WO2022000753A1 - 一种防止rna降解的保护方法、保护序列、组合物和试剂盒及其应用 - Google Patents

一种防止rna降解的保护方法、保护序列、组合物和试剂盒及其应用 Download PDF

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WO2022000753A1
WO2022000753A1 PCT/CN2020/112399 CN2020112399W WO2022000753A1 WO 2022000753 A1 WO2022000753 A1 WO 2022000753A1 CN 2020112399 W CN2020112399 W CN 2020112399W WO 2022000753 A1 WO2022000753 A1 WO 2022000753A1
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rnase
rna
protection
sequence
gene
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PCT/CN2020/112399
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French (fr)
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瞿佳
徐陶
王金固
陈江帆
徐良德
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温州医科大学附属眼视光医院
杭州拜赛思生物科技有限责任公司
温州瓯佳生物科技有限公司
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Priority to EP20942719.4A priority Critical patent/EP4163388A4/en
Publication of WO2022000753A1 publication Critical patent/WO2022000753A1/zh

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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the invention relates to the technical field of in vitro nucleic acid detection, in particular to a protection method, protection sequence, composition and kit for preventing RNA degradation, and applications thereof.
  • RNA Ribonucleic Acid
  • RNase Ribonucleic Acid
  • the present invention provides a protection method, a protection sequence, a composition and a kit for preventing RNA degradation, and applications thereof, to further improve the protection effect on viral RNA.
  • the technical solution adopted in the present invention is: a protection method for preventing RNA degradation, comprising the following steps: forming an RNA:DNA complex after the efficient combination of an RNase protection sequence (RNase-AP) and a target RNA, and the combination makes the RNA exposed The region is reduced, so that RNase cannot effectively cleave the target RNA to prevent RNA degradation.
  • RNase-AP RNase protection sequence
  • the binding position between the RNase protection sequence (RNase-AP) and the target RNA is the RNase recognition site on the target RNA.
  • a protection method for preventing RNA degradation comprising the following steps: forming a complex by combining RNase protection sequence (RNase-AP) with RAN enzyme, affecting the activity of RNase, inhibiting the ability of RNase to combine with target RNA, preventing The target RNA is degraded.
  • RNase-AP RNase protection sequence
  • the binding position of the RNase protection sequence (RNase-AP) and the RAN enzyme is the RNase recognition site on the RAN enzyme.
  • a protection method for preventing RNA degradation comprising the following steps: after combining with the target RNA through the RNase protection sequence (RNase-AP), the local structural change of the target RNA is caused, so that the binding efficiency of the RNase and the target RNA is reduced or the binding position Changed, so that RNase cannot cut and degrade the target RNA, preventing the target RNA from being degraded.
  • RNase-AP RNase protection sequence
  • the binding position between the RNase protection sequence (RNase-AP) and the target RNA is the RNase recognition site on the target RNA.
  • An RNase protection sequence using the sequence of the RNase recognition site as the RNase protection sequence.
  • a virus detection composition comprising the RNase protection sequence.
  • a virus detection kit comprising the virus detection reagent.
  • the present invention provides a protection method, a protection sequence, a composition and a kit for preventing RNA degradation, and applications thereof, so as to improve the protection effect on viral RNA.
  • the RNase AP protection sequence is clearly added, the three-dimensional structure of the target protected RNA and the structural allosteric properties of the RNA can achieve a better protection effect on the integrity of the RNA, and a high-level detection rate can be obtained by one-time amplification. Ensuring efficient enrichment of viral RNA, providing guarantee for subsequent reverse transcription and PCR amplification.
  • Figure 1 shows the fluorescence quantitative detection of plasmid cDNA at different concentrations, and the minimum detected copy number is 1ag (15 copies)/ ⁇ l.
  • Figure 2 shows the repeatability of virus cDNA: RT-PCR (15 copies/ml) detection, 1ag plasmid cDNA was repeated 10 times and negative control was repeated 10 times, and no false positives were found in 50 cycles of Q-PCR amplification.
  • Figure 3 shows that the minimum copy number of viral RNA standard RT-PCR detection is 80 copies/ml.
  • Figure 4 shows that the minimum copy number is 80 copies/ml by adding viral RNA standard to the mock sample of saliva.
  • Figure 5 compares the detection effects of commercial kit A and commercial kit B in saliva samples of viral RNA standards.
  • Figure 6 shows that common bacteria do not interfere with the detection of COVID-19 virus RNA.
  • Figure 7 shows that the CT value of the ORF gene detected in the extract of Wenzhou Medical University is significantly lower than that of the commercial extraction kit A extract.
  • Figure 8 is a schematic diagram of the secondary structure of the 500 nt fragment of the N gene before and after AP binding.
  • Figure 9 is a schematic diagram of the secondary structure of the 500 nt fragment of the ORF1ab gene before and after AP binding.
  • the SARS-CoV-2 new coronavirus is now used, and the protection method for preventing RNA degradation according to the present invention is used to further verify the effect:
  • a primer sequence for a new RNA protection site is designed for the target gene of the new coronavirus. And optimize the distribution ratio of lysis/protection components, compound reagent concentration, salt ion concentration, pH, etc. in the sample treatment solution, improve the reaction system of the new lysis solution, and further improve the protection effect of the lysis solution/protection solution on viral RNA.
  • an effective protective agent After adding an effective protective agent, the three-dimensional structure of the target-protected viral RNA and the structural allosteric properties of the viral RNA were determined, and the effective identification of the SARS-CoV-2 virus and the protective effect on the viral RNA of the sample treatment solution in the kit of this project were studied. and mechanism.
  • Composition ratio of sample treatment solution 1mmol/L 2-(N-morpholine)ethanesulfonic acid, 100mmol/L NaCl, 100mmol/L KCl, 10mmol/L Tris-HCl, 5mol/L guanidine hydrochloride, 1% Triton X-100, 0.1mg/ml proteinase K, 0.1mg/ml diatomaceous earth, specially designed 20nM N gene protection sequence 1 (anchor primer, AP, see the second part of the protection sequence information for AP-WHN-1, ) and a specially designed 20nM ORF1ab1ab protection sequence 2 (AP-WHORF1ab-1, see section II for protection sequence information).
  • RNASE1, RNASE3, RNASE7 had a strong binding probability with the core sequence of the N gene (ORF1ab gene), which may be its potential role Target, call catRAPID to predict the possible binding position of RNase RNASE1-7 and the core sequence of N gene (ORF1ab gene), and find that the protective sequence position can interact with RNase.
  • RNA structure was used to predict the RNA secondary structure before and after the combination of the protective sequence of the core sequence of the virus N gene (ORF1ab gene). Further, the RNA secondary structure local alignment algorithm RNAsmc was used to compare the structural similarity. The results showed that the RNA structure similarity score of the core sequence of the N gene (ORF1ab gene) before and after the combination of the protective sequence was 7.70 (1-10 scale) (ORF1ab gene, 9.12), indicating that the protective sequence was very important to the core sequence of the N gene (ORF1ab gene). The overall structural state of the sequence has a great influence.
  • the PSMAlign structure alignment tool was used to quantify the impact of the binding region on the RNA structure of the N gene (ORF1ab gene) core sequence ( Figure 8B and Figure 9B). ).
  • the PSMAlign result score of 0 represents no change in structure, on the contrary, the higher the score, the more obvious the difference.
  • the RNA structure similarity of the N gene core sequence before and after the combination of the protection sequence was scored as N gene 148 (ORF1ab gene, 18), indicating that the protection sequence has a greater impact on the structure of the N gene, and this result is consistent with the RNAsmc evaluation results.
  • the structural changes of the core sequence RNA of the N gene (ORF1ab gene) may cause abnormal molecular interactions.
  • RNase 1-7 has a strong binding probability with the core region of the N gene (ORF1ab gene) and can bind to the N gene (ORF1ab gene) at the protective sequence, and after the protective sequence is combined with the N gene (ORF1ab gene) , has a great impact on the structure of the N gene (ORF1ab gene), and the optimal interaction position with RNase has been significantly changed, indicating that the structural change of the core sequence RNA of the N gene (ORF1ab gene) may cause abnormal molecular interactions .
  • the kit's sample treatment solution By improving the compatibility of the kit's sample treatment solution and the kit, using specific reagents to directly inactivate the virus, and studying the effects of the lowest and optimal concentrations on the sensitivity and specificity of the kit's detection. Compare and analyze the advantages and disadvantages between the sample treatment solution of this kit and the current RNA preservation solution, such as storage time, storage conditions, storage effect, etc.
  • the sample treatment solution of this kit is intended to be used for the preservation of clinical harmful biological specimens (viruses, bacteria, etc.), and its inactivation effect on the virus must be clear; Virus inactivation effect under different temperature conditions in 30 minutes, avoiding the infection problem of testing personnel.
  • the sample treatment solution of this kit contains 5 mol/L guanidine hydrochloride. Guanidine salt will destroy the structure of the sugar coat protein of SARS-CoV-2 virus at room temperature and has the effect of inactivating SARS-CoV-2 virus. When the standard RNA was incubated at 60°C for 5, 10, 20, and 30 minutes, the RNA was not degraded. In addition, this kit has been found in clinical tests that the RNA of SARS-CoV-2 virus can still be extracted after incubation at 60°C for 30 minutes. In this way, this kit has the dual effects of guanidine salt and 60°C warm bath (the guidelines of the National Health Commission specify that 56°C, 30min can inactivate SARS-CoV-2 virus).
  • This kit adds SARS-CoV-2 viral plasmid DNA and viral RNA standard in saliva to obtain the minimum detection sensitivity of 15 copies/ml (cDNA) and 80 copies/ml (RNA standard; the current clinical kit is ⁇ 500 copies/ml), with high detection sensitivity.
  • test results of this kit in 252 clinically confirmed COVID-19 positive cases show that the Wen Medical University kit has a good protective effect on RNA integrity under the action of AP of a single gene (N gene), and realizes one-time expansion.
  • a high level of detection rate (80%) can be obtained by increasing.
  • This kit detects the interference of common flora (including Streptococcus pneumoniae, Streptococcus pyogenes, Klebsiella pneumoniae, Candida albicans, Staphylococcus aureus, Haemophilus influenzae and Aspergillus fumigatus) on the detection system. It was found that common bacteria did not interfere with the detection of SARS-CoV-2 viral RNA.
  • common flora including Streptococcus pneumoniae, Streptococcus pyogenes, Klebsiella pneumoniae, Candida albicans, Staphylococcus aureus, Haemophilus influenzae and Aspergillus fumigatus
  • the sample treatment solution of this kit can process a variety of samples, increase the ability of the sample treatment solution to lyse viruses in complex samples and release viral RNA, and improve the protection effect of viral RNA; at the same time, the adaptation conditions of this kit are optimized. Improve the corresponding components of the washing solution, effectively remove the interference of other components in complex samples, ensure efficient enrichment of viral RNA, and provide guarantee for subsequent reverse transcription and PCR amplification.
  • This kit can process a variety of SARS-CoV-2 samples, such as nasal swabs, throat swabs, sputum, urine, feces, conjunctival sac swabs and tears.
  • Clinical test findings such as nasal swabs, throat swabs, sputum, urine, feces, conjunctival sac swabs and tears.
  • Feces Pick a micro sample and put it into the sample treatment solution, incubate at 60°C for 30 minutes, centrifuge at 12,000 rpm for 1 minute, and take the supernatant for extraction on the column.
  • this kit can inactivate the virus in this step; on the other hand, it can effectively lyse the virus to release the SARS-CoV-2 viral RNA, and the AP in the sample treatment solution immediately exerts its protective effect.
  • Urine First, centrifuge the collected clinical urine samples at 12,000 rpm for 2 minutes, remove 200-300 ul of urine from the lower layer, and add 500 ul of sample treatment solution. This step will further concentrate the epithelial cells in the urine, degrading them all for sample processing. This is extremely critical for micro sample processing.
  • This kit sets a specific protection region for the protection sequence region, SARS-CoV-2.
  • Positive control 3 ⁇ l (full-length plasmid of N gene);
  • cycle parameter setting please refer to the instrument operating instructions for setting
  • the threshold setting principle is that the threshold line exceeds the highest point of the negative control and is in the initial stage of the exponential amplification phase of the positive control.
  • the quality control procedure is to monitor both negative and positive control assays.
  • the test results are valid only if the negative control and positive control results are in compliance with the interpretation of the test results.
  • Re-examination is required: 40 ⁇ Ct value ⁇ 45, if the Ct value ⁇ 45 is re-examined, it is judged as a positive result.
  • the high-precision novel coronavirus (SARS-CoV-2) detection kit is based on the development technology of the predecessor cancer early detection kit (RNA detection).
  • RNA detection cancer early detection kit
  • compatibility studies were carried out in throat swabs, nasal secretions and feces.
  • the reagents use guanidine salts to treat the samples. Incubating the samples at 600°C for 30 minutes can completely inactivate the virus. Subsequent studies will shorten the time to 10 minutes.
  • triple PCR or multiple PCR methods are used to simultaneously conduct multiple gene fragments such as the open reading frame and N gene of the coronavirus (SARS-CoV-2). Test to avoid false negatives caused by virus mutation.
  • SARS-CoV-2 coronavirus
  • the identification of multi-locus RNA sequences protects the integrity of RNA, avoids the problem of RNA degradation caused by the sample processing process, and solves the false positive problem by optimizing the primer probe, so that the system can determine the positive detection of any locus of the new coronavirus. positive for the initial screening.
  • Multi-target genes are used for protection, and multiple gene sequences are detected. At the same time, the mutation of the virus should be considered to prevent missed detection due to mutation.
  • This kit has released RNA into the sample treatment solution at the same time as the sample is obtained.
  • the sample treatment solution can protect the integrity of the RNA very efficiently, and at the same time, it is effective for multiple sites of the released RNA samples.
  • the design of effective RNA protection has also been carried out in subsequent detection operations, and has been verified in a number of products developed.
  • RNA samples have been specifically and effectively recognized multiple target gene loci during the sample processing process and formed complexes with them, stably targeting the new coronavirus RNA, and enriched by selective reverse transcription.
  • the self-designed primers and probes are significantly better than the probes and primer sequences published by the National Center for Disease Control (bioinformatics analysis found that there is no secondary structure such as hairpins). ); by designing primers and probes for the dual gene targets of ORF1ab and N gene, and detecting these two sites at the same time, the sensitivity and specificity of the kit will be greatly increased.
  • the sample treatment solution can directly store the collected virus samples, and the sample treatment solution contains guanidine salts, which can effectively and quickly inactivate the virus.
  • the sample treatment solution contains guanidine salts, which can effectively and quickly inactivate the virus.
  • self-designed materials, the three-dimensional structure of the target-protected RNA and the structural allosteric properties of the RNA are added to the sample treatment solution, so that the target gene can be specifically recognized and protected, and the detection sensitivity and specificity are increased.
  • the unique reaction system of the kit the use of innovative lysate and reagents makes this detection kit have four characteristics: high sensitivity (100 times higher than that of Shanghai “Zhijiang Bio"), high specificity, rapidity (from the sample 40-45 minutes from preparation to result) and double direct inactivation of virus to avoid infection of testing personnel. .
  • Rapid sample preparation to detection 40 minutes: The detection steps of the kit currently developed by our team have four steps: release, identification (5 minutes) - washing (1 minute) - elution (2 minutes) - signal amplification (35 minutes). The detection time of throat swab samples can achieve results in 45 minutes. The time can be shortened to 40 minutes if used in conjunction with an automatic nucleic acid extractor.
  • this kit can process various types of samples, such as nasal/pharyngeal swabs, sputum, saliva, feces and urine, etc.
  • trace virus samples that may exist in tears, conjunctival sac swabs, etc. are being verified middle. The detailed progress report is as follows:
  • the cooperative unit (Taizhou Enze Hospital) compared the "warmth" of 33 positive samples.
  • the RNA extracted from the lysate/protection solution of the Medical University and Zhijiang Biological kits were both amplified and detected by RT-PCR with the Zhijiang nucleic acid detection kit. The clinical test results showed that:
  • the sample treatment solution of this kit contains guanidine salts, which have the effect of inactivating viruses (proven by reports).
  • virus inactivation can be achieved at 60°C for 30 minutes. Virus inactivation can effectively reduce the exposure risk of testing personnel, protect the safety of operators, and reduce the risk of infection in testing laboratories.
  • the detection step of this kit has four steps: release, identification (5 minutes) - washing (2 minutes) - elution (1 minute) - signal amplification (35 minutes).
  • the detection time of throat swab samples can achieve results in 45 minutes.
  • the time can be shortened to 40 minutes if used in conjunction with an automatic nucleic acid extractor.

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Abstract

提供了一种防止RNA降解的保护方法、保护序列、组合物和试剂盒及其应用，其能够提高对病毒RNA的保护效应。加入RNase AP保护序列后，可以保护的RNA的三维结构以及RNA的结构变构特性，对RNA完整性有较好的保护作用，实现一次性扩增即能得到高水平的检出率，确保对病毒RNA的高效富集，为后续逆转录和PCR扩增提供保障。

Description

一种防止RNA降解的保护方法、保护序列、组合物和试剂盒及其应用 技术领域
本发明涉及体外核酸检测技术领域，具体涉及一种防止RNA降解的保护方法、保护序列、组合物和试剂盒及其应用。
背景技术
核糖核酸(Ribonucleic Acid,RNA)，存在于生物细胞以及部分病毒、类病毒中的遗传信息载体，RNA只有单链，不稳定，极易被RNA酶降解。RNA酶(RNase)活性非常强，自然界中存在有大量的RNA酶。为了防止RNA酶对RNA的降解，提高实验或检验过程中目的RNA的得率，研究和开发各种RNA提取技术和方法意义重大。
发明内容
为了解决现有技术存在的问题，本发明提供了一种防止RNA降解的保护方法、保护序列、组合物和试剂盒及其应用，进一步提高对病毒RNA的保护效应。
本发明采用的技术解决方案是：一种防止RNA降解的保护方法，包括以下步骤：通过RNA酶保护序列(RNase-AP)与目的RNA的高效结合后形成RNA:DNA复合物，结合使得RNA暴露区域减少，从而使RNA酶不能有效切割目的RNA防止RNA降解。
所述的RNA酶保护序列(RNase-AP)与目的RNA结合位置为目的RNA上的RNA酶识别位点。
一种防止RNA降解的保护方法,包括以下步骤：通过RNA酶保护序列(RNase-AP)与RAN酶结合形成复合物，影响了RNA酶的活性，抑制了RNA酶与目的RNA结合的能力，防止目的RNA被降解。
所述的RNA酶保护序列(RNase-AP)与RAN酶结合位置为RAN酶上的RNA酶识别位点。
一种防止RNA降解的保护方法,包括以下步骤：通过RNA酶保护序列(RNase-AP)与目的RNA结合后，引起目的RNA的局部结构改变，使得RNA酶与目的RNA结合的效率降低或结合位置改变，使得RNA酶不能对目的RNA进行切割降解，防止目的RNA被降解。
所述的RNA酶保护序列(RNase-AP)与目的RNA结合位置为目的RNA上的RNA酶识别位点。
一种RNA酶保护序列,采用RNA酶识别位点的序列作为RNA酶保护序列。
一种RNA酶保护序列在作为制备病毒检测试剂上的应用。
一种病毒检测组合物，包括所述的RNA酶保护序列。
一种病毒检测试剂盒，包括所述的病毒检测试剂。
本发明的有益效果是：本发明提供了一种防止RNA降解的保护方法、保护序列、组合物和试剂盒及其应用，提高对病毒RNA的保护效应。明确加入RNase AP保护序列后，目标保护的RNA的三维结构以及RNA的结构变构特性，实现对RNA完整性有较好的保护作用，实现一次性扩增即能得到高水平的检出率，确保对病毒RNA的高效富集，为后续逆转录和PCR扩增提供保障。
附图说明
图1为不同浓度的质粒cDNA的荧光定量检测，最低检出拷贝数1ag(15拷贝)/μl。
图2为病毒cDNA:RT-PCR(15拷贝/ml)检出的重复性，1ag质粒cDNA重复10次和阴性对照重复10次，Q-PCR扩增50个循环未见假阳性。
图3为病毒RNA标准品RT-PCR检测最低拷贝数为80拷贝/ml。
图4为在唾液的模拟样本中加入病毒RNA标准品检测最低拷贝数为80拷贝/ml。
图5为比较商业试剂盒A和商业试剂盒B在病毒RNA标准品在唾液样本中的检测效果。
图6为常见细菌对COVID-19病毒RNA检测无干扰作用。
图7为温医大”提取液检出的ORF基因的CT值显著低于商业提取试剂盒A提 取液。
图8为N基因500nt片段在AP结合前后的二级结构的示意图。
图9为ORF1ab基因500nt片段在AP结合前后的二级结构的示意图。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
现采用SARS-CoV-2新型冠状病毒，采用本发明所述的防止RNA降解的保护方法对效果进行进一步验证：
(一)新型样品处理液对SARS-CoV-2病毒的有效识别和对病毒RNA的保护作用及机制。
针对新型冠状病毒的目标靶点基因，设计新的RNA保护位点的引物序列。并优化样本处理液中裂解/保护各组分配比，复合试剂浓度，盐离子浓度，PH等，完善新型裂解液反应体系，进一步提高裂解液/保护液对病毒RNA的保护效应。明确加入有效保护剂后，目标保护的病毒RNA的三维结构以及病毒RNA的结构变构特性，研究本项目试剂盒中样本处理液对SARS-CoV-2病毒的有效识别和对病毒RNA的保护作用及机制。
1.样品处理液的组分配比：1mmol/L 2-(N-吗啡啉)乙磺酸,100mmol/L NaCl，100mmol/L KCl,10mmol/L Tris-HCl，5mol/L盐酸胍，1％Triton X-100，0.1mg/ml蛋白酶K，0.1mg/ml硅藻土,特定设计的20nM的N基因保护序列1(anchor primer,AP，保护序列信息详见第二部分AP-WHN-1,)和特定设计的20nM的ORF1ab1ab保护序列2(AP-WHORF1ab-1，保护序列信息详见第二部分)。
2.作用机制：首先，AP与SARS-CoV-2病毒RNA结合后，引起病毒RNA的局部结构改变，使得RNA酶与病毒RNA结合的效率降低或结合位置改变，使得RNA酶不能对病毒RNA进行切割降解。其次，RNase-AP首先与SARS-CoV-2病毒RNA的高效结合后形成RNA:DNA复合物，这样的竞争结合使得RNA暴露区域减少，RNA酶不能有效切割。最后，RNase-AP可能与RAN酶结合形 成复合物，这样的结合竞争抑制了与病毒RNA结合的能力，影响了RNase的活性。
(二)引物/探针的特异性优化，提高试剂盒的特异性，有效减少假阳率，提高检出率；参照新型冠状病毒的结构，设计新型特异性引物/探针应对当前病毒可能出现变异的情况。
通过设计有针对SARS-CoV-2病毒的ORF1ab1ab和N基因双重基因靶点的探针，同时检测，实现对靶向病毒RNA的检测的特异性。增加对ORF1ab其他区域的引物设计，实现多基因、多位点的检测，有效提高试剂盒的检出率。同时，详细分析不同类型冠状病毒同源性，结合针对2019新型冠状病毒结构生物学和生物信息学的最新研究结果，针对其高度保守区域设计新的引物探针，应对2019新型冠状病毒可能发生的变异，实现检测试剂盒的即时更新换代，确保检测准确性。
通过生物信息学分析，我们首先针对SARS-CoV-2的N基因(基因坐标2889-2997,图9A)和ORF1ab1ab基因(基因坐标28548-28763,图8A)，设计了特定的保护序列，如表1所示AP-WHORF1ab-1,AP-WHN-1。
表1、
Figure PCTCN2020112399-appb-000001
1.RNA与RNase-AP结合后，SARS-CoV-2病毒RNA的生物信息学的结构预测(图8B和图8B)，SARS-CoV-2病毒RNA与RNA酶的结合预测研究(图1C和图2C)。
1)首先我们调用RPIseq随机森林和支持向量机算法，对保护序列与人类RNA 酶的结合特性进行评价。通过对RNA酶RNASE1-7与N基因(ORF1ab基因)核心序列的亲合力打分结果，发现RNASE1、RNASE3、RNASE7与N基因(ORF1ab基因)核心序列有较强的结合概率，可能是其潜在的作用靶点，调用catRAPID对RNA酶RNASE1-7与N基因(ORF1ab基因)核心序列可能的结合位置进行预测，发现保护序列位置可以与RNA酶进行互作。
进一步采用最小自由能预测算法，利用RNAstructure分别实现病毒N基因(ORF1ab基因)核心序列保护序列结合前后的RNA二级结构预测。进一步使用RNA二级结构局部比对算法RNAsmc进行结构相似度比较。结果显示，保护序列结合前后的N基因(ORF1ab基因)核心序列RNA结构相似性得分N基因为7.70(1-10分制)(ORF1ab基因，9.12)，表明保护序列对N基因(ORF1ab基因)核心序列整体结构状态影响较大，为进一步验证引物对N基因(ORF1ab基因)的作用，使用PSMAlign结构比对工具量化结合区域对N基因(ORF1ab基因)核心序列RNA结构的影响(图8B和图9B)。PSMAlign结果分值0代表结构无变化，相反，分值越高，差异越明显。经计算，保护序列结合前后的N基因核心序列RNA结构相似性打分为N基因148(ORF1ab基因,18)，表明保护序列对N基因结构有较大影响，且这一结果与RNAsmc评价结果一致，N基因(ORF1ab基因)核心序列RNA的结构变化可能引起分子互作异常。
2)进一步利用RNAComposer对N基因(ORF1ab基因)核心区域进行3D结构预测，使用HDOCK SERVER进行RNA酶RNASE1-7与N基因(ORF1ab基因)核心序列的互作区域进行预测(图8C和图9C)，结果表明在结合保护序列后，N基因(ORF1ab基因)核心序列与RNA酶的最优互作位置都发生了明显的改变，进一步表明N基因(ORF1ab基因)核心序列RNA的结构变化可能引起分子互作异常。结果表明RNA酶1-7与N基因(ORF1ab基因)核心区域与存在较强结合概率，可以在保护序列处与N基因(ORF1ab基因)进行结合，在保护序列与N基因(ORF1ab基因)结合后，对N基因(ORF1ab基因) 结构有较大影响，并且与RNA酶的最优互作位置都发生了明显的改变，表明N基因(ORF1ab基因)核心序列RNA的结构变化可能引起分子互作异常。
(三)实现试剂盒样本处理液对SARS-CoV-2病毒的灭活处理，将其开发成为一种新型的病毒RNA保存液；并探讨其在临床检验过程中生物安全性问题。
通过对试剂盒的样本处理液与试剂盒的兼容性进行改善，使用特定的试剂直接对病毒进行灭活，研究其最低和最佳浓度对试剂盒检测的灵敏性和特异性的影响。比较分析本试剂盒样本处理液和目前RNA保存液之间的优劣性，如保存时间，保存条件，保存效果等。拟将本试剂盒样本处理液用于临床有害生物标本(病毒，细菌等)的保存，必须明确其对病毒的灭活作用；针对2019新型冠状病毒，用通过60℃孵育5，10，20，30分钟不同温度条件下病毒灭活效果，避免检测人员的感染问题。
本试剂盒样本处理液含有5mol/L盐酸胍，胍盐在常温下会破坏SARS-CoV-2病毒的糖衣壳蛋白的结构，具有SARS-CoV-2病毒灭活作用。本试剂盒在对标准品RNA进行60℃孵育5，10，20，30分钟时，RNA无降解。此外，本试剂盒在临床测试中发现，60℃孵育30分钟仍能提取到SARS-CoV-2病毒的RNA。这样本试剂盒具有胍盐和60℃温浴(国家卫健委指南明确56℃，30min可灭活SARS-CoV-2病毒)的双重灭和作用。
本试剂盒在唾液中加入SARS-CoV-2病毒质粒DNA和病毒RNA标准品分别获得最低检测灵敏性为15拷贝/ml(cDNA)和80拷贝/ml(RNA标准品；目前临床试剂盒为～500拷贝/ml)，具有较高的检测灵敏性。
本试剂盒在252例临床确证的COVID-19阳性病例中测试结果中表明温医大试剂盒在单个基因(N基因)的AP作用下，对RNA完整性有较好的保护作用，实现一次性扩增即能得到高水平的检出率(80％)。
本试剂盒检测常见菌群(包括肺炎链球菌，化脓性链球菌，肺炎克雷伯菌，白色念珠菌，金黄色葡萄球，流感嗜血杆菌和烟曲霉菌)对检测体系的干扰作用。发现常见细菌对SARS-CoV-2病毒RNA检测无干扰作用。
(四)提高对病毒RNA的保护作用，实现对复杂样本的检测。
本试剂盒样本处理液对多种样本进行处理，增加样本处理液对复杂样本中病毒裂解及病毒RNA释放能力，提高对病毒RNA的保护功效；同时，对本试剂盒的适配条件做优化处理，改进洗涤液相应成分，有效去除复杂样本中其他成分的干扰，确保对病毒RNA的高效富集，为后续逆转录和PCR扩增提供保障。
1.本试剂盒可以处理多种SARS-CoV-2样本，如鼻拭子，咽拭子，痰液，尿液，粪便，结膜囊拭子及泪液等。临床测试发现
Figure PCTCN2020112399-appb-000002
2.特殊样本的处理：
1)粪便：挑取微量样本放入样本处理液中，60℃孵育30分钟，12000rpm离心1分钟，取上清液上柱提取。本试剂盒在此步骤一方面可以灭活病毒；另外一方面可以有效的裂解病毒释放SARS-CoV-2病毒RNA，样本处理液中的AP立即发挥其保护作用。
2)尿液：首先对收集到的临床尿液标本进行离心处理，12000rpm离心2分钟， 取下层200-300ul尿液，加入500ul样本处理液。此步骤将进一步浓缩尿液中的上皮细胞，降解全部进行样本处理。这对于微量样本处理异常关键。
(五)试剂盒的引物及探针。
本试剂盒针对保护序列区域设定特定保护区域，SARS-CoV-2。
N保护序列 cctcttctcgttcctcatcacgtagtcgcaacagttcaa
WHN1-F2 5’-CAAGCCTCTTCTCGTTCCT-3’
WHN1-R2 5’-GCAGCAGATTTCTTAGTGACAG-3’
N探针 5’-FAM-ATTCAACTCCAGGCAGCAGTAG-BHQ1-3’
ORF1ab保护序列 gtcctcactgccgtcttgttgaccaacagtttgttgact
WHO1-F2 5’-GCCACTTCTGCTGCTCTTC-3’
WHO1-R2 5’-tgattgtcctcactgccgtc-3’
ORF1ab探针 5’-VIC-CAACCTGAAGAAGAGCAAGAA-MGB-3’
检测步骤：
1.实验前准备：需确保实验设施器材具备，金属浴、水浴锅等温度务必经过校准验证。
2.样本处理
Figure PCTCN2020112399-appb-000003
Figure PCTCN2020112399-appb-000004
照：ddH 2O；
阳性对照：3μl(N基因全长质粒)；
均按上述处理流程第4和第5步操作。
*粪便标本需要先12000rpm离心1min，取上清液进行60℃孵育10min后续实验。
**洗脱液在使用前需在60℃温浴10～15分钟。
3.PCR扩增
在荧光定量PCR仪上进行如下程序：循环参数设定(请参考仪器操作说明进行设置)
Figure PCTCN2020112399-appb-000005
*阈值设定原则以阈值线超过阴性对照的最高点，并且处于阳性对照的指数扩增期初始阶段为准。
质量控制
质量控制程序为同时进行阴性对照和阳性对照检测监控。在阴性对照和阳性对照结果均符合检验结果的解释所规定的情况下，检测结果方为有效。
检验结果的解释：
阴性(-)：无Ct值或Ct值≥45
阳性(+)：Ct值≤40。
需复查：40＜Ct值＜45，若再次复查Ct值＜45，判断为阳性结果。
(1)病毒样本灭活处理
高精准新型冠状病毒(SARS-CoV-2)检测试剂盒基于前身癌症早期检测试剂盒(RNA检测)开发技术，前期对采集样本的生物安全性风险已做了技术上的优化和改进，在此基础上将现有的技术在咽拭子、鼻腔分泌物及粪便等中进行兼容性研究。试剂采用胍盐对样本进行处理，样本600℃，30分钟孵育可完全将病毒灭活，后续研究会将时间缩短至10分钟。
(2)试剂盒检测的稳定性
通过对裂解释放出的RNA样本进行多个位点有效识别并形成复合物，采用三重PCR或多重PCR方法同时对冠状病毒(SARS-CoV-2)的开放阅读框、N基因等多段基因片段进行检测，避免病毒因变异导致的假阴性存在。对多位点RNA序列识别保护RNA完整性，避免样本处理过程导致RNA降解问题，通过对引物探针的优化解决假阳性问题，使体系针对新冠状病毒任何一个基因位点检出阳性都可以判定为初筛阳性。采取多靶点基因进行保护，多个基因序列进行检测同时需考虑病毒的变异问题防止因变异导致的漏检。
RNA易降解问题：本试剂盒在样本获取的同时已将RNA释放在样本处理液中，样本处理液可非常高效的对RNA的完整性进行保护，同时对释放的RNA样本进行多个位点有效识别并形成复合物，在后续的检测操作中也均进行了RNA有效保护的设计，并在多个研发的产品中得以验证。
试剂盒的灵敏度及特异性：在样本处理过程中RNA样本已被特异有效识别多个靶点基因位点并与之形成复合物，稳定靶向新冠病毒RNA，通过选择性逆转录富集后特异的RNA复合物，实现对多靶点RNA扩增；自行设计的引物、探针明显优于国家疾病控制中心公布了的探针和引物序列(生物信息学分析发现没有发夹等二级结构存在)；通过设计ORF1ab和N基因双重基因靶点的引 物及探针，同时对这两个位点进行检测，试剂盒的敏感性和特异性会大大的增加。
本发明的优点：
(1)创新裂解液与试剂盒独特的反应体系：样本处理液可直接保存采集的病毒样本，样本处理液中包含了胍盐可有效快速灭活病毒。同时在样本处理液中加入可有自行设计的材料，目标保护的RNA的三维结构以及RNA的结构变构特性，使目的基因被特异识别及保护，增加检出的灵敏度及特异性。
(2)试剂盒独特的反应体系：创新裂解液与试剂配套使用使本检测试剂盒具有四大特点：高灵敏度(较上海“之江生物”提高100倍)、高特异性、快速(从样本制备到出结果40-45分钟)和双重直接灭活病毒以避免检测人员感染。。
(3)独特样本处理液可对复杂性样本(粪便和痰液)有效处理：在技术开发中已对痰液样本进行验证，技术开发初期在临床样本上处理效果非常理想，并采用优化前的试剂开发出一款产品：肺癌早期检测试剂盒(国械注准20173403247)。我们可将现有的样本处理方式在粪便等样品检测中临床应用。
(4)快速样本制备到检测(40分钟)：本我们团队目前开发的试剂盒检测步骤有四步：释放、识别(5分钟)-洗涤(1分钟)-洗脱(2分钟)-信号放大(35分钟)。对咽拭子样本检测时间可实现45分钟出结果。如果配套使用核酸自动提取仪使用可将时间缩短至40分钟。
结论：
(1)在唾液中加入COVID-19病毒质粒DNA和病毒RNA标准品分别获得最低检测灵敏性为15拷贝/ml(cDNA)和80拷贝/ml(RNA标准品；目前临床试剂盒为～500拷贝/ml)；
(2)临床252例COVID-19阳性病人标本检测结果表明温医大试剂盒对RNA完整性(N基因位点)有较好的保护作用，实现一次性扩增即能得到高水平的检出率(80％)。
(i)台州恩泽医疗中心(57例)(CT值有待重新统计分析)：“温医大”提取液检出的ORF基因的CT值显著低于“之江生物”提取液，ORF基因检出CT值平均降低2.27个循环，提示灵敏度提高～10倍(p＝0.0001配对t-test，n＝33)，而N基因与E基因也有所降低(p＝0.0074和p＝0.6894)；
(ii)温州CDC(59例)：“温医大”提取液+扩增液检出的N和E基因的CT值显著低于其他临床试剂盒(N基因降低3.74个循环，p＝0.0001，E基因降低2.0个循环，p＝0.002，n＝59)；
(iii)浙江省CDC(54例)：“温医大”提取液+扩增液检出的N和E基因的CT值显著低于其他临床试剂盒(N基因降低3.79个循环，p<0.0001，E基因降低XX个循环，p＝0.002，n＝59)；
(iv)温医大附一院附二院(54例轻症)：“温医大”提取液+之江扩增液检出的，以及转阴后复阳4例7.4％(4/54)。
(ⅴ)温医大附一院(11例尿液)：尿液检出率36.36％(4/11)。
(ⅵ)本试剂盒一次检出COVID-19(N基因)阳性率：台州70.18％(40/57)；温州CDC:N基因80.87％(97/115)，；浙江省CDC：N基因78.67％(59/75)。温医大附二院：54例临床轻症/普通型(恢复期转阳)：4例7.4％(4/54)。
(3)因此，本试剂盒可以处理各种类型的样本，如鼻/咽拭子，痰液，唾液，粪便和尿液等，另泪液，结膜囊拭子等可能存在的微量病毒标本正在验证中。详细进展汇报如下：
(一)伦理：获得温州医科大学附属眼视光医院伦理委员通过。
(二)COVID-19假病毒cDNA/RNA试验得出温医大试剂盒检测最低拷贝数约15拷贝/ml(cDNA标准品)和80拷贝/ml(RNA标准品)，显示其较目前临床试剂盒较高的灵敏性，可检测出病人样本中较低的病毒滴度，有效降低假阴性 率。
2.1.COVID-19假病毒质粒cDNA检出最低拷贝数：在痰液/唾液的模拟样本中，加入假病毒质粒cDNA(厦门致善生物科技股份有限公司提供)，经过整个裂解/提取和RT-PCR反应步骤，我们得出本试剂盒的质粒cDNA的最低拷贝数约1ag(15拷贝)/μl(图一)(目前临床试剂盒为～500拷贝/ml)。
2.2.COVID-19假病毒质粒cDNA最低拷贝数检出的重复性：在痰液/唾液的模拟样本中，加入最低检出假病毒质粒cDNA15拷贝/ml(10个重复管)，经过整个裂解/提取和RT-PCR反应步骤，本试剂盒在最低拷贝数时有良好重复性(图二)
2.3.用COVID-19病毒RNA标准品验证RT-PCR反应步骤的最低检出拷贝数：在RT-PCR反应体系(图三)和唾液的模拟样本中(图四)，加入新冠病毒RNA标准品(中国计量研究研究提供)验证RT-PCR反应步骤的最低检出拷贝数为80拷贝/ml(目前临床试剂盒为～500拷贝/ml)
2.4.平行比较研究发现：在痰液/唾液的模拟样本中加入新冠病毒RNA标准品(中国计量研究研究提供)8380拷贝/ul,比较“温医大”和“之江生物”试剂盒RT-PCR反应步骤，“温医大”检出CT值为20，荧光信号强度强；而“之江生物”检出CT值为26，并且荧光信号强度弱。
2.5.常见细菌对COVID-19病毒RNA检测无干扰作用
应用温医大提取液+之江RT-PCR扩增体系，检测常见菌群(包括肺炎链球菌，化脓性链球菌，肺炎克雷伯菌，白色念珠菌，金黄色葡萄球，流感嗜血杆菌和烟曲霉菌)对检测体系的干扰作用。实验结果(菌提取核酸重复3次)表明：强阳和弱性对照(红色)出现预计的扩增，而干扰菌均显示无扩增(绿色)。
(三)临床COVID-19病人样本收集统计：
Figure PCTCN2020112399-appb-000006
Figure PCTCN2020112399-appb-000007
3.2.温医大裂解液/保护液稳定保护COVID-19病毒RNA的效果研究
为检测创新裂解液/保护液能特异有效识别多个COVID-19靶点基因位点并形成复合物，稳定保护COVID-19病毒RNA，合作单位(台州恩泽医院)对33例阳性标本比较“温医大“和“之江生物”试剂盒裂解液/保护液提取的RNA，都用之江核酸检测试剂盒进行RT-PCR扩增检测，临床实验结果表明：
(1)临床实验结果表明：COVID-19N基因检出阳性率(95％)，E基因检出阳性率(100％)，ORF/RDRP基因检出阳性率(100％)。这表明本试剂盒对RNA完整性(多个基因位点)有较好的保护作用，为实现一次性扩增来检测多种基因，以提高样本的检出率和准确性(表1)。
住院号/门诊号 性别 年龄 标本类型 检测结果 之江提取/之江检测 温医大提取/之江检测
50151329 男 56岁 痰液 阳性(+) RDRP26.2932/E25.83/N26.29 RDRP 25.79/E 25.85/N 26.7
50151142 男 75岁 痰液 阳性(+) RDRP35.21/34.45/34.91 RDRP 37.35/E 0/N 39.57
50151377 女 67岁 咽拭子 阳性(+) RDRP38.05/E37.48/39.02 RDRP 35.74/E 37.03/N 34.96
50151088 男 55岁 粪便 阳性(+) RDRP37.68/E37.09/N38.70 RDRP 37.65/E 36.94/N 36.8
50151536 女 77岁 鼻咽拭子 阳性(+) rdrp33.3/E35/N33.07 RDRP 28.11/E 29.25/N 27.32
50151345 男 64岁 痰液 阳性(+) rdrp32.9/E30.3/N38.6 RDRP 32.87/E 32.7/N 31.86
50151329 男 56岁 痰液 阳性(+) RDRP30.38/E29.53/N30.21 RDRP 29.47/E 29.12/N 28.46
50151379 男 55岁 鼻咽拭子 阳性 RdRP 38.97/E 38.9/N 37 RDRP 0/E 39.71/N 37.71
50151118 女 65岁 鼻咽拭子 阳性 RdRP 35.25/E 35.21/N 33.51 RDRP 35.15/E 34.33/N 33.95
50151406 男 33岁 痰液 阳性(+) rdrp 35.6/E34.6/N33.5 RDRP 31.58/E 32.11/N 30.69
50150887 男 34岁 痰液 阳性 RDRP 33.9/E29.9/N 30.8 RDRP 32.81/E 32.58/N 32.3
50151409 女 51岁 痰液 待复查 E36、N35.9 RDRP 0/E 36.29/N 43.69
50150812 男 50岁 痰液 阳性(+) rdrp37.4/E35.5/N35 RDRP 35.07/E 34.2/N 33.9
50151541 女 48岁 痰液 阳性(+) rdrp36.99/E35.6/N35.6 RDRP 0/E 0/N 35.14
50151270 女 44岁 咽拭子 阳性 RDRP 35.22/E 31.68/N 32.97 RDRP 35.15/E 32.35/N 31.75
50151536 女 77岁 痰液 阳性(+) RDRP29.8/E28.0/E29.2 RDRP 21.43/E 22.17/N 23.09
50151307 女 68岁 痰液 待复查 E37.9/N38.7 RDRP 31.38/E 31.94/N 31.83
50150765 女 56岁 痰液 阳性(+) RDRP35.6/E32.7/N 33.3 RDRP 35.26/E 34.32/N 33.9
50151436 女 30岁 痰液 阳性(+) RDRP37.46/E34.5/N36.36 RDRP 35.25/E 34.54/N 34.06
50150798 男 43岁 痰液 阳性(+) rdrp36.34/E33.1/N33.03 RDRP 37.03/E 34.43/N 35.89
50151142 男 75岁 痰液 阳性(+) RDRP38.66/E 35.7/N 37.5 RDRP 38.95/E 39.82/N 28.03
50151345 男 64岁 痰液 阳性(+) rdrp34.91/E33.9/N 32.53 RDRP 37.08/E 37.22/N 36.98
9000548489 女 43岁 咽拭子 阴性 RdRP 41.7/E 36.56/N 36.9 RDRP 35.91/E 35.84/N 36.04
50150798 男 43岁 咽拭子 阳性 RdRP 38.30/E 38.14/N 35.78 非标准曲线
50151020 女 26岁 咽拭子 阳性(+) RdRP 38.79/E 37.07/N 37.71 RDRP 33.87/E 34.02/N 34.27
50150887 男 34岁 咽拭子 阳性(+) RdRP 31.61/E 28.56/N 28.78 RDRP 28.86/E 28.92/N 28.44
50151237 男 70岁 咽拭子 阳性 RDRP38.01/E35.94/N35.38 RDRP 33.6/E 35.07/N 34.09
50151166 男 44岁 咽拭子 阳性 RdRP 32.53/E 31.16/N 35.41 RDRP 33.74/E 33.65/N 33.64
50151065 男 51岁 痰液 阳性(+) RDRP37.70/E37.12/N37.94 RDRP 29.08/E 28.45/N 28.47
50150954 女 41岁 咽拭子 阳性 RdRP 33.77/E 32.98/N 39.01 RDRP 32.3/E 32.56/N 32.68
50151017 男 38岁 咽拭子 阳性 RdRP 39.91/E 39.93/N 38.21 非标准曲线
50151406 男 33岁 粪便 阳性 RDRP33.12/E30.88/N32.29 RDRP 23.98/E 25.39/N 24.04
50151406 男 33岁 痰液 阳性 RDRP35.53/E32.72/N34.12 非标准曲线
(2)比较“温医大”和“之江生物”提取液得到COVID-19三个基因片段(ORF，E和N)检测的CT值，结果表明：“温医大”提取液检出的ORF基因的CT值显著低于“之江生物”提取液(p＝0.0067，N基因，p＝0.057；E基因，p＝0.761，配对t-test，n＝33，图七)，ORF基因的CT值平均降低3个循环，经由假病毒标准曲线换算，检出COVID-19 RNA拷贝数提高了～10倍。
3.4.临床符合率
本试剂盒一次检出COVID-19(N基因)阳性率：台州70.18％(40/57)；温 州CDC:N基因80.87％(97/115)，；浙江省CDC：N基因78.67％(59/75)。温医大附二院：54例临床轻症/普通型(恢复期转阳)：4例7.4％(4/54)。3.5.“温医大”提取液可处理多种，复杂COVID-19病人样本：
Figure PCTCN2020112399-appb-000008
3.6.COVID-19病毒灭活与安全性
本试剂盒样本处理液中含有胍盐，胍盐具有灭活病毒作用(已有报道证实)。此外，样本处理阶段可实现60℃，30min对病毒的灭活作用。病毒灭活可以有效的减少检测人员的暴露风险，保护操作人员的安全性，降低检验实验室感染风险。
3.7.快速样本制备到检测(40分钟)：
本试剂盒检测步骤有四步：释放、识别(5分钟)-洗涤(2分钟)-洗脱(1分钟)-信号放大(35分钟)。对咽拭子样本检测时间可实现45分钟出结果。如果配套使用核酸自动提取仪使用可将时间缩短至40分钟。
3.8.检测敏感性
本试剂盒离体实验中发现COVID-19病毒质粒DNA和病毒RNA标准品分别获得最低检测灵敏性为15拷贝/ml(cDNA)和80拷贝/ml，具有较低的检测线。此外，在温州医科大学附属第一、二医院的临床检测发现，在54例COVID-19恢复期病人中，发现临床转阴病人检测为阳性。说明本试剂盒具有较高的检测灵敏性。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
各位技术人员须知：虽然本发明已按照上述具体实施方式做了描述，但是本发明的发明思想并不仅限于此发明，任何运用本发明思想的改装，都将纳入本专利专利权保护范围内。
以上所述仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种防止RNA降解的保护方法,其特征在于，包括以下步骤：通过RNA酶保护序列(RNase-AP)与目的RNA的高效结合后形成RNA:DNA复合物，结合使得RNA暴露区域减少，从而使RNA酶不能有效切割目的RNA防止RNA降解。
  2. 根据权利要求1所述的一种防止RNA降解的保护方法，其特征在于，所述的RNA酶保护序列(RNase-AP)与目的RNA结合位置为目的RNA上的RNA酶识别位点。
  3. 一种防止RNA降解的保护方法,其特征在于，包括以下步骤：通过RNA酶保护序列(RNase-AP)与RAN酶结合形成复合物，影响了RNA酶的活性，抑制了RNA酶与目的RNA结合的能力，防止目的RNA被降解。
  4. 根据权利要求3所述的一种防止RNA降解的保护方法，其特征在于，所述的RNA酶保护序列(RNase-AP)与RAN酶结合位置为RAN酶上的RNA酶识别位点。
  5. 一种防止RNA降解的保护方法,其特征在于，包括以下步骤：通过RNA酶保护序列(RNase-AP)与目的RNA结合后，引起目的RNA的局部结构改变，使得RNA酶与目的RNA结合的效率降低或结合位置改变，使得RNA酶不能对目的RNA进行切割降解，防止目的RNA被降解。
  6. 根据权利要求5所述的一种防止RNA降解的保护方法，其特征在于，所述的RNA酶保护序列(RNase-AP)与目的RNA结合位置为目的RNA上的RNA酶识别位点。
  7. 一种RNA酶保护序列,其特征在于，采用RNA酶识别位点的序列作为RNA酶保护序列。
  8. 一种权利要求7所述的RNA酶保护序列在作为制备病毒检测试剂上的应用。
  9. 一种病毒检测组合物，其特征在于，包括权利要求7所述的RNA酶保护序列。
  10. 一种病毒检测试剂盒，其特征在于，包括权利要求8所述的病毒检测试 剂。
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