LAMP detection results.
    [63] [63] The positive templates of different concentration gradients of SARS-CoV-2 were detected by using the RT-LAMP detection kit and method of the present disclosure. After isothermal amplification at 63’C, the reaction solutions of the 1# to 8# tubes were all detected bright green fluorescence, while the 9# to 11# tubes were not detected green fluorescence. It shows that the detection sensitivity of the RT-LAMP detection kit and detection method of the present disclosure for SARS-CoV-2 is the concentration gradient of the 8# tube, i.e. 80 copies/uL. Although all the 1# to 8# tubes could display bright green fluorescence, the time that the positive result can be detected is different. The corresponding detection time of each tube is 20, 20, 20, 25, 25, 30, 35, 40 minutes (see Table 1 for details). The detection results show that the RT-LAMP detection kit and detection method of the present disclosure can detect the results in 20 minutes at the earliest, and the minimum detection limit can be detected in 40 minutes at the latest. In order to ensure that trace amounts of SARS-CoV-2 nucleic acid can be detected, the detection time should not be less than 40 minutes. EXAMPLE 4: Specificity verification of RT-LAMP for detecting novel coronavirus SARS-CoV-2
    [64] [64] The viral nucleic acids used in the experiment included severe acute respiratory syndrome coronavirus (SARS-Cov), Middle East respiratory syndrome coronavirus (MERS-Cov), endemic human coronavirus (HKUI, OC43, NL63 and 229E), high pathogenic avian influenza virus (H7N9, H5N1), influenza virus (A HINI, B Victoria), respiratory syncytial virus, rhinovirus, and parainfluenza virus. The nucleic acids of the coronavirus SARS-Cov and MERS-Cov used in the experiment were the artificially synthesized full-length nucleotide sequences of orf8 gene connected to the positive recombinant plasmid screened and identified by the pUC57 vector. The nucleic acids of the influenza viruses H7N9 and H5N1 used in the experiment were the artificially synthesized full-length nucleotide sequences of HA gene connected to the positive recombinant plasmid screened and identified by the pUCS57 vector. Other viral nucleic acids were extracted from inactivated virus liquid.
    [65] [65] The RT-LAMP reaction system was the same as the reaction system in Example 2. Positive control group and blank control group were set in the experiment. The reaction temperature was set to 63°C, the reaction time was set to 50 minutes, and then the reaction was terminated after reacting at 85 C for 5 minutes. The results show that only the novel coronavirus RT-LAMP experiment is positive, while the experiment results of other types of viral RNA samples are all negative, indicating that the RT-LAMP detection method and kit of the present disclosure have good detection specificity and do not interfered by other viruses.
    [66] [66] The clinical validation trial was conducted in Jinan Novel Coronavirus Pneumonia designated diagnosis and treatment hospital. Clinical sample was divided into two parts, and each parts was detected with the kit of the present disclosure and the fluorescent RT-PCR kit respectively, the coincidence rate and the sensitivity of the two detection results were compared.
    [67] [67] The sample types included throat swabs, sputum, alveolar lavage fluid, etc., with a total of 182 samples, including 130 throat swabs, 34 sputa, and 18 alveolar lavage fluids. The samples were inactivated in a dry bath at 56°C for 30 minutes before nucleic acid extraction and detection. Viral nucleic acid was extracted from 1404uL of sample by one-step lysis method, and viral RNA was extracted by using QIAGEN extraction kit or directly detected after treatment with nucleic acid lysate. If the sample was viscous, it could be mixed with an equal volume of 1% pancreatin before nucleic acid extraction, and incubated at 37°C with shaking liquefaction for 15min.
    [68] [68] The RT-LAMP reaction system referred to Example 2, the reaction temperature was set to 63°C, the reaction time was set to 50 minutes, and then the reaction was terminated after reacting at 85°C for 5 minutes. The fluorescent RT-PCR method was performed according to the methods recommended in the Technical Guidelines for Laboratory Testing of Pneumonia Infected by Novel Coronavirus. The recommended novel coronavirus (2019 ORF lab/N gene) dual channel fluorescence RT-PCR nucleic acid detection kit (batch number: 2020002) was used for viral nucleic acid detection. The system was prepared according to the instructions of the kit, and the reaction procedure was as follows: 45°C for 10min, 95°C for 10min; 95°C for 15 s, 55°C for Imin, 40 cycles. The amplified samples were placed in ABI7500 fluorescence quantitative PCR instrument for detection, single-point fluorescence detection at 55°C, FAM/none was selected for fluorescence detection channel, ROX was selected for passive reference. Judgment criteria: positive (Ct value<37, typical amplification curve), gray area (37>Ct value<40), negative (Ct value>40 or none, no amplification curve).
    [69] [69] The detection results of 182 samples show that the detection rates of the kit of the present disclosure and the fluorescent RT-PCR kit are 35.2% (64/182) and 34.6% (63/182), respectively. The consistency rate of the detection results of the two kits is 99.45%. The detection sensitivities of the present disclosure and the fluorescent RT-PCR method are 2 copies/uL and 5 copies/uL, respectively. Compared with the fluorescence RT-PCR kit, the present disclosure has obvious advantages in terms of the independence of the detection on the instrument, the technical requirements for the operation, simplicity of operation and the economic cost. The kit of the present disclosure is suitable for primary detection, diagnosis and treatment institutions and field detection applications.
    [70] [70] The experimental results of the above examples show that the novel coronavirus SARS-CoV-2 nucleic acid RT-LAMP detection kit provided by the present disclosure has strong specificity, high sensitivity and convenient operation, which can realize rapid detection of the novel coronavirus.
    SEQLTXT-1
    SEQUENCE LISTING <110> Qilu University of Technology <120> RT-LAMP primer set for detecting novel coronavirus SARS-CoV-2 and kit thereof <141> 2020-03-06 <160> 6 <170> SIPOSequenceListing 1.0 <2105 1 <211> 24 <212> DNA <213> artificial sequence <400> 1 tggtatatta gagtaggagc taga 24 <210> 2 <211> 21 <212> DNA <213> artificial sequence <400> 2 agtcttcata gaacgaacaa c 21 <2105 3 <211> 48 <212> DNA <213> artificial sequence <400> 3 tcgatgtact gaatgggtga tttagtcagc acctttaatt gaattgtg 48 <210> 4 <211> 46 <212> DNA <213> artificial sequence <400> 4 tacagtttcc tgttcacctt ttacagcact acaagactac ccaatt 46 <210> 5 <211> 19 <212> DNA <213> artificial sequence <400> 5 attaattgcc aggaaccta 19 <210> 6 <2115 17 <212> DNA Pagina 1
    SEQLTXT-1 <213> artificial sequence <400> 6 aaccagcctc atccacg 17
    Pagina 2
    权利要求:
    Claims (7)
    [1]
    An RT-LAMP primer set for detecting novel coronavirus SARS-CoV-2, the primer set comprising 6 primers, the sequences of which are as follows: primer F3: 5'-TGGTATATTAGAGTAGGAGCTAGA-3' (SEQ ID NO: 1 ), primer B3: 5'-AGTCTTCATAGAACGAACAAC-3' (SEQ ID NO: 2), primer FIP: 5'-TCGATGTACTGAATGGGTGATTTAGTCAGCACCTTTAATTGAATTGTG-3' (SEQ ID NO: 3), primer BIP: 5'-TACAGTTTCCTGTTCACCTTTTACAGCACTAATTAAGACT (SECCQATATTAAGACT IDNO:4, primer LF: 5'-ATTAATTGCCAGGAACCTA-3' (SEQ ID NO:5), primer LB: 5'-AACCAGCCTCATCCACG-3' (SEQ ID NO:8).
    [2]
    The primer set of claim 1, wherein the molar ratio of outer primers (F3, B3): inner primers (FIP, BIP): loop primers (LF, LB) is 1:8:4, each 25 µl of the RT-LAMP reaction solution contains 5 pmol of primer F3, 5 pmol of primer B3, 40 pmol of primer FIP, 40 pmol of primer BIP, 20 pmol of primer LF and 20 pmol of primer LB.
    [3]
    A kit, wherein the kit comprises the primer set of claim 1 or 2 and 2 x RT-LAMP reaction solution, positive control, negative control, and DEPC water.
    [4]
    The kit of claim 3, wherein the 2 x RT-LAMP reaction solution contains 40 mM Tris-HCl (pH 8.8), 20 mM KCl, 16 mM MgSO 4 , 20 mM (NH:)250.4, 0, 2% Tween 20, 1.6 M betaine, 2.8 mM dNTPs, 1.28 U/uL Bst DNA polymerase, 16 U/uL AMV reverse transcriptase, 1.6 U/uL RNAase inhibitor, 0.1 mM calcein, 1.2 mM MnCl.
    [5]
    The kit of claim 3, wherein the positive control is obtained by linking the full length DNA of the ORF8 gene of SARS-CoV-2 to the positive recombinant plasmid screened and identified by the pUC57 vector, the transcribing in vitro to obtain an RNA fragment, and finally purifying by HPLC; the copy number of 1 µg of plasmid 3.157 x 10°! is.
    [6]
    A detection method using the kit of claim 3, wherein the detection steps are as follows: adding 7 µl primer set, 12.5 µl 2x RT-LAMP reaction solution, 1 HI positive template or 1-3 µl sample RNA to be tested to the reaction tube in the refrigerator, add DEPC water to bring the volume to 25 µL, close the lid of the reaction tube, evenly mix the reaction solution, immediately spin to remove the reaction solution at the bottom of the tube, placing the reaction tube in a water bath, metal bath, thermostat or PCR machine and isothermal multiplication at a fixed temperature in the range of 58-65°C for 40-60 minutes, preferably 63°C for 50 minutes, and then reacting at 85°C for 5 minutes to finish the reaction, and observing the color of the reaction solution under natural light or an ultraviolet lamp; like the color
    -16 - the reaction solution turns green, the sample to be tested is determined to contain the novel coronavirus SARS-CoV-2; if the color of the reaction solution remains the original brown, it is determined that the sample to be tested does not contain the novel coronavirus SARS-CoV-2.
    [7]
    The use of the kit according to claim 3, for detecting the novel coronavirus SARS-CoV-2 in clinical samples.
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    同族专利:
    公开号 | 公开日
    CN111518947A|2020-08-11|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    CN1813064B|2003-06-27|2011-11-02|荣研化学株式会社|Method of detecting SARS coronavirus|
    CN102286633B|2010-01-08|2013-08-14|江苏省家禽科学研究所|Avian infectious bronchitis virus quick detection kit based on loop-mediated isothermal amplification technology and application method thereof|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    CN202010150527.8A|CN111518947A|2020-03-06|2020-03-06|RT-LAMP primer group and kit for detecting novel coronavirus SARS-CoV-2|
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