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CN-116286801-B - Probe set, probe set for detecting novel coronavirus multiple nucleic acid targets, kit and detection method thereof

CN116286801BCN 116286801 BCN116286801 BCN 116286801BCN-116286801-B

Abstract

The invention discloses a probe set, a probe set for detecting novel coronavirus multiple nucleic acid targets, a kit and a detection method thereof. The invention is based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) technology, combines endonuclease and a medium-universal probe (reporting probe) signal reporting mode, and achieves novel coronavirus double-target gene specific analysis in a fluorescent probe system which does not depend on target sequence design within 90 minutes. The sensitivity of the double genes of the system reaches 100 copies/reaction, the system specificity is good, and no cross signal exists. The method is helpful for accurately and rapidly screening the patients infected by the novel coronavirus, and has important significance for diagnosis and treatment of the novel coronavirus and epidemic prevention and control.

Inventors

  • LIU XIAOLONG
  • XU HAIPO
  • ZHAO BIXING
  • CAI ZHIXIONG
  • ZENG YONGYI
  • SUN YUPENG
  • ZHANG XIAOLONG

Assignees

  • 福建医科大学孟超肝胆医院(福州市传染病医院)

Dates

Publication Date
20260505
Application Date
20211203

Claims (12)

  1. 1. A probe set for detecting multiple nucleic acid targets of a novel coronavirus, comprising a primer set for detecting an N gene of the novel coronavirus SAR-CoV-2, or/and a primer set for detecting an E gene of the novel coronavirus SAR-CoV-2; The primer group for detecting the E gene of the novel coronavirus SAR-CoV-2 comprises a first probe group, a second probe group, an E gene outer primer E-WF/N-WR, an E gene inner primer E-FIP/N-BIP and an E gene annular primer E-LF/N-LB; The primer group for detecting the N gene of the novel coronavirus SAR-CoV-2 comprises a third probe group, an N gene outer primer N-WF/N-WR, an N gene inner primer N-FIP/N-BIP and an N gene annular primer N-LF/N-LB; A first probe set, a loop primer of an E gene of a targeted novel coronavirus SAR-CoV-2, which comprises a loop mediator probe E-LMP with a nucleotide sequence shown as SEQ ID No.7 and one of 2 reporting probes UP1/UP2 respectively shown as SEQ ID No.8 and 18, wherein the 5 'end of the reporting probe is marked with a fluorescent group and the 3' end of the reporting probe is marked with a quenching group; A second probe set, an inner primer of an E gene of a targeted new coronavirus SAR-CoV-2, which comprises an inner mediator probe E-IMP with a nucleotide sequence shown as SEQ ID No.9 and one of 2 reporting probes UP1/UP2 respectively shown as SEQ ID No.8 and 18, wherein the 5 'end of the reporting probe is marked with a fluorescent group and the 3' end of the reporting probe is marked with a quenching group; A third probe set, a loop primer of N gene of targeting new coronavirus SAR-CoV-2, which comprises a loop mediator probe N-LMP with a nucleotide sequence shown as SEQ ID No.17, and one of 2 reporting probes UP1/UP2 shown as SEQ ID No.8 and 18 respectively, wherein the 5 'end of the reporting probe is marked with a fluorescent group, and the 3' end of the reporting probe is marked with a quenching group; also included is a reporter probe, and at least one mediator probe, wherein, The vector probe comprises a vector sequence, an oligonucleotide sequence and a target specific sequence from 5' to 3', wherein the oligonucleotide sequence and the target specific sequence comprise sequences complementary to partial control sequences of nucleic acid target sequences of novel coronaviruses, the partial control sequences of the nucleic acid target sequences are sequences amplified along the edges after the nucleic acid target sequences of the novel coronaviruses are complementarily paired with a circular primer or an inner primer in a loop-mediated isothermal amplification reaction, the vector sequence comprises sequences which are not complementary to the nucleic acid target sequences or the control sequences, one base in the oligonucleotide sequence is modified into RNA bases, the rest is DNA bases, and the RNA bases are positioned at 2-3 bases at the 5' end of the target specific sequence; The reporter probe comprises, in the 3 'to 5' direction, a capture sequence complementary to the mediator sequence or a portion thereof, and a template sequence, and the target-specific sequence at the RNA base and RNA base end is not complementarily paired with the reporter probe, the reporter probe being labeled at the 5 'end with a reporter group and at the 3' end with a quencher group, the reporter probe emitting a different signal upon hybridization to its complementary sequence than without hybridization to its complementary sequence, the reporter group and quencher group being separated by a distance of 10-80nt or more.
  2. 2. The probe set of claim 1, wherein the nucleic acid target sequence of the novel coronavirus is selected from the group consisting of conserved gene sequences of the novel coronavirus.
  3. 3. The probe set of claim 2, wherein the nucleic acid target sequence of the novel coronavirus is selected from the group consisting of a conserved gene sequence of SARS-CoV-2 virus, N gene, E gene.
  4. 4. The probe set of claim 1, wherein the probe set comprises a plurality of probes, The outer primer of the N gene is primer N-WF with the nucleotide sequence shown as SEQ ID No.11, and primer N-WR with the nucleotide sequence shown as SEQ ID No. 12; the inner primer of the N gene is primer N-FIP with the nucleotide sequence shown as SEQ ID No.13, and primer N-BIP with the nucleotide sequence shown as SEQ ID No. 14; The nucleotide sequence of the N-gene cyclic primer is shown as SEQ ID No.15, and the nucleotide sequence of the primer N-LB is shown as SEQ ID No. 16.
  5. 5. The probe set of claim 1, wherein the probe set comprises a plurality of probes, The outer primer of the E gene is primer E-WF, the nucleotide sequence of which is shown as SEQ ID No.1, and primer E-WR, the nucleotide sequence of which is shown as SEQ ID No. 2; the primer E-FIP on the inner side of the E gene has a nucleotide sequence shown as SEQ ID No.3, and the primer E-BIP has a nucleotide sequence shown as SEQ ID No. 4; The nucleotide sequence of the E gene cyclic primer is shown as SEQ ID No.5, and the nucleotide sequence of the primer E-LB is shown as SEQ ID No. 6.
  6. 6. A kit for detecting a novel coronavirus multiplex nucleic acid target is characterized by comprising a solution containing the primer set according to any one of claims 1 to 4, wherein the solution of the primer set is a solution obtained by diluting each primer or probe in the primer set to 0.05 to 1.6 mu mol/L.
  7. 7. The kit of claim 6, further comprising 6 mmol MgSO 4 , 1.4 mmol dNTP (A/G/C/T), 0.32U/. Mu.L Bst DNA polymerase, 0.5. Mu.L reverse transcriptase, 0.04U/. Mu. L RNASE HII, 5. Mu.L template gene, 1X Isothermal Amplification Buffer, the total volume of the kit detection system being 25. Mu.L, the remainder being made up to 25. Mu.L with nuclease-free water.
  8. 8. The kit according to claim 6 or 7, comprising a solution containing a primer set for detecting the N gene of novel coronavirus SAR-CoV-2 according to claim 6, wherein the primer set is used as a solution 0.2 μmol N-WF,0.2 μmol N-WR,1.6 μmol N-FIP,1.6 μmol N-BIP,0.8 μmol N-LF,0.4 μmol N-LB,0.4 μmol N-LMP,0.2 μmol UP1 or UP2, further comprising 6 mmol MgSO 4 , 1.4 mmol dNTP (a/G/C/T), 0. 0.32U/. Mu.l Bst DNA polymerase, 0.5. Mu.l reverse transcriptase, 0.04U/. Mu. L RNASE HII, 5. Mu.l template gene, 1 x Isothermal Amplification Buffer, the total volume of the kit detection system being 25. Mu.l, the remainder being made UP to 25. Mu.l with nuclease-free water.
  9. 9. The kit according to claim 6 or 7, comprising a solution containing a primer set for detecting the E gene of novel coronavirus SAR-CoV-2 according to claim 6, wherein said primer set is in the form of a solution 0.2 μmol E-WF,0.2 μmol E-WR,1.6 μmol E-FIP,0.8 μmol E-BIP,0.8 μmol E-LF,0.8 μmol E-LB,0.8 μmol E-LMP,0.2 μmol UP1 or UP2, further comprising 6 mmol MgSO 4 , 1.4 mmol dNTP (a/G/C/T), 0. 0.32U/. Mu.l Bst DNA polymerase, 0.5. Mu.l reverse transcriptase, 0.04U/. Mu. L RNASE HII, 5. Mu.l template gene, 1 x Isothermal Amplification Buffer, wherein the total volume of the kit detection system is 25. Mu.l, and the remainder is made UP to 25. Mu.l with nuclease-free water.
  10. 10. Kit according to claim 6 or 7, comprising a solution containing the primer set for detecting the N gene of the novel coronavirus SAR-CoV-2 and a solution containing the primer set for detecting the E gene of the novel coronavirus SAR-CoV-2 according to claim 6; The solution of the primer group for detecting the N gene of the novel coronavirus SAR-CoV-2 is ：0.05 μmol N-WF,0.05 μmol N-WR,0.4 μmol N-FIP,0.4 μmol N-BIP,0.2 μmol N-LF,0.1 μmol N-LB,0.1 μmol N-LMP,0.2 μmol UP1 or UP2; the solution of the primer group for detecting the E gene of the novel coronavirus SAR-CoV-2 is ：0.2 μmol E-WF,0.2 μmol E-WR,1.6 μmol E-FIP,1.6 μmol E-BIP,0.8 μmol E-LF,0.4 μmol E-LB,0.4 μmol E-LMP,0.2 μmol UP1 or UP2; Also included are 6 mmol MgSO 4 , 1.4 mmol dNTP (A/G/C/T), 0.32U/. Mu.L Bst DNA polymerase, 0.5. Mu.L reverse transcriptase, 0.04U/. Mu. L RNASE HII,40 mmol guanidine hydrochloride, 5. Mu.L template gene, 1X Isothermal Amplification Buffer, the total volume of the kit assay system being 25. Mu.L, the remainder being made up to 25. Mu.L with nuclease-free water.
  11. 11. A method for detecting a novel coronavirus multiplex nucleic acid target for non-disease diagnosis, comprising the steps of: S1, sampling; S2, preparing an LAMP reaction system by using the kit according to any one of claims 6-10 and an obtained sample, placing the LAMP reaction system into a reaction tube, uniformly mixing and centrifuging; S3, placing the reaction tube on a fluorometer with a constant temperature function to perform a reaction at 60-65 ℃, collecting fluorescence every 1min, and collecting 60-90 cycles; S4, reading a Ct value, wherein the detection result judging standard is that the Ct value of a sample is less than or equal to 60, and judging that the sample is positive; The Ct value of the sample is less than or equal to 75 and is 60, if the amplification curve is a logarithmic amplification curve, the sample is judged to be a suspicious positive sample, otherwise, the sample is judged to be negative; Rechecking the suspicious positive sample, judging that the suspicious positive sample is positive if the Ct value of the rechecked sample is less than or equal to 60, otherwise, judging that the sample is negative; And judging that the sample is negative if the sample has no Ct value or Ct value is more than 75.
  12. 12. The method according to claim 11, wherein in step S3, the isothermal amplification is performed at 65℃when the kit of step S2 contains only the primer set for detecting the N or E gene, and at 60℃when the kit of step S2 contains both the primer sets for detecting the N and E genes.

Description

Probe set, probe set for detecting novel coronavirus multiple nucleic acid targets, kit and detection method thereof Technical Field The invention belongs to the field of biomedical engineering, and mainly relates to a probe set, a probe set for detecting novel coronavirus multiple nucleic acid targets, a kit and a detection method thereof. Background Currently, the diagnosis of novel coronavirus infection is mainly carried out by real-time fluorescent quantitative RT-PCR (RT-qPCR) for detecting viral nucleic acid load or by second-generation sequencing for analyzing viral nucleic acid sequence. However, the RT-qPCR virus nucleic acid detection needs to make the DNA template to be detected, a specific primer, a fluorescent dye and a PCR amplification buffer solution circulate for a certain number of times under the PCR amplification reaction condition to enable the fluorescent signal to reach a set threshold value, and the second generation sequencing needs to add dNTPs to catalyze a substrate to excite fluorescence through enzymatic cascade reaction when generating a new DNA complementary strand, or directly add the dNTPs or semi-degenerate primer marked by the fluorescence to release the fluorescent signal when synthesizing or connecting the complementary strand and generate a complementary strand, and convert the fluorescent signal into a sequencing peak value to obtain complementary strand sequence information, so that a series of problems of long detection time and period, high environmental requirement, high technical strength requirement, high hardware equipment requirement, high price and the like exist in the RT-qPCR and the second generation sequencing. In recent years, isothermal amplification techniques have found widespread use in pathogen nucleic acid detection. Among them, reverse transcription/loop-mediated isothermal amplification RT-/LAMP is most prominent in application due to its low requirements on use environment, no need of expensive thermal cycler, short reaction time, high sensitivity and specificity, low cost and the like. LAMP can efficiently amplify targets through at least six target primer areas (four primers) at a constant temperature (60-65 ℃), the detection sensitivity can reach 100 copies/reaction, and the reaction time is about 20-45 min. Meanwhile, bst DNA polymerase used in LAMP has better tolerance to reaction inhibitor, and can be used for amplification detection through sample simple treatment such as heating and the like. At present, the LAMP method is applied to detection of the novel coronavirus, but most of the LAMP method belongs to a single-target detection mode. The novel coronavirus belongs to the genus single-stranded RNA coronavirus, has the characteristics of easy variation (such as B1.1.7, B1.351 and the like), and the like, and can easily generate false negative results in single-target detection. The existing loop-mediated isothermal amplification detection method for SARS-CoV-2 virus comprises a LAMP primer group introduced by China patent ：CN202010150878.9、CN202011429713.1、CN202011167529.4、CN202010312576.7、CN202011045783.7、CN202010150527.8, the known primer group is only four-six primers designed for six target index primer areas of a target gene of SARS-CoV-2 virus, the LAMP method has high sensitivity and short reaction time (the reaction can be completed within 30-60 min), no special instrument is needed in clinic use (a real-time turbidimeter is recommended in a reagent kit research and development stage), the operation is simple (no matter DNA or RNA is needed to be mixed in a PCR tube in the detection step, the reaction liquid, enzyme and template are mixed in a water bath pot or a constant temperature oven for about 63 ℃, the temperature is kept for 30-60 min, and the visual observation result) besides turbidity detection, after the LAMP reaction, mn 2+ is combined with a reaction product pyrophosphate to release the calcein so as to release the quenched state and take on yellowish Green fluorescence, but the detection sensitivity is reduced by an order of magnitude due to the addition of Mn 2+; in the improvement of LAMP detection method for other target genes, also Ball et al propose a simple RT-LAMP end point detection technology to realize detection of West Nile virus and chikungunya virus RNA, the method firstly marks fluorescent dye on a loop primer or an inner primer, then hybridizes with the fluorescent dye by a short chain marked with a quenching group, so that when the whole reaction system does not carry out LAMP amplification, the system does not have strong fluorescent signals, and LAMP amplification begins under the condition of the existence of a target template, the primer marked with the fluorescent dye is combined with the specific part of the target template, so that the short chain of the quenching group is dissociated in the solution, and the reaction system presents a strong fluorescence signal, but the method needs to add