WO2022266946A1 - 检测病原体rna、及动物或人类基因rna的试剂盒及其应用 - Google Patents

检测病原体rna、及动物或人类基因rna的试剂盒及其应用 Download PDF

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WO2022266946A1
WO2022266946A1 PCT/CN2021/102134 CN2021102134W WO2022266946A1 WO 2022266946 A1 WO2022266946 A1 WO 2022266946A1 CN 2021102134 W CN2021102134 W CN 2021102134W WO 2022266946 A1 WO2022266946 A1 WO 2022266946A1
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rna
cov
primer pair
sars
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黄婉秋
黄健
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黄婉秋
黄健
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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
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention belongs to the technical field of bioengineering, in particular to a kit for detecting pathogens based on RT-nest RPA (RT-nestRPA) technology and an application thereof.
  • RT-nestRPA RT-nest RPA
  • COVID-19 is a global epidemic caused by a novel coronavirus SARS-CoV-2 infection. COVID-19 is characterized by a long incubation period, strong infectivity and high mortality. The epidemic has spread rapidly and has caused enormous human health damage and significant socioeconomic harm.
  • Coronavirus is an enveloped single-stranded RNA virus that spreads widely among humans, other mammals and birds, and can cause respiratory, intestinal, hepatic and nervous system diseases. Seven coronaviruses are known to cause disease in humans, four of which, Cov-229E, Cov-OC43, Cov-NL63 and Cov-HKU1, are prevalent in humans and often cause common cold symptoms. The other 3 coronaviruses SARS-CoV, MERS-CoV and SARS-CoV-2 are highly dangerous and can cause severe pneumonia and even death.
  • SARS-CoV-2 nucleic acid detection is an important means for rapid diagnosis of new coronary pneumonia.
  • the commonly used nucleic acid detection method is fluorescent quantitative PCR (qPCR) technology.
  • qPCR fluorescent quantitative PCR
  • Viral nucleic acid detection from the collection of samples from patients to the completion of test reports, has to go through many steps, including sample collection, sample storage, sample delivery to testing laboratories, virus inactivation, cell lysis, nucleic acid extraction, testing and issuance of test reports. A slight problem with any of these steps can lead to false negative results. Among them, high-sensitivity, high-specificity and high-repeatability detection technologies are crucial to improving the quality of SARS-CoV-2 nucleic acid detection and reducing the false negative rate of experiments.
  • Recombinase Polymerase Amplification is based on the principle of recombinase polymerase-mediated amplification, simulating the enzymatic reaction process of DNA replication in vivo, relying on specific enzyme and protein combinations (recombinase, single-chain binding protein and DNA polymerase) to specifically amplify the DNA template, which can achieve rapid specific amplification (37°C-42°C, 5-30min) under conditions close to body temperature. As an isothermal technology, it reduces the need for high-precision and expensive instruments.
  • RT-RPA reverse transcriptase-recombinase polymerase amplification
  • the present invention will solve the technical problem of lack of rapid and accurate detection kits for pathogens such as SARS-CoV-2 new coronavirus, and provide a kit for detecting pathogens based on RT-nest RPA (RT-nestRPA) technology.
  • RT-nestRPA technology simultaneously detects pathogen gene RNA and internal reference gene RNA, and can quickly, accurately, sensitively and specifically detect SARS-CoV-2 virus and other pathogen RNA.
  • a kit for detecting pathogen RNA and animal or human gene RNA comprising a primer pair for detecting pathogen RNA and animal or human gene RNA with RT-nested RPA, And the primer pair for detecting internal reference gene RNA by RT-nested RPA, the primer pair for detecting pathogen RNA, animal or human gene RNA and the primer pair for detecting internal reference gene RNA are simultaneously carried out in the same reaction tube for detection reaction.
  • the pathogen RNA includes RNA of viruses, bacteria, chlamydia, mycoplasma, rickettsia, spirochetes, fungi, or parasites
  • the animal or human gene RNA includes human tumor-related gene mRNA and genetic disease-related gene mRNA .
  • the pathogen is SARS-CoV-2 new coronavirus
  • the primer pair for detecting SARS-CoV-2 new coronavirus with RT-nested RPA is aimed at SARS shown in SEQ ID NO: 1 -CoV-2 new coronavirus N gene sequence or a primer pair designed for its partial sequence.
  • the primer pair for detecting SARS-CoV-2 new coronavirus with RT-nested RPA includes: the pair of outer primers of sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and The inner primer pair of the sequence shown in SEQ ID NO:6.
  • the internal reference gene is the human housekeeping gene RP gene.
  • the primer pair for detecting RP gene with RT-nested RPA includes: the pair of outer primers of sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10, the pair of outer primers of sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12 inner primer pair.
  • the kit also includes a probe designed specifically for the N gene sequence of SARS-CoV-2 novel coronavirus shown in SEQ ID NO: 1 or a partial sequence thereof, and specifically for the RP shown in SEQ ID NO: 7 Probes designed for gene sequences or partial sequences.
  • a primer pair for detecting SARS-CoV-2 new coronavirus is also provided, said primer pair is a primer pair for detecting the N gene of SARS-CoV-2 new coronavirus with RT-nested RPA, It comprises an outer primer pair and an inner primer pair.
  • the primer pair is a primer pair designed for the SARS-CoV-2 new coronavirus N gene sequence shown in SEQ ID NO: 1 or a partial sequence thereof, and the outer primer pair has such as SEQ ID NO: 3 and SEQ ID The base sequence shown in NO: 4, the inner primer pair has the base sequence shown in SEQ ID NO: 5 and SEQ ID NO: 6.
  • a probe for detecting SARS-CoV-2 new coronavirus is also provided, and the probe is specific for SARS-CoV-2 new coronavirus N gene sequence shown in SEQ ID NO: 1 Probes designed for their partial sequences.
  • the probe has a base sequence as shown in SEQ ID NO:2.
  • kits for detecting SARS-CoV-2 novel coronavirus comprising the above-mentioned primer pair.
  • the kit also includes a primer pair for detecting the internal reference gene with RT-nested RPA, and the primer pair for detecting the SARS-CoV-2 new coronavirus N gene and the primer pair for detecting the internal reference gene are simultaneously in the same reaction tube Perform a detection reaction.
  • the kit also includes the above-mentioned probes and probes specific for internal reference genes.
  • the kit for detecting pathogens of the present invention uses the RT-nestRPA method to simultaneously perform nucleic acid detection on pathogen RNA and internal reference gene RNA, thereby achieving the purpose of simultaneously detecting target genes and internal reference genes in the same tube, and making the interpretation of detection results more accurate.
  • the kit for detecting SARS-CoV-2 new coronavirus in the embodiment of the present invention can detect at least 1 copy/uL of SARS-CoV-2 nucleic acid fragments, which has ultra-high sensitivity, and the RT-nestRPA in the present invention operates at a constant temperature of 39°C It can be carried out without expensive detection equipment, and the detection time is as short as 15 minutes. It provides new technical support for the early detection, early isolation, and early treatment of the new coronavirus SARS-CoV-2, and has a very good application prospect.
  • Fig. 1 is the SARS-CoV-2 genome mutation distribution figure of the embodiment 1 of the present invention.
  • Fig. 2 is the LOD result figure of the preferred primers for detection of SARS-CoV-2N gene by RT-RPA in Example 1 of the present invention
  • Fig. 3 is that embodiment of the present invention 2 utilizes RT-nestRPA to detect the LOD result comparison figure of SARS-CoV-2 pseudovirus RNA;
  • Fig. 4 is the preferred primer result figure of screening RP gene by RT-RPA method in Example 3 of the present invention.
  • Fig. 5 is the comparative figure of the detection ability of human blood cell RNA RP gene with RT-RPA and RT-nestRPA method in embodiment 3 of the present invention
  • Fig. 6 is the result figure of detecting simulated sample RNA by RT-nestRPA double-gene detection method in Example 4 of the present invention
  • Example 7 is a schematic diagram of the grouping of clinical samples of new coronary pneumonia in Example 5 of the present invention.
  • Fig. 8 is a graph showing the results of the N gene and RP gene detection of the samples of healthy persons returning to work by using the RT-nestRPA method in Example 5 of the present invention
  • Fig. 9 is a statistical chart of the results of detecting clinical samples by RT-nestRPA and qPCR in Example 5 of the present invention.
  • the present invention proposes the concept of RT-nestRPA, and develops an ultra-high-sensitivity pathogen nucleic acid detection technology based on a fluorescence detection analyzer to realize simultaneous detection of pathogen target genes and internal reference genes in the same tube, and the interpretation of detection results is more accurate.
  • the kit for detecting the SARS-CoV-2 new coronavirus is specifically described, which simultaneously detects the SARS-CoV-2 virus N gene and the human housekeeping gene RP gene, and 1 copy can be detected under laboratory conditions /uL of SARS-CoV-2 nucleic acid fragments with ultra-high sensitivity.
  • the RT-nestRPA technology adopted in the present invention has the advantage that it can detect target gene and internal reference gene RNA in the same reaction tube, and use internal reference gene to perform sample quality control.
  • RT-nestRPA technology can quickly detect the sample to be tested with a concentration as low as 1 copy/uL, and accurately and reliably interpret positive and negative results through the significant difference in the fluorescence value of the amplification product in a short period of time (15 minutes). It has the characteristics of high sensitivity, high specificity and high repeatability.
  • Appropriate primer sequence is an important part of RPA high-efficiency amplification.
  • T thymines
  • Ten pairs of primers (Table 1) were designed at different positions at both ends of the probe to screen out the most efficient combination of primers and probes.
  • F forward primer
  • R reverse primer
  • reverse primers are screened, and the first reverse primer (R) is paired with the first to tenth forward primers (F) respectively.
  • the new coronavirus pseudovirus RNA quality control product take the pseudovirus culture medium rewarmed and mixed at room temperature, use the column extraction method to extract the pseudovirus RNA, and dissolve the RNA with enzyme-free water.
  • the pseudovirus RNA was diluted to 1 ⁇ 10 5 copies/uL, 1 ⁇ 10 4 copies/uL, 1 ⁇ 10 3 copies/uL, 500 copies/uL, 100 copies/uL, 10 copies/uL and 1 copy/uL.
  • 1 ⁇ 10 5 copies/uL pseudoviral RNA as a template, after preliminary screening, the results showed that the amplification of FN-1/RN-2, FN1-/RN-3, FN6/RN-5, FN-6/RN-7 The efficiency is higher.
  • the above primer pairs are 1 ⁇ 10 5 copies/uL, 1 ⁇ 10 4 copies/uL, 1 ⁇ 10 3 copies/uL, 500 copies/uL, 100 copies/uL, 10 copies/uL and 1 copy/uL Pseudoviral RNA was used as a template, and the RT-RPA method was used to set the reaction time to 30 minutes. Observe whether the difference in fluorescence value between the sample to be tested and the blank control (enzyme-free water) was statistically significant, and the judgment standard was p ⁇ 0.05. Positive, p>0.05 is negative, and the lowest RNA concentration with a positive detection result is taken as the sensitivity of the primer pair.
  • the RT-RPA reaction system is: lyophilized enzyme powder (including reverse transcriptase, RNase inhibitor, recombinase, single-stranded DNA binding protein and polymerase), 25uL buffer V, 2.1uL N gene forward outer primer ( 10uM), 2.1uL N gene reverse outer primer (10uM), 0.6uL specific probe (10uM), 1uL test sample, 2.5uL magnesium acetate (280mM). Once magnesium acetate is added to the reaction system, the reaction starts immediately.
  • lyophilized enzyme powder including reverse transcriptase, RNase inhibitor, recombinase, single-stranded DNA binding protein and polymerase
  • 25uL buffer V 2.1uL N gene forward outer primer ( 10uM), 2.1uL N gene reverse outer primer (10uM), 0.6uL specific probe (10uM), 1uL test sample, 2.5uL magnesium acetate (280mM).
  • Example 2 RT-nestRPA and RT-RPA compare the detection ability of SARS-CoV-2 pseudovirus RNA
  • the SARS-CoV-2 pseudovirus RNA quality control product (including ORF1, E and N genes), that is, the new coronavirus pseudovirus culture medium, verifies the sensitivity of the preferred primers for the N gene.
  • the judgment standard is that p ⁇ 0.05 is positive, and p>0.05 is negative, and the minimum detectable RNA concentration is used as the method the LODs.
  • RT-nestRPA can indeed significantly improve the detection sensitivity of SARS-CoV-2 virus N gene.
  • the present invention detects the method for SARS-CoV-2 pseudovirus with RT-nestRPA (RT-nested RPA), comprises the following steps:
  • Step 1 Using a pair of outer primers of the N gene of the SARS-CoV-2 virus, perform an RT-RPA reaction on the template RNA to pre-amplify the first fragment of the N gene of the target gene;
  • Step 2 Use a pair of internal primers of the N gene of SARS-CoV-2 virus, use the amplified first fragment of the target gene as a template, and perform an RPA reaction under the condition of adding differently labeled signal probes of the N gene, Amplify the second fragment of the target gene N gene. By detecting the probe signals of different labels, the detection result of the N gene of the viral nucleic acid is obtained.
  • the signal probes with different labels are probes with different signal labels, usually probes with different fluorescein labels, such as FAM, HEX, Cy3, Cy5, VIC and other fluorescein-labeled probes.
  • fluorescein labels such as FAM, HEX, Cy3, Cy5, VIC and other fluorescein-labeled probes.
  • the RT-RPA reaction system in step 1 is: lyophilized enzyme powder (including reverse transcriptase, RNase inhibitor, recombinase, single-stranded DNA binding protein and polymerase), 25uL buffer V, 2.1uL forward outer primer (10uM), 2.1uL reverse outer primer (10uM), 5uL test sample, 2.5uL magnesium acetate (280mM). Once magnesium acetate is added to the reaction system, the reaction starts immediately.
  • the RPA reaction system in step 2 is: lyophilized enzyme powder (including recombinase, single-stranded DNA binding protein, polymerase and exonuclease), 25uL buffer VI, 2.1uL forward internal primer (10uM), 2.1uL Reverse internal primer (10uM), 0.6uL probe (10uM), all pre-reaction products, 2.5uL magnesium acetate (280mM). Once magnesium acetate is added to the reaction system, the reaction starts immediately.
  • the RT-RPA reaction in Step 1 can be carried out at 37-42°C, and the reaction time is 5-20min.
  • the reaction temperature is 39° C., and the reaction time is 15 minutes.
  • the RPA reaction in the second step can be carried out at 37-42° C., and the reaction time is 1-30 min.
  • the reaction temperature is 39° C., and the reaction time is 15 minutes.
  • the human ribonuclease P Homo sapiens ribonuclease P, RP
  • the probe was designed at the sequence position with three consecutive thymines (T) according to the gene sequence (NM_001104546.1) , and the probe was modified with HEX fluorescein.
  • Ten pairs of primers were designed at different positions at both ends of the probe to screen out the most efficient combination of primers and probes (Table 4).
  • primer screening pair the first forward primer (F) with the first to tenth reverse primers (R) respectively.
  • reverse primers are screened, and the first reverse primer (R) is paired with the first to tenth forward primers (F) respectively.
  • the best primer combination with the largest slope and shortest onset time was found.
  • Peripheral blood samples from healthy people collected in the laboratory (the samples were tested negative for SARS-CoV-2 virus N gene by nestRPA and serum IgG/IgM antibody test results were all negative), and blood cells were obtained by high-speed centrifugation at 3000rpm.
  • the extraction method was used to extract total RNA, which was used to screen RP gene primers and test the minimum detection ability of optimal primers.
  • the blood cell total RNA was sequentially diluted 10 times, 100 times, 1000 times, 5000 times and 10000 times by 10 times serial dilution method. Using the RT-RPA method, at the end of the 30-minute reaction, observe whether the difference in fluorescence between the test sample and the blank control is statistically significant.
  • the judgment standard is that p ⁇ 0.05 is positive, and p>0.05 is negative.
  • the primer pairs RP-F7/RP-R10 and RP-F2/RP-R6 satisfied the positional relationship between the inner and outer primer pairs, and the slope of the amplification curve was relatively large (Table 5, Fig. 4), the minimum detectable blood cell total RNA sample diluted 100 times (Fig. 5 left).
  • the RP gene can be detected in a blood cell total RNA sample diluted 5000 times, which significantly improves the detection sensitivity of the RP gene ( Figure 5 right).
  • the present invention detects the method for RP internal reference gene with RT-nestRPA (RT-nested RPA), comprises the following steps:
  • Step 1 Use a pair of outer primers of the RP gene to perform an RT-RPA reaction on the template RNA to pre-amplify the first fragment of the RP gene of the target gene;
  • Step 2 Use a pair of internal primers of the RP gene of the SARS-CoV-2 virus, use the amplified first fragment of the target gene as a template, and perform an RPA reaction under the condition of adding differently labeled signal probes of the RP gene, Amplify the second fragment of the RP gene of interest. By detecting the signals of probes with different labels, the detection results of the internal reference RP gene were obtained.
  • the signal probes with different labels are probes with different signal labels, usually probes with different fluorescein labels, such as FAM, HEX, Cy3, Cy5, VIC and other fluorescein-labeled probes.
  • fluorescein labels such as FAM, HEX, Cy3, Cy5, VIC and other fluorescein-labeled probes.
  • the RT-RPA reaction system in step 1 is: lyophilized enzyme powder (including reverse transcriptase, RNase inhibitor, recombinase, single-stranded DNA binding protein and polymerase), 25uL buffer V, 2.1uL forward outer primer (10uM), 2.1uL reverse outer primer (10uM), 5uL test sample, 2.5uL magnesium acetate (280mM). Once magnesium acetate is added to the reaction system, the reaction starts immediately.
  • the RPA reaction system in step 2 is: lyophilized enzyme powder (including recombinase, single-stranded DNA binding protein, polymerase and exonuclease), 25uL buffer VI, 2.1uL forward internal primer (10uM), 2.1uL Reverse internal primer (10uM), 0.6uL probe (10uM), all pre-reaction products, 2.5uL magnesium acetate (280mM). Once magnesium acetate is added to the reaction system, the reaction starts immediately.
  • the RT-RPA reaction in Step 1 can be carried out at 37-42°C, and the reaction time is 5-20min.
  • the reaction temperature is 39° C., and the reaction time is 15 minutes.
  • the RPA reaction in the second step can be carried out at 37-42° C., and the reaction time is 1-30 min.
  • the reaction temperature is 39° C., and the reaction time is 15 minutes.
  • RPA or RT-RPA detection methods usually only detect one target gene in one reaction. If it is necessary to clarify the expression of the internal reference gene of the sample to be tested, another reaction tube is required for detection.
  • the SARS-CoV-2 virus is an RNA virus
  • the present invention proposes the concept of RT-nestRPA for the first time, and innovatively integrates the SARS-CoV-2 virus N gene and The human housekeeping gene RP gene is detected in the same reaction tube, and the RNA sample is directly used to quickly detect the SARS-CoV-2 virus N gene and the internal reference RP gene with high sensitivity.
  • the monitoring system includes the internal reference gene RP gene to monitor the quality of the experimental process. control to avoid false negative results.
  • SARS-CoV-2 virus RNA quality control prseudovirus culture medium, containing N gene, Guangzhou Bangdesheng Biotechnology Co., Ltd.
  • SARS-CoV-2 virus RNA quality control prseudovirus culture medium, containing N gene, Guangzhou Bangdesheng Biotechnology Co., Ltd.
  • peripheral blood samples from healthy people were collected (the samples were tested negative by RT-nestRPA N gene and new crown IgG/IgM antibodies), and blood cells were obtained by high-speed centrifugation at 3000rpm. The two samples were mixed to simulate the real SARS-CoV- 2.
  • Nucleic acid composition and structure of virus-infected positive samples sample contains both SARS-CoV-2 viral RNA and RP gene).
  • the simulated sample was mixed with pseudovirus culture medium and healthy human blood cells at a volume ratio of 1:1, and the total RNA of the mixed sample was extracted by column extraction.
  • the pseudovirus culture solution RNA and human blood cell RNA were extracted for RT-nestRPA double gene detection.
  • RT-nestRPA dual-gene detection consists of two steps:
  • Step 1 In the same reaction tube, use a pair of outer primers of the SARS-CoV-2 virus N gene and a pair of outer primers of the RP gene at the same time to perform an RT-RPA reaction on the template RNA, and simultaneously pre-amplify the target gene N gene and The first segment of the RP gene.
  • Step 2 In a new reaction tube, use a pair of internal primers of the SARS-CoV-2 virus N gene and a pair of internal primers of the RP gene at the same time, use the first fragment of the amplified target gene as a template, and An RPA reaction is performed under the condition of adding differently labeled signal probes of the N gene and the RP gene at the same time, and the second fragments of the target gene N gene and the RP gene are simultaneously amplified.
  • the signal probes with different labels are probes with different signal labels, usually probes with different fluorescein labels, such as FAM, HEX fluorescein-labeled probes.
  • the RT-RPA reaction system in step 1 is: lyophilized enzyme powder (including reverse transcriptase, RNase inhibitor, recombinase, single-stranded DNA binding protein and polymerase), 25uL buffer V, 1.0uL N gene forward Outer primer (10uM), 1.0uL N gene reverse outer primer (10uM), 1.0uL RP gene forward outer primer (10uM), 1.0uL RP gene reverse outer primer (10uM), 5uL test sample, 2.5uL acetic acid Magnesium (280mM). Once magnesium acetate is added to the reaction system, the reaction starts immediately.
  • lyophilized enzyme powder including reverse transcriptase, RNase inhibitor, recombinase, single-stranded DNA binding protein and polymerase
  • the RPA reaction system in step 2 is: lyophilized enzyme powder (including recombinase, single-stranded DNA binding protein, polymerase and exonuclease), 25uL buffer VI, 1.0uL N gene forward internal primer (10uM), 1.0uL N gene reverse internal primer (10uM), 1.0uL RP gene forward internal primer (10uM), 1.0uL RP gene reverse internal primer (10uM), 0.3uL N gene probe (10uM), 0.3uL RP gene Probe (10 uM), total pre-reaction product (about 37 uL), 2.5 uL magnesium acetate (280 mM). Once magnesium acetate is added to the reaction system, the reaction starts immediately.
  • lyophilized enzyme powder including recombinase, single-stranded DNA binding protein, polymerase and exonuclease
  • 25uL buffer VI 1.0uL N gene forward internal primer (10uM), 1.0uL N gene reverse internal primer (10uM), 1.0uL RP gene forward internal
  • the RT-RPA reaction in Step 1 can be carried out at 37-42°C, and the reaction time is 5-20min.
  • the reaction temperature is 39° C.
  • the reaction time is 15 minutes.
  • the RPA reaction in the second step can be carried out at 37-42° C., and the reaction time is 1-30 min.
  • the reaction temperature is 39° C.
  • the reaction time is 15 minutes.
  • RT-nestRPA Using RT-nestRPA technology to detect the simulated sample RNA, pseudoviral RNA and total blood cell RNA, it was found that the results of the N gene detection channel (FAM fluorescence signal) showed that the simulated sample RNA and pseudoviral RNA presented a typical amplification curve, while the total blood cell There was no obvious amplification curve for RNA (Fig. 6 left).
  • the results of the RP gene detection channel (HEX fluorescent signal) showed that the simulated sample RNA and the total RNA of blood cells presented a typical amplification curve, while the pseudovirus RNA had no obvious amplification curve ( Figure 6 right).
  • the test result of the SARS-CoV-2 virus nucleic acid of the sample to be tested is positive. If there is no obvious amplification curve in the N gene detection channel of the sample to be tested, and a typical exponential amplification curve appears in the internal reference RP gene detection channel, it is determined that the nucleic acid test result of the sample to be tested is negative for SARS-CoV-2 virus.
  • the test result of the sample to be tested is determined to be suspicious, and a second test is performed if necessary.
  • Example 5 RT-nestRPA performs common detection of N gene and RP gene on clinical samples
  • nucleic acid samples were extracted nucleic acid (including DNA and RNA) using an automatic nucleic acid extraction instrument according to the operating procedures of the nucleic acid extraction kit (Huayin Biotech Co., Shenzhen, China). All nucleic acid samples were tested by real-time quantitative PCR (qPCR) to determine whether they were positive samples for SARS-CoV-2 infection. Between February 2020 and April 2020, a total of 111 samples (nasopharyngeal swabs or sputum) from 84 patients were collected at Shenzhen Third People's Hospital.
  • qPCR real-time quantitative PCR
  • the RT-nestRPA described in Example 4 was used to jointly detect the N gene and the RP gene on the RNA extracted from the clinical sample.
  • the clinical samples with positive qPCR (N gene and internal reference gene) detection results were detected by the RT-nestRPA method of the present invention, and one of the positive samples with qPCR (N gene and internal reference gene) detection results was serially diluted 11 times by 10 times. The serially diluted samples were then tested using the RT-nest RPA dual-gene assay. Samples that are negative for qPCR (N gene) detection results are also detected using the RT-nestRPA dual-gene method of the present invention.
  • RT-nestRPA method of the present invention Fifteen nasal swabs or sputum samples from 15 confirmed COVID-19 patients were all positive for the SARS-CoV-2N gene using the qPCR method.
  • the detection by the RT-nestRPA method of the present invention is consistent with the detection result of the N gene by the qPCR method, and the positive coincidence rate is 100% (15/15).
  • the RT-nestRPA method of the present invention was used to detect the internal reference gene (RP gene) of 15 samples, and the detection results were all positive (Table 6).
  • the RT-nestRPA nucleic acid detection method of the present invention detects the N gene of the new coronavirus and the RP gene of the internal reference gene in a total of 111 clinical samples.
  • the detection results of the RT-nestRPA method of the present invention are all positive (15/15) to 15 qPCR positive samples, the consistency rate with the qPCR detection method is 100%, and the false negative rate is 0%.
  • the RT-nestRPA method of the present invention detected positive results for the N gene of the new coronavirus in 22 cases (22.91%) of the 96 qPCR-negative samples ( Figure 9). Based on the above results, the present invention has higher sensitivity than the qPCR method, and has a good application prospect for the early detection of patients infected with the new coronavirus.

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Abstract

一种检测病原体RNA、及动物或人类基因RNA的试剂盒，试剂盒包含以RT-巢式RPA检测病原体RNA、及动物或人类基因RNA的引物对，及以RT-巢式RPA检测内参基因的引物对，检测病原体RNA、及动物或人类基因RNA的引物对与检测内参基因的引物对在同一反应管中同时进行检测反应。检测病原体RNA、及动物或人类基因RNA的试剂盒，实现同管检测病原体目的基因和内参基因的RNA水平，增加内参基因对实验过程的质量控制，避免假阴性结果。检测新冠病毒的试剂盒最低可检测到1拷贝/uL的SARS-CoV-2病毒RNA片段，具有超高灵敏度，为新冠病毒SARS-CoV-2的早期发现、早期隔离、早期治疗提供新的技术支撑，具有非常好的应用前景。

Description

检测病原体RNA、及动物或人类基因RNA的试剂盒及其应用 技术领域
本发明属于生物工程技术领域，尤其涉及一种基于RT-巢式RPA(RT-nestRPA)技术检测病原体的试剂盒及其应用。
背景技术
从2019年12月底以来，在全球爆发了大规模的新型冠状病毒感染性肺炎(COVID-19)的疫情。COVID-19是由一种新型冠状病毒SARS-CoV-2感染所致的全球性流行病。COVID-19的特点是潜伏期长、传染性强及死亡率高。该流行病传播迅速，已造成巨大的人身健康损害和重大的社会经济危害。
冠状病毒(Coronavirus,CoV)是一种包膜的单链RNA病毒，在人类、及其它哺乳动物和鸟类中广泛传播，能引起呼吸系统、肠、肝及神经系统疾病。已知有7种冠状病毒使人类致病，其中4种如Cov-229E、Cov-OC43、Cov-NL63和Cov-HKU1在人群中流行，常引起普通感冒症状。另外3种冠状病毒SARS-CoV,MERS-CoV和SARS-CoV-2具有高度的危险性，会导致严重的肺炎甚至死亡。
截至2021年6月8日，全球累计已有超过1.73亿人被确诊为新冠肺炎(COVID-19)，死亡病例数达373万。目前，中国新冠肺炎防控工作已步入常态化，不同城市零星散发病例的报告此起彼伏，境外输入病例报告连续不断。与此同时，全球其他国家的疫情仍在持续蔓延。美国、巴西、印度累计确诊病例远超1600万人。SARS-CoV-2在环境中存活的机会大大增加，冷链运输工作人员、车辆甚至肉制品样品，检测出SARS-CoV-2阳性结果的报道屡见不鲜。新冠肺炎防控措施中，早诊断、早治疗是关键。SARS-CoV-2核酸检测是快速诊断新冠肺炎的重要手段，目前常用的核酸检测方法是荧光定量PCR(qPCR)技术。根据中国在线数据，中国115家公司已经开发了116种检测SARS-CoV-2核酸的试剂盒，其中98种(84.5％)试剂盒采用qPCR技术。
但是基于qPCR的SARS-CoV-2核酸检测技术在临床应用中屡屡出现“假阴性”结果。我国的多家医院先后报道了基于qPCR检测方法的核酸“假阴性”结果。最近的新闻显示，有患者经过6次核酸检测后才出现核酸阳性结果，此前，发现有些患者经过正规治疗后两次核酸检测阴性，然而在隔离和观察期间，他们的核酸检测结果又重新阳性。这些假阴性结果大大增加COVID-19疫情防控的复杂性。
病毒核酸检测，从患者采集样本到完成检测报告，要经历许多步骤，包括样本采集、样本保存、样本传送至检测实验室、病毒灭活、细胞溶解、核酸提取、检测并出具检测报告。 其中任何步骤出现一点问题都可能导致假阴性结果。其中，高灵敏度、高特异性和高重复性的检测技术，对于提高SARS-CoV-2核酸检测质量和减少实验假阴性率至关重要。
重组酶聚合酶扩增(Recombinase Polymerase Amplification，RPA)是基于重组酶聚合酶介导的扩增原理，模拟体内DNA复制的酶反应过程，依赖特定的酶和蛋白组合(重组酶、单链结合蛋白和DNA聚合酶)对DNA模板进行特异性扩增，可在接近体温条件下实现快速特异性扩增(37℃-42℃，5-30min)，作为一种等温技术减少了对高精度昂贵仪器、稳定电力供应设施及高级别实验室的依赖，只需要一个恒温装置就可完成，且可通过荧光分析装置实时判断是否存在扩增产物，具有对设施和操作要求均较为简单的特点，已应用于生命科学研究、医学检测、农业、食品安全、转基因检测等多个领域。
目前已有基于RPA原理发展出的RT-RPA(逆转录酶-重组酶聚合酶扩增)技术，即在RPA反应体系中增加逆转录酶和RNA酶抑制剂，使得RNA核酸样本也可实现恒温条件下的快速扩增，省去了RNA样本还需经逆转录为cDNA才能进行检测的弊端。
传统RPA或RT-RPA检测，往往一个反应仅检测1个目的基因，若需检测待测样本的内参基因则需要再进行一个新的反应。这无疑增加了检测的时间成本和经济成本，并且增加了结果解读的不确定性。
虽然目前全球各国都在大力推广新冠病毒疫苗接种的防疫工作，但实现完全的群体免疫仍需很长时间。目前，由SARS-CoV-2病毒引起的COVID-19疫情，在全球多个国家仍持续蔓延，SARS-CoV-2病毒的传染性更强、传播速度更快，疫情并未得到有效控制。并且国内多个城市出现了小范围聚集性病例的出现，今年4月，我国云南省瑞丽市爆发了新冠疫情。因此，为了实现COVID-19疫情的有效防控，急需开发出具有高灵敏度高特异性和高重复性的新型快速核酸检测技术。
发明内容
本发明要解决目前缺乏快速、准确检测SARS-CoV-2新冠病毒等病原体试剂盒的技术问题，提供一种基于RT-巢式RPA(RT-nestRPA)技术检测病原体的试剂盒，该试剂盒利用RT-nestRPA技术同时检测病原体基因RNA和内参基因RNA，能快速、准确、灵敏、特异地对SARS-CoV-2病毒等病原体RNA进行检测。
为了解决上述技术问题，本发明通过如下技术方案实现:
在本发明的一个方面，提供了一种检测病原体RNA、及动物或人类基因RNA的试剂盒，所述试剂盒包含以RT-巢式RPA检测病原体RNA、及动物或人类基因RNA的引物对，及以RT-巢式RPA检测内参基因RNA的引物对，所述检测病原体RNA、及动物或人类基因RNA的引物对与检测内参基因RNA的引物对在同一反应管中同时进行检测反应。
优选的，所述病原体RNA包括病毒、细菌、衣原体、支原体、立克次体、螺旋体、真菌、或寄生虫的RNA，所述动物或人类基因RNA包括人类肿瘤相关基因mRNA以及遗传病相关基因mRNA。
在本发明的具体实施例中，所述病原体为SARS-CoV-2新冠病毒，所述以RT-巢式RPA检测SARS-CoV-2新冠病毒的引物对是针对SEQ ID NO：1所示SARS-CoV-2新冠病毒N基因序列或其部分序列设计的引物对。更优选的，所述以RT-巢式RPA检测SARS-CoV-2新冠病毒的引物对包括：SEQ ID NO：3和SEQ ID NO：4所示序列的外引物对，SEQ ID NO：5和SEQ ID NO：6所示序列的内引物对。
优选的，所述内参基因为人类管家基因RP基因。所述以RT-巢式RPA检测RP基因的引物对包括：SEQ ID NO：9和SEQ ID NO：10所示序列的外引物对，SEQ ID NO：11和SEQ ID NO：12所示序列的内引物对。
优选的，所述试剂盒还包含特异性针对SEQ ID NO：1所示SARS-CoV-2新冠病毒N基因序列或其部分序列设计的探针，及特异性针对SEQ ID NO：7所示RP基因序列或其部分序列设计的探针。
在本发明的另一方面，还提供了一种检测SARS-CoV-2新冠病毒的引物对，所述引物对是以RT-巢式RPA检测SARS-CoV-2新冠病毒N基因的引物对，其包含外引物对和内引物对。
优选的，所述引物对是针对SEQ ID NO：1所示SARS-CoV-2新冠病毒N基因序列或其部分序列设计的引物对，所述外引物对具有如SEQ ID NO：3和SEQ ID NO：4所示的碱基序列，所述内引物对具有如SEQ ID NO：5和SEQ ID NO：6所示的碱基序列。
在本发明的另一方面，还提供了一种检测SARS-CoV-2新冠病毒的探针，所述探针是特异性针对SEQ ID NO：1所示SARS-CoV-2新冠病毒N基因序列或其部分序列设计的探针。
优选的，所述探针具有如SEQ ID NO：2所示的碱基序列。
在本发明的另一方面，还提供了一种检测SARS-CoV-2新冠病毒的试剂盒，所述试剂盒包含上述的引物对。
优选的，所述试剂盒还包含以RT-巢式RPA检测内参基因的引物对，所述检测SARS-CoV-2新冠病毒N基因的引物对与检测内参基因的引物对在同一反应管中同时进行检测反应。
所述试剂盒还包含上述的探针，以及特异性针对内参基因的探针。
在本发明的另一方面，还提供了上述试剂盒在制备检测病原体的产品中的应用。
本发明检测病原体的试剂盒，以RT-nestRPA方法同时对病原体RNA和内参基因RNA进行核酸检测，实现同管同时检测目的基因和内参基因的目的，使检测结果的判读更准确。本发 明实施例检测SARS-CoV-2新冠病毒的试剂盒最低可检测到1拷贝/uL的SARS-CoV-2核酸片段，具有超高灵敏度，且本发明中的RT-nestRPA在39℃恒温条件下进行，无需昂贵检测设备，检测时间短仅需15min，为新冠病毒SARS-CoV-2的早期发现、早期隔离、早期治疗提供新的技术支撑，具有非常好的应用前景。
附图说明
下面结合附图和具体实施方式对本发明作进一步详细的说明。
图1是本发明实施例1的SARS-CoV-2基因组突变分布图；
图2是本发明实施例1利用RT-RPA检测SARS-CoV-2N基因优选引物的LOD结果图；
图3是本发明实施例2利用RT-nestRPA检测SARS-CoV-2假病毒RNA的LOD结果比较图；
图4是本发明实施例3利用RT-RPA方法筛选RP基因优选引物结果图；
图5是本发明实施例3用RT-RPA与RT-nestRPA方法对人血细胞RNA RP基因的检测能力比较图；
图6是本发明实施例4利用RT-nestRPA双基因检测方法检测模拟样本RNA结果图；
图7是本发明实施例5新冠肺炎临床样本分组情况示意图；
图8是本发明实施例5利用RT-nestRPA方法检测复工健康体检者样本N基因和RP基因结果图；
图9是本发明实施例5利用RT-nestRPA和qPCR检测临床样本的结果统计图。
具体实施方式
本发明提出RT-nestRPA概念，并基于荧光检测分析仪研发出超高灵敏度的病原体核酸检测技术，实现同管同时检测病原体目的基因和内参基因，检测结果判读更加准确。在本发明下述实施例中，具体阐述检测SARS-CoV-2新冠病毒的试剂盒，其同时检测SARS-CoV-2病毒N基因和人类管家基因RP基因，实验室条件下可以检出1拷贝/uL的SARS-CoV-2核酸片段，具有超高灵敏度。
本发明采用的RT-nestRPA技术与传统RPA和RT-RPA技术相比，优势在于其可以在同一反应管内检测目的基因和内参基因RNA，利用内参基因进行样本质量控制。同时，RT-nestRPA技术可快速检测出浓度低至1拷贝/uL的待测样本，在较短时间内(15min)通过显著的扩增产物荧光值差异，准确可靠地判读阳性结果和阴性结果，具有高灵敏度、高特异性和高重复性的特点。
实施例1 SARS-CoV-2新冠病毒N基因的引物和探针设计及筛选
通过检索国家生物信息库(CNCB)公布的SARS-CoV-2基因突变信息，发现新冠病毒基因组中各基因的突变频率较为平均一致，在0.78％-1％范围(图1)。其中ORF1基因的核苷酸突变数达17396个(图1)，是新冠病毒基因组中突变数最多的基因，作为国家CDC推荐的检测基因，这可能是目前临床应用中ORF1基因检测效果较差的重要原因之一。目前实验室可获得的SARS-CoV-2假病毒标准品RNA仅包含ORF1、E和N基因的核酸序列，经过序列分析E基因的序列结构不适合设计RPA探针，而N基因的变异数均较低，因此本发明针对N基因，按照RPA引物和探针设计原则进行设计。
合适的引物序列是RPA高效扩增的重要一环，首先寻找N基因有连续3个胸腺嘧啶(T)的序列设计探针，以FAM荧光素修饰探针。在探针两端不同位置分别设计10对引物(表1)，以筛选出最高效的引物和探针组合。在引物筛选时，将第一条正向引物(F)分别与第一至第十条反向引物(R)配对。接着再筛选反向引物，将第一条反向引物(R)分别与第一至第十条正向引物(F)配对。通过两轮引物筛选，找到具有最大斜率和最短起峰时间的最佳引物组合。
表1 N基因引物和探针设计
Figure PCTCN2021102134-appb-000001
Figure PCTCN2021102134-appb-000002
将新冠假病毒RNA质控品，取室温复温混匀的假病毒培养液，使用柱式抽提法提取假病毒RNA，用无酶水溶解RNA。采用系列稀释法将假病毒RNA稀释为1×10 5拷贝/uL,1×10 4拷贝/uL,1×10 3拷贝/uL,500拷贝/uL,100拷贝/uL,10拷贝/uL和1拷贝/uL。以1×10 5拷贝/uL假病毒RNA作为模板，经过初步筛选，结果显示FN-1/RN-2、FN1-/RN-3、FN6/RN-5、FN-6/RN-7的扩增效率较高。进一步上述引物对，分别以1×10 5拷贝/uL,1×10 4拷贝/uL,1×10 3拷贝/uL,500拷贝/uL,100拷贝/uL,10拷贝/uL和1拷贝/uL假病毒RNA为模板，使用RT-RPA方法，设置反应时间为30分钟，观察待测样本与空白对照(无酶水)之间的荧光值差异是否有统计学意义，判断标准是p<0.05为阳性，p>0.05为阴性，检测结果为阳性的最低RNA浓度作为该引物对的灵敏度。结果显示引物对FN-6/RN-7和FN-1/RN-2具有更高的灵敏度(表2)，其最低检测限均为500拷贝/uL(图2)。但这一结果与目前的qPCR方法相比，检测方法的灵敏度并没有明显优势。
表2 N基因的优选引物对和探针组合
Figure PCTCN2021102134-appb-000003
RT-RPA反应体系为：酶的冻干粉(包括逆转录酶、RNA酶抑制剂、重组酶、单链DNA结合蛋白及聚合酶)，25uL缓冲液V，2.1uL N基因正向外引物(10uM)，2.1uL N基因反向外引物(10uM)，0.6uL特异性探针(10uM)，1uL待测样品，2.5uL乙酸镁(280mM)。醋酸镁一旦加入反应体系中，反应立即开始。
实施例2 RT-nestRPA和RT-RPA对SARS-CoV-2假病毒RNA的检测能力比较
SARS-CoV-2假病毒RNA质控品(含ORF1、E和N基因)，即新冠假病毒培养液，验证N基因优选引物的灵敏度。取室温复温混匀的假病毒培养液，使用柱式抽提法提取假病毒RNA，溶解于无酶水。将假病毒RNA提取液依次稀释为500拷贝/uL,100拷贝/uL,10拷贝/uL和1拷贝/uL作为模板，分别使用RT-nestRPA和RT-RPA方法进行检测，设置反应时间为30 分钟，观察待测样本与空白对照(无酶水)之间的荧光值差异是否有统计学意义，判断标准是p<0.05为阳性，p>0.05为阴性，其最小可检测的RNA浓度作为该方法的LOD。
利用RT-RPA和RT-nestRPA方法检测上述不同浓度RNA样本，发现RT-nestRPA方法针对新冠病毒N基因，最低可检测到假病毒RNA浓度为1拷贝/uL(图3，表3)，而RT-RPA方法仅能检测到假病毒RNA浓度为500拷贝/uL(图3，表3)。RT-nestRPA确实可显著提高SARS-CoV-2病毒N基因检测灵敏度。
表3 RT-nestRPA与RT-RPA方法对SARS-CoV-2假病毒N基因检出能力比较
Figure PCTCN2021102134-appb-000004
本发明以RT-nestRPA(RT-巢式RPA)检测SARS-CoV-2假病毒的方法，包括以下步骤：
步骤一：使用SARS-CoV-2病毒N基因的一对外引物，对模板RNA进行一次RT-RPA反应，预扩增目的基因N基因的第一片段；
步骤二：使用SARS-CoV-2病毒N基因的一对内引物，以扩增出的目的基因第一片段为模板，并在加入N基因不同标记的信号探针的条件下进行一次RPA反应，扩增目的基因N基因的第二片段。通过检测不同标记的探针信号，获得病毒核酸N基因的检测结果。
所述不同标记的信号探针为带有不同信号标记的探针，通常为带有不同荧光素标记的探针，如FAM、HEX、Cy3、Cy5、VIC等荧光素标记探针。
步骤一中RT-RPA反应体系为：酶的冻干粉(包括逆转录酶、RNA酶抑制剂、重组酶、单链DNA结合蛋白及聚合酶)，25uL缓冲液V，2.1uL正向外引物(10uM)，2.1uL反向外引物(10uM)，5uL待测样品，2.5uL乙酸镁(280mM)。醋酸镁一旦加入反应体系中，反应立即开始。
步骤二中RPA反应体系为：酶的冻干粉(包括重组酶、单链DNA结合蛋白、聚合酶及核酸外切酶)，25uL缓冲液VI，2.1uL正向内引物(10uM)，2.1uL反向内引物(10uM)，0.6uL探针(10uM),全部预反应产物，2.5uL乙酸镁(280mM)。醋酸镁一旦加入反应体系中，反应立即开始。
步骤一的RT-RPA反应可在37-42℃进行，反应时间为5-20min。优选反应温度为39℃，反应时间15min。
步骤二的RPA反应可在37-42℃进行，反应时间为1-30min。优选反应温度为39℃，反应时间15min。
通过恒温荧光扩增仪实时观察，在上机检测15min内可见典型指数型扩增曲线，曲线斜率大于20，且荧光值差值与空白/阴性对照具有显著统计学差异(p<0.05)，判断为阳性结果，待测样本新冠核酸N基因检测结果为阳性。
实施例3内参RP基因的引物和探针的设计筛选及RT-nestRPA灵敏度分析
根据CDC指南的推荐，选择人核糖核酸酶P(Homo sapiens ribonuclease P，RP)基因作为内参基因，根据基因序列(NM_001104546.1)，在有连续3个胸腺嘧啶(T)的序列位置设计探针，并以HEX荧光素修饰探针。在探针两端不同位置分别设计10对引物，以筛选出最高效的引物和探针组合(表4)。在引物筛选时，将第一条正向引物(F)分别与第一至第十条反向引物(R)配对。接着再筛选反向引物，将第一条反向引物(R)分别与第一至第十条正向引物(F)配对。通过两轮引物筛选，找到具有最大斜率和最短起峰时间的最佳引物组合。
表4 RP基因引物和探针设计
Figure PCTCN2021102134-appb-000005
Figure PCTCN2021102134-appb-000006
利用实验室收集的健康人外周血样本(样本经nestRPA检测SARS-CoV-2病毒N基因和试纸条血清IgG/IgM抗体检测结果均为阴性)，经3000rpm高速离心分离得到血细胞，采用柱式抽提法提取总RNA，用于筛选RP基因引物并测试优选引物最低检出能力。将血细胞总RNA用10倍梯度稀释法依次稀释10倍、100倍、1000倍、5000倍、10000倍。用RT-RPA方法，在30分钟反应终点，观察待测样本与空白对照之间的荧光度差异是否有统计学意义，判断标准是p<0.05为阳性，p>0.05为阴性。经筛选，发现检测相同浓度血细胞总RNA样本，引物对RP-F7/RP-R10和RP-F2/RP-R6满足内外引物对的位置关系，并且扩增曲线斜率值较大(表5、图4)，其最低可检测到稀释100倍的血细胞总RNA样本(图5左)。利用RT-nestRPA方法，RP基因可检测到稀释5000倍的血细胞总RNA样本,显著提高了RP基因检测灵敏度(图5右)。
表5 RP基因的优选引物对和探针组合
Figure PCTCN2021102134-appb-000007
本发明以RT-nestRPA(RT-巢式RPA)检测RP内参基因的方法，包括以下步骤：
步骤一：使用RP基因的一对外引物，对模板RNA进行一次RT-RPA反应，预扩增目的基因RP基因的第一片段；
步骤二：使用SARS-CoV-2病毒RP基因的一对内引物，以扩增出的目的基因第一片段为模板，并在加入RP基因不同标记的信号探针的条件下进行一次RPA反应，扩增目的RP基因的第二片段。通过检测不同标记的探针信号，获得内参RP基因的检测结果。
所述不同标记的信号探针为带有不同信号标记的探针，通常为带有不同荧光素标记的探针，如FAM、HEX、Cy3、Cy5、VIC等荧光素标记探针。
步骤一中RT-RPA反应体系为：酶的冻干粉(包括逆转录酶、RNA酶抑制剂、重组酶、单链DNA结合蛋白及聚合酶)，25uL缓冲液V，2.1uL正向外引物(10uM)，2.1uL反向外引物(10uM)，5uL待测样品，2.5uL乙酸镁(280mM)。醋酸镁一旦加入反应体系中，反应立即开始。
步骤二中RPA反应体系为：酶的冻干粉(包括重组酶、单链DNA结合蛋白、聚合酶及核酸外切酶)，25uL缓冲液VI，2.1uL正向内引物(10uM)，2.1uL反向内引物(10uM)，0.6uL探针(10uM),全部预反应产物，2.5uL乙酸镁(280mM)。醋酸镁一旦加入反应体系中，反应立即开始。
步骤一的RT-RPA反应可在37-42℃进行，反应时间为5-20min。优选反应温度为39℃，反应时间15min。
步骤二的RPA反应可在37-42℃进行，反应时间为1-30min。优选反应温度为39℃，反应时间15min。
通过恒温荧光扩增仪实时观察，在上机检测15min内可见典型指数型扩增曲线，曲线斜率大于20，且荧光值差值与空白/阴性对照具有显著统计学差异(p<0.05)，判断为阳性结果，待测样本内参RP基因检测结果为阳性。
实施例4模拟SARS-CoV-2病毒感染阳性样本的制备及RT-nestRPA双基因检测结果
RPA或RT-RPA检测方法通常一个反应仅能检测一个目的基因，如需明确待测样本的内参基因表达情况，则需要另外一个反应管进行检测。考虑到SARS-CoV-2病毒是RNA病毒的特点，为了简化实验步骤、降低检测成本同时提高检测灵敏度，本发明首次提出RT-nestRPA概念，并创新性地将SARS-CoV-2病毒N基因和人类管家基因RP基因在同一反应管中进行检测，直接针对RNA样本，进行高灵敏度地快速检测SARS-CoV-2病毒N基因和内参RP基因，监测体系中包含内参基因RP基因对实验过程进行质量控制，避免假阴性结果。
由于SARS-CoV-2病毒具有极强的传染性，因此我们购买SARS-CoV-2病毒RNA质控品(假病毒培养液,含N基因，广州邦德盛生物科技有限公司)用于RT-nestRPA技术的引物筛选和灵敏度测试。同时收集健康人外周血样本(样本经RT-nestRPA N基因检测和新冠IgG/IgM抗体检测结果均为阴性)，经3000rpm高速离心分离得到血细胞，将两种样本混合以模拟符合真实SARS-CoV-2病毒感染阳性样本的核酸组成结构(样本中同时含有SARS-CoV-2病毒RNA和RP基因)。模拟样本由假病毒培养液和健康人血细胞按1：1体积比混合，采用柱式抽提法提取混合样本总RNA。同时分别提取假病毒培养液RNA和人血细胞RNA，用于RT-nestRPA双基因检测。
RT-nestRPA双基因检测包含两个步骤：
步骤一：在同一反应管中，同时使用SARS-CoV-2病毒N基因的一对外引物和RP基因的一对外引物，对模板RNA进行一次RT-RPA反应，同时预扩增目的基因N基因和RP基因的第一片段。
步骤二：在一个新的反应管中，同时使用SARS-CoV-2病毒N基因的一对内引物和RP基因的一对内引物，以扩增出的目的基因第一片段为模板，并在同时加入N基因和RP基因不同标记的信号探针的条件下进行一次RPA反应，同时扩增目的基因N基因和RP基因的第二片段。
通过检测不同标记的探针信号，获得病毒核酸和内参基因的检测结果。
所述不同标记的信号探针为带有不同信号标记的探针，通常为带有不同荧光素标记的探针，如FAM、HEX荧光素标记探针。
步骤一中RT-RPA反应体系为：酶的冻干粉(包括逆转录酶、RNA酶抑制剂、重组酶、单链DNA结合蛋白及聚合酶)，25uL缓冲液V，1.0uL N基因正向外引物(10uM)，1.0uL N基因反向外引物(10uM)，1.0uL RP基因正向外引物(10uM)，1.0uL RP基因反向外引物(10uM)，5uL待测样品，2.5uL乙酸镁(280mM)。醋酸镁一旦加入反应体系中，反应立即开始。
步骤二中RPA反应体系为：酶的冻干粉(包括重组酶、单链DNA结合蛋白、聚合酶及核酸外切酶)，25uL缓冲液VI，1.0uL N基因正向内引物(10uM)，1.0uL N基因反向内引物(10uM)，1.0uL RP基因正向内引物(10uM)，1.0uL RP基因反向内引物(10uM)，0.3uL N基因探针(10uM),0.3uL RP基因探针(10uM)，全部预反应产物(约37uL)，2.5uL乙酸镁(280mM)。醋酸镁一旦加入反应体系中，反应立即开始。
步骤一的RT-RPA反应可在37-42℃进行，反应时间为5-20min。
优选反应温度为39℃，反应时间15min。
步骤二的RPA反应可在37-42℃进行，反应时间为1-30min。
优选反应温度为39℃，反应时间15min。
通过恒温荧光扩增仪实时观察，在上机检测15min内可见典型指数型扩增曲线，曲线斜率大于20，且荧光值差值与空白/阴性对照具有显著统计学差异(p<0.05)，则为阳性结果。
利用RT-nestRPA技术对模拟样本RNA、假病毒RNA和血细胞总RNA进行检测，发现在N基因检测通道(FAM荧光信号)的结果显示模拟样本RNA和假病毒RNA呈现典型扩增曲线，而血细胞总RNA未见明显扩增曲线(图6左)。在RP基因检测通道(HEX荧光信号)的结果显示模拟样本RNA和血细胞总RNA呈现典型扩增曲线，而假病毒RNA未见明显扩增曲线(图6右)。该结果说明RT-nestRPA双基因检测系统不会造成不同通道间的显著荧光信号干扰，不同引物间不会产生可检测到的交叉反应。
当待测样本的N基因检测通道和内参RP基因检测通道同时出现典型指数型扩增曲线，则判定该待测样本SARS-CoV-2病毒核酸检测结果为阳性。若待测样本的N基因检测通道未 见明显扩增曲线，同时内参RP基因检测通道出现典型指数型扩增曲线，则判定该待测样本SARS-CoV-2病毒核酸检测结果为阴性。若待测样本的N基因检测通道出现明显扩增曲线，同时内参RP基因检测通道未见典型指数型扩增曲线，则判定该待测样本检测结果为可疑，必要时进行第二次检测。
实施例5 RT-nestRPA对临床样本进行N基因和RP基因共同检测
所有临床样本均按照核酸提取试剂盒的操作步骤(Huayin Biotech Co.,Shenzhen,China)使用自动核酸提取仪，提取核酸(包含DNA和RNA)。所有核酸样本均通过实时定量PCR(qPCR)核酸检测，判断是否为SARS-CoV-2感染阳性样本。2020年2月至2020年4月期间，在深圳第三人民医院共收集到来自84位患者的111例样本(鼻咽拭子或痰液)。包括来自15位新冠肺炎确诊患者的qPCR检测结果阳性样本15例，来自37位新冠肺炎确诊患者qPCR检测结果阴性样本64例，以及来自32位复工健康体检者样本32例(图7，图7中数值为病例数)。
用实施例4所述的RT-nestRPA对临床样本提取的RNA进行N基因和RP基因共同检测。将qPCR(N基因和内参基因)检测结果阳性的临床样本用本发明RT-nestRPA方法检测，并将其中1例qPCR(N基因和内参基因)检测结果阳性样本进行连续11次10倍比稀释。随后使用RT-nestRPA双基因检测方法对该系列稀释样本进行检测。对qPCR(N基因)检测结果为阴性的样本也使用本发明RT-nestRPA双基因方法进行检测。
来自15位COVID-19确诊患者的15份鼻拭子或痰样本，这些样本是使用qPCR方法检测SARS-CoV-2N基因均为阳性。使用本发明RT-nestRPA方法检测，与qPCR方法N基因的检测结果一致，阳性符合率为100％(15/15)。同时使用本发明RT-nestRPA方法检测15例样本的内参基因(RP基因)，检测结果均为阳性(表6)。
表6 RT-nestRPA在qPCR阳性样本的N基因和内参基因的检测结果比较
Figure PCTCN2021102134-appb-000008
Figure PCTCN2021102134-appb-000009
选取其中一个阳性样本，进行连续11次10倍比稀释并分别使用本发明RT-nestRPA与qPCR方法进行同步检测，比较本发明RT-nestRPA与qPCR方法的灵敏度。结果显示，该SARS-CoV-2核酸阳性样本在第10次连续稀释后，使用本发明RT-nestRPA方法的检测结果仍为阳性，而qPCR方法对第4次稀释后的样本的检测结果已呈现阴性(表7)。
表7 RT-nestRPA与qPCR方法对一阳性样本的N基因检测结果比较
Figure PCTCN2021102134-appb-000010
收集来自37位COVID-19确诊患者的64例鼻拭子、肛拭子或痰样本，这些样本使用qPCR方法检测SARS-CoV-2核酸结果均为阴性，用本发明RT-nestRPA方法检测发现其中29.69％(19/64)样本结果为阳性(表8)。该结果表明使用qPCR方法检测COVID-19确诊患者样本SARS-CoV-2核酸结果存在假阴性，使用本发明RT-nestRPA方法的检测灵敏度优于qPCR方法。
表8 RT-nestRPA在qPCR阴性样本的N基因和内参基因的检测结果比较
Figure PCTCN2021102134-appb-000011
Figure PCTCN2021102134-appb-000012
Figure PCTCN2021102134-appb-000013
为了观察在一般健康人群中是否存在SARS-CoV-2核酸阳性的无症状感染者，收集复工健康体检人员的咽拭子样本共32份，所有样本使用qPCR方法检测SARS-CoV-2核酸结果均为阴性。然而，用本发明RT-nestRPA方法检测发现3例(9.37％)样本N基因结果为阳性(表9)，同时这3例样本的内参基因RP基因结果也为阳性(图8)。这一结果表明本发明RT-nestRPA的核酸检测方法比qPCR方法具有更高灵敏度，对于早期发现已携带SARS-CoV-2的无症状感染者具有重要意义。
表9 RT-nestRPA在复工健康体检者样本N基因和内参基因的检测结果
Figure PCTCN2021102134-appb-000014
Figure PCTCN2021102134-appb-000015
本发明RT-nestRPA的核酸检测方法共计对111例临床样本进行新冠病毒N基因及内参基因RP基因的检测。本发明RT-nestRPA方法对15例qPCR阳性样本的检测结果均为阳性(15/15)，与qPCR检测方法的一致率为100％，假阴性率为0％。此外，本发明RT-nestRPA方法在96例qPCR阴性样本中，检测到22例(22.91％)样本新冠病毒N基因结果阳性(图9)。综合以上结果，本发明相比qPCR方法具有更高的灵敏度，对于新冠病毒感染者的早期检测具有较好的应用前景。
以上所述实施例仅表达了本发明的实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (20)

  1. 一种检测病原体RNA、及动物或人类基因RNA的试剂盒，其特征在于，所述试剂盒包含以RT-巢式RPA检测病原体RNA、及动物或人类基因RNA的引物对，及以RT-巢式RPA检测内参基因RNA的引物对，所述检测病原体RNA、及动物或人类基因RNA的引物对与检测内参基因RNA的引物对在同一反应管中同时进行检测反应。
  2. 根据权利要求1所述的检测病原体RNA、及动物或人类基因RNA的试剂盒，其特征在于，所述病原体RNA包括病毒、细菌、衣原体、支原体、立克次体、螺旋体、真菌、或寄生虫的RNA，所述动物或人类基因RNA包括人类肿瘤相关基因mRNA以及遗传病相关基因mRNA。
  3. 根据权利要求2所述的检测病原体RNA、及动物或人类基因RNA的试剂盒，其特征在于，所述病原体为SARS-CoV-2新冠病毒。
  4. 根据权利要求3所述的检测病原体RNA、及动物或人类基因RNA的试剂盒，其特征在于，所述以RT-巢式RPA检测SARS-CoV-2新冠病毒RNA的引物对是针对SEQ ID NO：1所示SARS-CoV-2新冠病毒N基因序列或其部分序列设计的引物对。
  5. 根据权利要求4所述的检测病原体RNA、及动物或人类基因RNA的试剂盒，其特征在于，所述以RT-巢式RPA检测SARS-CoV-2新冠病毒的引物对包括：SEQ ID NO：3和SEQ ID NO：4所示序列的外引物对，SEQ ID NO：5和SEQ ID NO：6所示序列的内引物对。
  6. 根据权利要求3所述的检测病原体RNA、及动物或人类基因RNA的试剂盒，其特征在于，所述内参基因为人类管家基因RP基因。
  7. 根据权利要求6所述的检测病原体RNA、及动物或人类基因RNA的试剂盒，其特征在于，所述以巢式RPA检测RP基因的引物对包括：SEQ ID NO：9和SEQ ID NO：10所示序列的外引物对，SEQ ID NO：11和SEQ ID NO：12所示序列的内引物对。
  8. 根据权利要求3所述的检测病原体RNA、及动物或人类基因RNA的试剂盒，其特征在于，所述试剂盒还包含特异性针对SEQ ID NO：1所示SARS-CoV-2新冠病毒N基因序列或其部分序列设计的探针。
  9. 根据权利要求6所述的检测病原体RNA、及动物或人类基因RNA的试剂盒，其特征在于，所述试剂盒还包含特异性针对SEQ ID NO：7所示RP基因序列或其部分序列设计的探针。
  10. 一种检测SARS-CoV-2新冠病毒的引物对，其特征在于，所述引物对是以RT-巢式RPA检测SARS-CoV-2新冠病毒N基因的引物对，其包含外引物对和内引物对。
  11. 根据权利要求10所述的检测SARS-CoV-2新冠病毒的引物对，其特征在于，所述引物对是针对SEQ ID NO：1所示SARS-CoV-2新冠病毒N基因序列或其部分序列设计的引物 对，所述外引物对具有如SEQ ID NO：3和SEQ ID NO：4所示的碱基序列，所述内引物对具有如SEQ ID NO：5和SEQ ID NO：6所示的碱基序列。
  12. 一种检测SARS-CoV-2新冠病毒的探针，其特征在于，所述探针是特异性针对SEQ ID NO：1所示SARS-CoV-2新冠病毒N基因序列或其部分序列设计的探针。
  13. 根据权利要求12所述的检测SARS-CoV-2新冠病毒的探针，其特征在于，所述探针具有如SEQ ID NO：2所示的碱基序列。
  14. 一种检测SARS-CoV-2新冠病毒的试剂盒，其特征在于，所述试剂盒包含权利要求10或11所述的引物对。
  15. 根据权利要求14所述的检测SARS-CoV-2新冠病毒试剂盒，其特征在于，所述试剂盒还包含以RT-巢式RPA检测内参基因的引物对，所述检测SARS-CoV-2新冠病毒N基因的引物对与检测内参基因的引物对在同一反应管中同时进行检测反应。
  16. 根据权利要求15所述的检测SARS-CoV-2新冠病毒试剂盒，其特征在于，所述试剂盒还包含权利要求12或13所述的探针，以及特异性针对内参基因的探针。
  17. 根据权利要求15或16所述的检测SARS-CoV-2新冠病毒试剂盒，其特征在于，所述内参基因为人类管家基因RP基因。
  18. 根据权利要求17所述的检测SARS-CoV-2新冠病毒试剂盒，其特征在于，所述以RT-巢式RPA检测RP基因的引物对包括：SEQ ID NO：9和SEQ ID NO：10所示序列的外引物对，SEQ ID NO：11和SEQ ID NO：12所示序列的内引物对。
  19. 根据权利要求17所述的检测SARS-CoV-2新冠病毒试剂盒，其特征在于，所述以RT-巢式RPA检测该RP基因的探针是特异性针对SEQ ID NO：7所示RP基因序列或其部分序列设计的探针，其具有如SEQ ID NO：8所示的碱基序列。
  20. 权利要求1-9中任一项所述试剂盒在制备检测病原体RNA的产品中的应用。
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