Novel coronavirus sars-cov-2 nucleic acid visual detection kit

专利摘要:
The present disclosure provides a novel coronavirus SARS-CoV—2 nucleic acid visual detection kit. The kit of the disclosure includes primer set, RT—LAMP reaction solution, positive control, negative control, DEPC water. 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 NO.] to SEQ ID NO.6. The primer set and kit mentioned above can accurately detect the S gene of SARS-CoV—Z, without cross-reacting with other coronaviruses and other pneumonia-causing viruses. SARS-CoV—2 may be detected by isothermal amplification at 63 DC in 15 minutes at the earliest. The detection sensitivity reaches 1.6fg/uL. 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 under natural light without using eXpensive instruments, can realize rapid and accurate detection of SARS-CoV—Z, and is suitable for on-site detection and clinical detection in primary hospitals.
公开号:NL2027316A
申请号:NL2027316
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 disclosure claims the priority of Chinese Patent Application NO. 202010150791.1 entitled “Novel coronavirus SARS-CoV-2 nucleic acid visual detection kit” 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 present disclosure belongs to the technical field of viral nucleic acid detection, and relates to a nucleic acid isothermal amplification visual detection kit for the novel coronavirus SARS-CoV-2.BACKGROUND ART
[03] [03] Novel coronavirus pneumonia (short for COVID-19) has strong infectivity, characterized by fever, dry cough and pneumonic lesions, which leads to death under serious condition.
[04] [04] The pathogen of novel coronavirus pneumonia is the novel coronavirus SARS-CoV-2. Severe acute respiratory syndrome coronavirus (SARS-Cov), Middle East respiratory syndrome coronavirus (MERS-Cov) and SARS-CoV-2 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 respiratory infections in humans, the representative strains are HKU1, OC43, NL63 and 229E.
[05] [05] 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, HSN, 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, wherein the influenza virus, respiratory syncytial virus, and rhinovirus are the most common and serious.
[06] [06] 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.
[07] [07] At present, the methods used to detect SARS-CoV-2 include virus isolation and culture, electron microscopy observation, immunological detection, and molecular biology detection, 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 requires the acquisition of viruses, which takes a long time, and most institutions have no such conditions. The specificity and sensitivity of immunoassay method are lower than that of molecular biology detection method. Molecular biology detection method has the advantages in specificity, sensitivity and accuracy. At present, the molecular biology methods used to identify SARS-CoV-2 include fluorescent PCR and genome sequencing. Both methods need expensive professional instruments, and have high requirement in 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.
[09] [09] Compared with the existing 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 ORFlab, 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.
[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 a LAMP detection primer set, kit and detection method for loop-mediated isothermal amplification of SARS-CoV-2 with high detection sensitivity.
[12] [12] The present disclosure is realized in the following technical solutions:
[13] [13] A novel coronavirus SARS-CoV-2 nucleic acid visual detection kit, includes 2xRT-LAMP reaction solution, positive control, negative control, DEPC water and primer set.
[14] [14] The primer set in the novel coronavirus SARS-CoV-2 nucleic acid visual detection kit includes 6 of specific RT-LAMP primers, and the sequence of the primers are set forth in as follows:
[5] [5] Primer F3: 5’-TGTGTTAATCTTACAACCAGAAC-3’ (SEQ ID NO:1),
[16] [16] Primer B3: 5’-ACCATTGGTCCCAGAGAC-3" (SEQ ID NO:2),
[17] [17] Primer FIP: 5’-GGATCTGAAAACTTTGTCAGGGTAATCAATTACCCCCTGCAAAC-3> (SEQ ID NO:3),
[18] [18] Primer BIP: 5-TCAGTTTTACATTCAACTCAGGACTATGTATAGCATGGAACCAAGT-3 (SEQ ID NO:4),
[19] [19] Primer LF: S°-GTTCTTACCTTTCTTTTCCAATG-3’ (SEQ ID NO:5),
[20] [20] Primer LB: 5’-TAAACACCACGTGTGAAAGAATTAG-3" (SEQ ID NO:6).
[21] [21] 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 25 uL of RT-LAMP reaction solution contains Spmol of primer F3, Spmol of primer B3, 40pmol of primer FIP, 40pmol of primer BIP, 20pmol of primer LF and 20pmol of primer LB.
[22] [22] The 2#RT-LAMP reaction solution in the novel coronavirus SARS-CoV-2 nucleic acid visual detection kit, wherein the components contained in the 2*RT-LAMP reaction solution and concentrations thereof are as follows: 40 mM Tris-HCI (pH 8. 8), 20 mM KCI, 16 mM MgSO, 20 mM (NH4)2SO04, 0.2% Tween 20, 1.6M betaine, 2.8 mM dNTPs, 1.28U/uL Bst DNA polymerase, 16U/uL AMV reverse transcriptase, 1.6U/uL RNAase inhibitor, 0.5mM metal ion indicator HNB.
[23] [23] The positive control in the novel coronavirus SARS-CoV-2 nucleic acid visual detection kit, wherein the full-length DNA of the S gene of SARS-CoV-2 is connected to the positive recombinant plasmid screened and identified by the pUCS7 vector, and the plasmid with S gene is transcribed in vitro to obtain the RNA fragments, the RNA fragments is purified by HPLC to obtain the positive control substance; the copy number of lug of plasmid is
[24] [24] The novel coronavirus SARS-CoV-2 nucleic acid visual detection kit for use in detecting the presence of a novel coronavirus SARS-CoV-2 in clinical samples, the specific operation process is: adding 7 pL of primer, 12.5 uL of 2=RT-LAMP reaction solution, 1 pL of positive template or 1-3 pL of sample RNA to the reaction tube in the ice box, adding DEPC water to make up the volume to 25uL, closing the lid of the reaction tube, mixing the reaction solution,
[25] [25] The beneficial effects of the present disclosure are:
[26] [26] (1) The novel coronavirus SARS-CoV-2 nucleic acid visual detection kit of the present disclosure is simple to operate, good for practical use and low in cost. There is no need for expensive specialized instruments, just use 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, CDCs and other institutions.
[27] [27] (2) The novel coronavirus SARS-CoV-2 nucleic acid visual detection kit of the present disclosure is fast and efficient. Due to the use of isothermal amplification, there is 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.
[28] [28] (3) The reaction solution of novel coronavirus SARS-CoV-2 nucleic acid visual detection kit of the present disclosure is added with HNB dye beforehand, so as to directly judge the result according to the colour change of the reaction tube, thus avoiding the needs for second addition of dyes since the observation of results after the reaction, which 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 is optimized, and the inhibition of RT-LAMP reaction by adding dye beforehand is avoided, and the one-step rapid colour development RT-LAMP detection of SARS-CoV-2 is realized.
[29] [29] (4) The RT-LAMP primer set of the novel coronavirus SARS-CoV-2 nucleic acid visual detection kit of the present disclosure is designed for the specific regions of the S gene sequence in the novel coronavirus SARS-CoV-2, which is the key technology of the present disclosure. The RT-LAMP primer set includes 6 specific primers including outer primers F3 and B3, inner primers FIP and BIP, loop primers LF and LB. The primer set can only specifically amplify the nucleic acid of the novel coronavirus SARS-CoV-2, while the nucleic acid of other viruses cannot be amplified.
[30] [30] (5) The RT-LAMP primer set of the novel coronavirus SARS-CoV-2 nucleic acid visual detection kit of the present disclosure and the kit 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
[31] [31] FIG 1 shows the determination of the LAMP experiment result of the novel coronavirus SARS-CoV-2 (the left tube shows sky blue, which is positive; the right tube keeps the original blue-purple colour, which is negative);
[32] [32] FIG 2 shows the LAMP sensitivity experiment results of the novel coronavirus SARS-CoV-2 (the 1-7# tubes are positive, and the concentration of the corresponding positive template is 1.6ng/uL, 0. 16ng/uL, 0.016ng/uL, 1.6pg/uL, 0.16pg/uL, 0.016pg/uL, 1.6fg/uL in turn; the 8-9# tubes are negative, and the corresponding positive template concentration is
[33] [33] The present disclosure will be further described in detail below in combination with examples, but the protection scope of the present disclosure is not limited thereto. EXAMPLE 1: Preparation of the novel coronavirus SARS-CoV-2 nucleic acid visual detection kit
[34] [34] (1) Design and synthesis of RT-LAMP primers and preparation of primer set solutions
[35] [35] 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, HKU1, OC43, NL63 and 229E, etc. were comparatively analyzed, and SARS-CoV-2 specific gene sequence region suitable for designing RT-LAMP primers was screened. At the same time, the sequence of the screened template region analyzed by BLAST online tool should only be specifically aligned to the gene sequence of SARS-CoV-2, not to other coronavirus gene sequences, nor to influenza virus, respiratory syncytial virus, rhinovirus, adenovirus, parainfluenza virus, enterovirus, herpes simplex virus, human metapneumovirus, reovirus, measles virus, cytomegalovirus and other gene sequences. The quality of RT-LAMP primer determined the specificity and sensitivity of the RT-LAMP detection method. The formation of primer dimers should be avoided when designing primers. The 6 regions identified by the primers were specific regions matching the S gene of SARS-CoV-2, which could identify all the S genes of SARS-CoV-2, so it could detect all novel coronavirus SARS-CoV-2. The present disclosure used the S gene of the novel coronavirus SARS-CoV-2 as template, and used the online software Primer Explore V5 to design RT-LAMP specific amplification primers. The
[36] [36] Primer F3: S’-TGTGTTAATCTTACAACCAGAAC-3’ (SEQ ID NO:1),
[37] [37] Primer B3: 5’-ACCATTGGTCCCAGAGAC-3" (SEQ ID NO:2),
[38] [38] Primer FIP: 5’-GGATCTGAAAACTTTGTCAGGGTAATCAATTACCCCCTGCAAAC-3° (SEQ ID NO:3),
[39] [39] Primer BIP: S’-TCAGTTTTACATTCAACTCAGGACTATGTATAGCATGGAACCAAGT-3 (SEQ ID NO:4),
[40] [40] Primer LF: 5’-GTTCTTACCTTTCTTTTCCAATG-3’ (SEQ ID NO:5),
[41] [41] Primer LB: 5’-TAAACACCACGTGTGAAAGAATTAG-3’ (SEQ ID NO:6).
[42] [42] The primers were synthesized by Sangon Biotech (Shanghai) Co, Ltd. and purified by HPLC.
[43] [43] 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 10 pM respectively, and the primers FIP, BIP, LF, and LB were prepared into an aqueous solution with a molar concentration of 20 uM. 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.
[44] [44] During detection, 7 uL of primer set solution was added to each 25 uL reaction system, so that the moles of each primer in each reaction system were: primers F3 and B3 were Spmol, FIP and BIP were 40pmol, and LF and LB were 20pmol.
[45] [45] (2) Preparation of 2XxRT-LAMP Reaction Solution
[46] [46] The components and concentrations thereof in the 2xRT-LAMP reaction solution were: 40 mM Tris-HCI (pH 8.8), 20 mM KCI, 16 mM MgSO,4, 20 mM (NH4):S04, 0.2% Tween 20,
[47] [47] During detection, 12.5uL of 2xRT-LAMP reaction solution was added to each reaction system (25 pL), so that each component and the final concentration thereof were 20 mM
[48] [48] (3) Assembly of the kit
[49] [49] The detection kit was consisted of 0.5mL of primer set aqueous solution (step (1)), | mL of 2xRT-LAMP reaction solution (step (2)), 0.1 mL of positive control, 0.5 mL of negative control and 1 mL of DEPC water.
[50] [50] Positive control of SARS-CoV-2: full-length DNA of synthetic S gene was prepared by conventional methods in the field, the full-length DNA was inserted into pUC57 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. EXAMPLE 2: Optimization of the detection temperature of the novel coronavirus SARS-CoV-2 nucleic acid visual detection kit
[51] [51] 12.5 pL of 2xLAMP reaction solution, 7 uL of primer set aqueous solution, 1 uL of positive template were added to each RT-LAMP reaction system (25 pL), and sterilized DEPC water was added to make up the volume to 25uL.. The final concentrations of each component in each 25 uL RT-LAMP reaction system were 20 mM Tris-HCI (pH 8.8), 10 mM KCI, 8 mM MgSOs, 10 mM (NH4):S04, 0.1% Tween 20, 0.8M betaine, 1.4 mM dNTPs, 0.64U/uL Bst DNA polymerase, 8U/uL AMV reverse transcriptase, 0.8U/uL RNAase inhibitor, 0.25mM HNB, the molar number of each primer were: primer F3 and B3 each 5pmol, primer FIP and BIP each 40pmol. Negative control and blank control were set in the experiment.
[52] [52] The reaction temperatures of RT-LAMP were set to 58 °C, 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 5 minutes, 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.
[53] [53] In the present example, a water bath was used as a heating device for RT-LAMP reaction.
[54] [54] The colour of the reaction solution was observed under natural light. If the colour of the reaction solution changed to sky blue, it was determined that the sample to be tested contained the novel coronavirus SARS-CoV-2; if the colour of the reaction solution remained the original blue-purple, it was determined that the sample to be tested did not contain the novel coronavirus SARS-CoV-2. See attached FIG. 1 for details.
[55] [55] The novel coronavirus SARS-CoV-2 nucleic acid visual detection kit of the present disclosure was used to detect the positive control of SARS-CoV-2, the reaction solution appeared sky blue after reacting at 58-65 °C for 50 minutes, but the colour of the amplified product at 63 °C was slightly stronger than the products amplified at other temperatures, so the reaction temperature was fixed at 63 °C. EXAMPLE 3: Optimization of detection time and sensitivity determination of SARS-CoV-2 nucleic acid visual detection kit
[56] [56] The positive template was diluted 10-folds with DEPC water. The concentrations of positive templates in the 1# to 9# tubes were 1.6ng/uL, 0.16ng/uL, 0.016ng/pL, 1.6pg/uL,
[57] [57] The RT-LAMP reaction system was the same as the reaction system in Example 2. That is, 12.5 uL of 2xL AMP reaction solution, 7 uL of primer set aqueous solution, 1 uL of positive template with different concentration gradients were added to each RT-LAMP reaction system (25 pL), and sterilized DEPC water was added to make up the volume to 25uL. The final concentrations of each component in each 25 uL RT-LAMP reaction system were 20 mM Tris-HCI (pH 8.8), 10 mM KCI, 8 mM MgSO4, 10 mM (NH:)2S04, 0.1% Tween 20, 0.8M betaine, 1.4 mM dNTPs, 0.64U/uL Bst DNA polymerase, 8U/uL AMV reverse transcriptase,
[58] [58] In the present example, a water bath was used as a heating device for RT-LAMP reaction.
[59] [59] The reaction temperature of RT-LAMP was set to 63 °C, and the colour of the liquid in each reaction tube was observed every 5 minutes, 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.
[60] [60] The colour of the reaction solution under natural light was observed. If the colour of the reaction solution changed to sky blue, it was determined that the sample to be tested contained the novel coronavirus SARS-CoV-2; if the colour of the reaction solution was remained the original blue-purple, it was determined that the sample to be tested did not contain the novel coronavirus SARS-CoV-2.
[61] [61] Table 1 Correspondence between the RNA concentration of SARS-CoV-2 and the occurrence time of RT-LAMP detection results nd [ee [| tube number | 1# 24 3# 44 S5# 6# 7# 8# O# control) | e control) concentration 16 | 0.16 1 001 | 16 | 0.16 001 | 16 | 0.16 | 0.01 of RNA/uL ng ng | ong | pg peg | 6pg | fe fg | 6fg ’ oe | + + + + + + + results occurrence time of u 15 20 20 25 25 30 35 55 55 55 55 positive result (min)
[62] [62] The positive templates of different concentration gradients of SARS-CoV-2 were detected by using the novel coronavirus SARS-CoV-2 nucleic acid visual detection kit of the present disclosure, and after isothermal amplification at 63 °C, the reaction solutions of the 1# to 7# tubes were all appeared sky blue, while the reaction solution of the 8# tube, negative control tube and blank control tube still remained the original blue purple. 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 positive control 7# tube, i.e.
[63] [63] 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 (HKU1, OC43, NL63 and 229E), high pathogenic avian influenza virus (H7N9, HSNI), 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 S gene connected to the positive recombinant plasmid screened and identified by the pUCS7 vector. The nucleic acids of the influenza viruses H7N9 and HSN1 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 pUC57 vector. Other viral nucleic acids were extracted from inactivated virus liquid.
[64] [64] 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.
[65] [65] 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 part was detected with the kit of the present disclosure and the fluorescent RT-PCR kit respectively, the coincidence rate of the two detection results was compared.
[66] [66] 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 140uL 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.
[67] [67] 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 ORF1ab/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 10 min, 95 °C for 10 min; 95 °C for 15's, 55 'C for 1 min, 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).
[68] [68] 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 34.07% (62/182) and 34.6% (63/182), respectively. The consistency rate of the detection results of the two kits is 99.45%. 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.
[69] [69] The experimental results of the above examples show that the novel coronavirus SARS-CoV-2 nucleic acid visual 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
SEQUENCE LISTING <110> Qilu University of Technology <120> Novel coronavirus SARS-CoV-2 nucleic acid visual detection kit <141> 2020-03-06 <160> 6 <170> SIPOSequenceListing 1.0 <2105 1 <211> 23 <212> DNA <213> artificial sequence <400> 1 tgtgttaatc ttacaaccag aac 23 <210> 2 <211> 18 <212> DNA <213> artificial sequence <400> 2 accattggtc ccagagac 18 <210> 3 <211> 44 <212> DNA <213> artificial sequence <400> 3 ggatctgaaa actttgtcag ggtaatcaat taccccctgc aaac 44 <210> 4 <211> 46 <212> DNA <213> artificial sequence <400> 4 tcagttttac attcaactca ggactatgta tagcatggaa ccaagt 46 <210> 5 <211> 23 <212> DNA <213> artificial sequence <400> 5 gttcttacct ttcttttcca atg 23 <210> 6 <211> 25 <212> DNA <213> artificial sequence Pagina 1
SEQLTXT <400> 6 taaacaccac gtgtgaaaga attag 25
Pagina 2

权利要求:
Claims (7)
[1]
1. A novel coronavirus SARS-CoV-2 nucleic acid visual detection kit, includes a novel coronavirus SARS-CoV-2 nucleic acid visual detection primer set, 2 x RT-LAMP reaction solution, positive control, negative control and DEPC water.
[2]
The novel coronavirus SARS-CoV-2 nucleic acid visual detection kit for coronavirus according to claim 1, wherein the novel coronavirus SARS-CoV-2 nucleic acid visual detection primer set comprises 6 specific RT-LAMP primers, the sequence of the primers of which is as follows: Primer F3: 5'-TGTGTTAATCTTACAACCAGAAC-3'(SEQ ID NO: 1), Primer B3: 5'-ACCATTGGTCCCAGAGAC-3'(SEQ ID NO: 2}, Primer FIP: 5'-GGATCTGAAAACTTTGTCAGGGTAATCAATTACCCCCTGCAAAC-3'(SEQ ID NO: 3), Primer BIP: 5'-TCAGTTTTACATTCAACTCAGGACTATGTATAGCATGGAACCAAGT-3'(SEQ ID NO: 4), Primer LF: 5'-GTTCTTACCTTTCTTTTCCAATG-3'(SEQ ID NO: 5), Primer LB: 5'-TAAACACCACGTGTGAAAGAATTAG-3'( SEQ ID NO: 8).
[3]
The novel coronavirus SARS-CoV-2 nucleic acid visual detection kit according to claim 1 or 2, wherein the molar ratio of outer primers (F3, B3): inner primers (FIP, BIP}: loop primers (LF, LB) 1:8:4 each 25 µl of RT-LAMP reaction solution contains 5 pmoles of primer F3, 5 pmoles of primer B3, 40 pmoles of primer FIP, 40 pmoles of primer BIP, 20 pmoles of primer LF and 20 pmoles of primer LB.
[4]
The novel SARS-CoV-2 nucleic acid visual detection kit for coronavirus according to claim 1, wherein the components in the 2 x RT-LAMP reaction solution and the concentrations thereof are as follows: 40 mM Tris-HCl (pH 8.8 ), 20 mM KCl, 16 mM MgSO 4 , 20 mM (NH 4 ) 2 SO 4 , 0.2% Tween 20, 1.6 M betaine, 2.8 mM dNTPs, 1.28 U/uL Bst DNA polymerase, 16 U /pL AMV reverse transcriptase, 1.6 U/uL RNAase inhibitor and 0.5 mM metal ion indicator HNB.
[5]
The novel coronavirus SARS-CoV-2 nucleic acid visual detection kit according to claim 1, wherein the positive control is prepared as follows: joining the entire DNA of the S gene of SARS-CoV-2 to the positive recombinant plasmid. screened and identified by the pUC57 vector, and transcribed in vitro to obtain the RNA fragments, purifying the fragments by HPLC to obtain the positive control substance; the copy number of 1 µg of plasmid is 1.492 x 10.
[6]
The novel coronavirus SARS-CoV-2 nucleic acid visual detection kit according to claim 1 for use in detecting the presence of a novel coronavirus SARS-CoV-2 in clinical samples, wherein the specific process of action is: adding 7 HL primer set, 12.5 µl 2x RT-LAMP reaction solution, 1 µl positive template or 1-3 µl sample
-16 - RNA in the reaction tube in the ice tray, adding DEPC water to bring the volume to 25 µl, closing the lid of the reaction tube, mixing the reaction solution, centrifuging immediately to settle 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 15-60 minutes, then 5 reacting at 85°C for minutes to terminate the reaction, and observing the color of the reaction solution under natural light, if the color of the reaction solution changes to sky blue, then it is determined that the sample to be tested has the novel coronavirus SARS-CoV -2 contains; if the color of the reaction solution remains the original blue-purple, it is determined that the sample to be tested does not contain the novel coronavirus SARS-CoV-2.
[7]
The novel coronavirus SARS-CoV-2 nucleic acid visual detection kit according to claim 8, wherein the isothermal multiplication condition is 83°C for 5 minutes.

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同族专利:

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公开号 | 公开日

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CN111378784A|2020-07-07|

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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CN202010150791.1A|CN111378784A|2020-03-06|2020-03-06|Novel coronavirus SARS-CoV-2 nucleic acid visual detection kit|

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