CN113891944A

CN113891944A - Kit for detecting pathogen RNA, animal or human gene RNA and application thereof

Info

Publication number: CN113891944A
Application number: CN202180001625.8A
Authority: CN
Inventors: 黄婉秋; 黄健
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-06-24
Filing date: 2021-06-24
Publication date: 2022-01-04
Also published as: WO2022266946A1

Abstract

The invention discloses a kit for detecting pathogen RNA and animal or human gene RNA, which comprises a primer pair for detecting the pathogen RNA and the animal or human gene RNA by using RT-nested RPA and a primer pair for detecting an internal reference gene by using RT-nested RPA, wherein the primer pair for detecting the pathogen RNA and the animal or human gene RNA and the primer pair for detecting the internal reference gene simultaneously carry out detection reaction in the same reaction tube. The kit for detecting the RNA of the pathogen and the RNA of the animal or human gene realizes the simultaneous detection of the RNA level of the target gene and the reference gene of the pathogen, increases the quality control of the reference gene on the experimental process, and avoids false negative results. Moreover, the kit for detecting the new coronavirus can detect the SARS-CoV-2 virus RNA fragment of 1 copy/uL at the lowest, has ultrahigh sensitivity, provides new technical support for early discovery, early isolation and early treatment of the new coronavirus SARS-CoV-2, and has very good application prospect.

Description

Kit for detecting pathogen RNA, animal or human gene RNA and application thereof

Technical Field

The invention belongs to the technical field of biological engineering, and particularly relates to a kit for detecting pathogens based on an RT-nested RPA (RT-nestRPA) technology and application thereof.

Background

Since the end of 12 months in 2019, a large-scale epidemic of novel coronavirus infectious pneumonia (COVID-19) has emerged globally. COVID-19 is a global epidemic caused by infection with a novel coronavirus SARS-CoV-2. COVID-19 is characterized by long latent period, strong infectivity and high death rate. The epidemic is spread rapidly, and has caused huge harm to human health and great social and economic hazards.

Coronavirus (CoV), an enveloped single-stranded RNA virus, is widely transmitted in humans, and other mammals and birds, and can cause respiratory, intestinal, hepatic and nervous system diseases. 7 coronaviruses are known to cause disease in humans, 4 of which, such as Cov-229E, Cov-OC43, Cov-NL63 and Cov-HKU1, are prevalent in the human population and often cause common cold symptoms. In addition, the 3 coronaviruses SARS-CoV, MERS-CoV and SARS-CoV-2 are highly dangerous and can cause severe pneumonia and even death.

By 6 months and 8 days 2021, more than 1.73 million people have been diagnosed with new crown pneumonia (COVID-19) and the number of death cases reaches 373 ten thousand. At present, the work of preventing and controlling new coronary pneumonia in China is normalized, sporadic reports of cases in different cities are frequent, and the overseas input of case reports is continuous. At the same time, the epidemic in other countries around the world is still spreading continuously. The total confirmed cases in the United states, Brazil and India are far more than 1600 ten thousand. The survival chance of SARS-CoV-2 in environment is greatly increased, and the reports of detecting SARS-CoV-2 positive result by cold chain transportation staff, vehicles and even meat product samples are frequently available. In the new prevention and control measures of coronary pneumonia, early diagnosis and early treatment are the key. SARS-CoV-2 nucleic acid detection is an important means for quickly diagnosing new coronary pneumonia, and the nucleic acid detection method commonly used at present is fluorescence quantitative PCR (qPCR) technology. Based on the Chinese online data, 115 companies in China have developed 116 kits for detecting SARS-CoV-2 nucleic acid, 98 (84.5%) of which employ qPCR technology.

However, the nucleic acid detection technique of SARS-CoV-2 based on qPCR frequently results in "false negative" in clinical applications. Nucleic acid 'false negative' results based on a qPCR detection method are reported in sequence by a plurality of hospitals in China. Recent news shows that a positive nucleic acid result is obtained after 6 nucleic acid tests of patients, and before now, some patients are found to be negative after two nucleic acid tests of regular treatment, but the nucleic acid test results of the patients are positive again during isolation and observation. These false negative results greatly increase the complexity of the control of COVID-19 epidemics.

Viral nucleic acid testing, from the time a sample is taken from a patient to the completion of a test report, proceeds through a number of steps including sample collection, sample storage, sample transport to a testing laboratory, virus inactivation, cell lysis, nucleic acid extraction, testing and reporting of the test report. Any one of these steps may be somewhat problematic and may lead to false negative results. Wherein, the detection technology with high sensitivity, high specificity and high repeatability is very important for improving the detection quality of SARS-CoV-2 nucleic acid and reducing the false negative rate of experiments.

Recombinase Polymerase Amplification (RPA) is based on a Recombinase Polymerase-mediated Amplification principle, simulates an enzyme reaction process of in vivo DNA replication, depends on specific enzymes and protein combinations (Recombinase, single-stranded binding protein and DNA Polymerase) to perform specific Amplification on a DNA template, can realize rapid specific Amplification (37-42 ℃ for 5-30min) under the condition of approaching body temperature, is used as an isothermal technology to reduce the dependence on high-precision expensive instruments, stable power supply facilities and high-level laboratories, can be completed by only one thermostatic device, can judge whether Amplification products exist in real time by a fluorescence analysis device, has the characteristic of simpler facility and operation requirements, and is applied to multiple fields of life science research, medical detection, agriculture, food safety, transgenic detection and the like.

At present, an RT-RPA (reverse transcriptase-recombinase polymerase amplification) technology developed based on an RPA principle is available, namely reverse transcriptase and an RNase inhibitor are added in an RPA reaction system, so that an RNA nucleic acid sample can be rapidly amplified under a constant temperature condition, and the defect that the RNA sample can be detected only by reverse transcription into cDNA is overcome.

In the traditional RPA or RT-RPA detection, only 1 target gene is often detected in one reaction, and a new reaction is needed if an internal reference gene of a sample to be detected needs to be detected. This undoubtedly increases the time and economic costs of detection and increases the uncertainty of the interpretation of the results.

Although epidemic prevention work of vaccination against new coronavirus is vigorously popularized in various countries around the world at present, it still takes a long time to achieve complete population immunity. At present, the COVID-19 epidemic caused by SARS-CoV-2 virus is still spread continuously in a plurality of countries all over the world, the SARS-CoV-2 virus has stronger infectivity and faster propagation speed, and the epidemic is not effectively controlled. In addition, a plurality of cities in China have small-scale aggregated cases, and in 4 months this year, new crown epidemic situation is developed in the Reuli city in Yunnan province in China. Therefore, in order to realize effective prevention and control of the COVID-19 epidemic situation, a novel rapid nucleic acid detection technology with high sensitivity, high specificity and high repeatability is urgently needed to be developed.

Disclosure of Invention

The invention aims to solve the technical problem that a kit for quickly and accurately detecting pathogens such as SARS-CoV-2 new coronavirus and the like is lacked at present, and provides a kit for detecting pathogens based on an RT-nested RPA (RT-nestRPA) technology, wherein the kit simultaneously detects pathogen gene RNA and reference gene RNA by utilizing the RT-nestRPA technology, and can quickly, accurately, sensitively and specifically detect the pathogen RNA such as SARS-CoV-2 virus and the like.

In order to solve the technical problems, the invention is realized by the following technical scheme:

in one aspect of the invention, a kit for detecting pathogen RNA and animal or human gene RNA is provided, the kit comprises a primer pair for detecting pathogen RNA and animal or human gene RNA by RT-nested RPA and a primer pair for detecting internal reference gene RNA by RT-nested RPA, and the primer pair for detecting pathogen RNA and animal or human gene RNA and the primer pair for detecting internal reference gene RNA carry out detection reaction simultaneously in the same reaction tube.

Preferably, the pathogen RNA includes viral, bacterial, chlamydia, mycoplasma, rickettsia, spirochete, fungal, or parasitic RNA, and the animal or human gene RNA includes human tumor-associated gene mRNA and genetic disease-associated gene mRNA.

In a specific embodiment of the invention, the pathogen is SARS-CoV-2 new coronavirus, and the primer pair for detecting SARS-CoV-2 new coronavirus by RT-nested RPA is a primer pair directed to the nucleotide sequence shown in SEQ ID NO: 1 or a primer pair designed by the partial sequence of the N gene sequence of the SARS-CoV-2 new coronavirus. More preferably, the primer pair for detecting SARS-CoV-2 new coronavirus by RT-nested RPA comprises: SEQ ID NO: 3 and SEQ ID NO: 4, the sequence shown in SEQ ID NO: 5 and SEQ ID NO: 6.

Preferably, the reference gene is a human housekeeping gene, RP gene. The primer pair for detecting the RP gene by the RT-nested RPA comprises: SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

Preferably, the kit further comprises a nucleic acid sequence specific for SEQ ID NO: 1 or partial sequence thereof, and a probe specifically designed for the N gene sequence of the SARS-CoV-2 new coronavirus or partial sequence thereof, wherein the probe is specifically designed for the N gene sequence of the SARS-CoV-2 new coronavirus or partial sequence thereof shown in SEQ ID NO: 7 or a partial sequence thereof.

In another aspect of the present invention, there is also provided a primer pair for detecting SARS-CoV-2 new coronavirus, which is a primer pair for detecting SARS-CoV-2 new coronavirus N gene in RT-nested RPA, comprising an outer primer pair and an inner primer pair.

Preferably, the primer pair is directed against SEQ ID NO: 1 or a partial sequence thereof, wherein the outer primer pair has the sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4, and the inner primer pair has a nucleotide sequence shown as SEQ ID NO: 5 and SEQ ID NO: 6 in a sequence listing.

In another aspect of the present invention, there is also provided a probe for detecting SARS-CoV-2 novel coronavirus, which is a probe specific for the polypeptide of SEQ ID NO: 1 or partial sequence of N gene sequence of SARS-CoV-2 new coronavirus.

Preferably, the probe has the nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence shown in the figure.

In another aspect of the present invention, there is also provided a kit for detecting SARS-CoV-2 novel coronavirus, which comprises the above primer pair.

Preferably, the kit also comprises a primer pair for detecting the internal reference gene by using 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 carry out detection reaction simultaneously in the same reaction tube.

The kit also comprises the probe and a probe specific to the reference gene.

In another aspect of the invention, the application of the kit in the preparation of products for detecting pathogens is also provided.

The kit for detecting the pathogen simultaneously carries out nucleic acid detection on the RNA of the pathogen and the RNA of the internal reference gene by an RT-nestRPA method, realizes the purpose of simultaneously detecting the target gene and the internal reference gene in the same tube, and ensures that the judgment of a detection result is more accurate. The kit for detecting the SARS-CoV-2 new coronavirus in the embodiment of the invention can detect 1 copy/uL of SARS-CoV-2 nucleic acid fragment at the lowest, has ultrahigh sensitivity, and the RT-nestRPA in the invention is carried out at the constant temperature of 39 ℃, does not need expensive detection equipment, has short detection time and only needs 15min, provides new technical support for early discovery, early isolation and early treatment of the new coronavirus SARS-CoV-2, and has very good application prospect.

Drawings

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

FIG. 1 is a SARS-CoV-2 genome mutation profile of example 1 of the present invention;

FIG. 2 is a graph showing the LOD results of the preferred primers for SARS-CoV-2N gene detection using RT-RPA in example 1 of the present invention;

FIG. 3 is a graph comparing the LOD results of SARS-CoV-2 pseudoviral RNA detection using RT-nestRPA in example 2 of the present invention;

FIG. 4 is a graph showing the results of screening for preferred primers for RP gene using RT-RPA method in example 3 of the present invention;

FIG. 5 is a graph showing comparison of the detection ability of RNA RP gene in human blood cells by RT-RPA and RT-nestRPA methods in example 3 of the present invention;

FIG. 6 is a graph showing the results of detection of RNA in a mock sample by RT-nestRPA double gene detection method in example 4 of the present invention;

FIG. 7 is a schematic diagram of grouping clinical samples of the novel coronary pneumonia according to example 5 of the present invention;

FIG. 8 is a graph showing the results of detecting N gene and RP gene in a sample of a physical examination of a double worker using RT-nestRPA in example 5 of the present invention;

FIG. 9 is a statistical chart of the results of testing clinical samples using RT-nestRPA and qPCR in example 5 of the present invention.

Detailed Description

The invention provides an RT-nestRPA concept, and develops an ultrahigh-sensitivity pathogen nucleic acid detection technology based on a fluorescence detection analyzer, so that the purpose gene and the reference gene of a pathogen can be simultaneously detected in the same tube, and the detection result can be more accurately interpreted. In the following examples of the present invention, a kit for detecting SARS-CoV-2 novel coronavirus, which can detect both SARS-CoV-2 virus N gene and human housekeeping gene RP gene, can detect SARS-CoV-2 nucleic acid fragment of 1 copy/uL under laboratory conditions, and has an ultrahigh sensitivity, is specifically described.

Compared with the traditional RPA and RT-RPA technologies, the RT-nestRPA technology adopted by the invention has the advantages that the RNA of a target gene and an internal reference gene can be detected in the same reaction tube, and the internal reference gene is utilized to carry out sample quality control. Meanwhile, the RT-nestRPA technology can quickly detect a sample to be detected with the concentration as low as 1 copy/uL, and accurately and reliably judges a positive result and a negative result through obvious fluorescence value difference of amplification products in a short time (15min), so that the kit has the characteristics of high sensitivity, high specificity and high repeatability.

EXAMPLE 1 primer and Probe design and screening of SARS-CoV-2 New coronavirus N Gene

Through searching SARS-CoV-2 gene mutation information published by the national biological information library (CNCB), the mutation frequencies of all genes in the new coronavirus genome are relatively uniform on average and are in the range of 0.78-1% (figure 1). Among them, the nucleotide mutation number of ORF1 gene is 17396 (FIG. 1), which is the gene with the most mutation number in the genome of new coronavirus, and it is one of the important reasons for the poor detection effect of ORF1 gene in clinical application. The SARS-CoV-2 pseudovirus standard product RNA available in laboratory only contains nucleic acid sequences of ORF1, E and N genes, and the sequence structure of E gene is not suitable for designing RPA probe through sequence analysis, and the variation number of N gene is low, so that the invention is designed according to the RPA primer and probe design principle for N gene.

An appropriate primer sequence is an important circle for efficient amplification of RPA, and a probe is designed by firstly searching a sequence of N genes with continuous 3 thymines (T) and modifying the probe with FAM fluorescein. 10 pairs of primers (Table 1) were designed at different positions at both ends of the probe, respectively, to screen out the most efficient combinations of primers and probes. In the primer screening, the first forward primer (F) is paired with the first to tenth reverse primers (R), respectively. Then, the reverse primers are screened again, and the first reverse primer (R) is paired with the first to tenth forward primers (F), respectively. Through two rounds of primer screening, the optimal primer combination with the largest slope and the shortest peak time was found.

TABLE 1N Gene primer and Probe design

Taking the pseudovirus culture solution which is re-warmed and uniformly mixed at room temperature for the new corona pseudovirus RNA quality control product, extracting the pseudovirus RNA by using a column type extraction method, and dissolving the RNA by using non-enzyme water. Pseudoviral RNA was diluted 1X 10 by serial dilution⁵copy/uL, 1X 10⁴copy/uL, 1X 10³copy/uL, 500 copies/uL, 100 copies/uL, 10 copies/uL and 1 copy/uL. At 1 × 10⁵The copy/uL pseudovirus RNA is used as a template, and the result of primary screening shows that the amplification efficiency of FN-1/RN-2, FN1-/RN-3, FN6/RN-5 and FN-6/RN-7 is higher. Further, the above primer sets were each at 1X 10⁵copy/uL, 1X 10⁴copy/uL, 1X 10³Using the RNA of the pseudoviruses with copy/uL, 500 copy/uL, 100 copy/uL, 10 copy/uL and 1 copy/uL as templates, using an RT-RPA method, setting the reaction time to be 30 minutes, observing whether the difference of fluorescence values between a sample to be detected and a blank control (without enzyme water) has statistical significance or not, and judging that the standard is p<0.05 is positive, p>0.05 is negative, and the lowest RNA concentration at which the detection result is positive is taken as the sensitivity of the primer pair. The results showed that the primer pairs FN-6/RN-7 and FN-1/RN-2 had higher sensitivity (Table 2), with the lowest detection limit of 500 copies/uL (FIG. 2). However, compared with the current qPCR method, the sensitivity of the detection method has no obvious advantage.

TABLE 2 preferred primer pairs and Probe combinations for the N Gene

The RT-RPA reaction system is as follows: the kit comprises enzyme freeze-dried powder (comprising reverse transcriptase, an RNase inhibitor, a recombinase, a single-stranded DNA binding protein and polymerase), 25uL buffer solution V, 2.1uL N gene forward outer primer (10uM), 2.1uL N gene reverse outer primer (10uM), 0.6uL specific probe (10uM), 1uL sample to be detected and 2.5uL magnesium acetate (280 mM). The reaction started immediately upon addition of magnesium acetate to the reaction system.

Example 2 comparison of the detection Capacity of RT-nestRPA and RT-RPA for SARS-CoV-2 pseudoviral RNA

SARS-CoV-2 pseudovirus RNA quality control (containing ORF1, E and N gene), i.e. new coronaviruses culture solution, verifies the sensitivity of N gene optimized primer. Taking the pseudovirus culture solution which is re-warmed and uniformly mixed at room temperature, extracting pseudovirus RNA by using a column type extraction method, and dissolving in enzyme-free water. Sequentially diluting the pseudovirus RNA extract to 500 copies/uL, 100 copies/uL, 10 copies/uL and 1 copy/uL as templates, respectively using RT-nestRPA and RT-RPA methods to detect, setting the reaction time to 30 minutes, observing whether the difference of fluorescence values between a sample to be detected and a blank control (without enzyme water) has statistical significance, and judging whether the standard is that p <0.05 is positive, p >0.05 is negative, and taking the minimum detectable RNA concentration as the LOD of the method.

The RT-nestRPA method detects RNA samples with different concentrations, and the RT-nestRPA method is found to aim at the N gene of the new coronavirus, the lowest detectable pseudoviral RNA concentration is 1 copy/uL (figure 3 and table 3), and the RT-RPA method can only detect the pseudoviral RNA concentration of 500 copy/uL (figure 3 and table 3). RT-nestRPA can obviously improve the detection sensitivity of SARS-CoV-2 virus N gene.

TABLE 3 comparison of RT-nestRPA and RT-RPA methods for detecting SARS-CoV-2 pseudovirus N Gene

The invention relates to a method for detecting SARS-CoV-2 pseudovirus by RT-nestRPA (RT-nested RPA), which comprises the following steps:

the method comprises the following steps: using a pair of external primers of SARS-CoV-2 virus N gene to perform RT-RPA reaction on template RNA for one time, and pre-amplifying a first segment of target gene N gene;

step two: using a pair of internal primers of SARS-CoV-2 virus N gene, taking the amplified first segment of the target gene as a template, and carrying out one time of RPA reaction under the condition of adding signal probes of different markers of the N gene, thereby amplifying the second segment of the target gene N gene. And detecting the probe signals of different labels to obtain the detection result of the virus nucleic acid N gene.

The signal probes with different labels are probes with different signal labels, and are usually probes with different fluorescein labels, such as fluorescein labeled probes of FAM, HEX, Cy3, Cy5, VIC, and the like.

In the first step, the RT-RPA reaction system is as follows: lyophilized enzyme powder (including reverse transcriptase, RNase inhibitor, recombinase, single-stranded DNA binding protein and polymerase), 25uL buffer solution V, 2.1uL forward outer primer (10uM), 2.1uL reverse outer primer (10uM), 5uL sample to be tested, and 2.5uL magnesium acetate (280 mM). The reaction started immediately upon addition of magnesium acetate to the reaction system.

In the second step, the RPA reaction system is as follows: lyophilized enzyme powder (including recombinase, single-stranded DNA binding protein, polymerase and exonuclease), 25uL buffer VI, 2.1uL forward inner primer (10uM), 2.1uL reverse inner primer (10uM), 0.6uL probe (10uM), all pre-reaction products, 2.5uL magnesium acetate (280 mM). The reaction started immediately upon addition of magnesium acetate to the reaction system.

The RT-RPA reaction in the first step can be carried out at 37-42 ℃ for 5-20 min. The reaction temperature is preferably 39 ℃ and the reaction time is preferably 15 min.

The RPA reaction in the second step can be carried out at 37-42 ℃ for 1-30 min. The reaction temperature is preferably 39 ℃ and the reaction time is preferably 15 min.

And (3) observing in real time by using a constant-temperature fluorescent amplification instrument, wherein a typical exponential amplification curve can be seen within 15min of on-line detection, the slope of the curve is greater than 20, the difference value of the fluorescence value and the blank/negative control has obvious statistical difference (p is less than 0.05), the result is judged to be a positive result, and the detection result of the N gene of the new crown nucleic acid of the sample to be detected is positive.

Example 3 design and screening of primers and probes for reference RP Gene and sensitivity analysis of RT-nestRPA

Human Ribonuclease P (RP) gene was selected as an internal reference gene according to the CDC guidelines, probes were designed at sequence positions with 3 consecutive thymines (T) according to the gene sequence (NM-001104546.1), and the probes were modified with HEX fluorescein. 10 pairs of primers were designed at different positions on both ends of the probe to screen out the most efficient primer and probe combinations (Table 4). In the primer screening, the first forward primer (F) is paired with the first to tenth reverse primers (R), respectively. Then, the reverse primers are screened again, and the first reverse primer (R) is paired with the first to tenth forward primers (F), respectively. Through two rounds of primer screening, the optimal primer combination with the largest slope and the shortest peak time was found.

TABLE 4 RP Gene primer and Probe design

The method comprises the steps of utilizing healthy human peripheral blood samples collected in a laboratory (the sample is negative in the detection results of SARS-CoV-2 virus N gene and test strip serum IgG/IgM antibody through the nestRPA), obtaining blood cells through high-speed centrifugal separation at 3000rpm, and extracting total RNA through a column extraction method for screening RP gene primers and testing the lowest detection capability of preferred primers. The total RNA of the blood cells is diluted by 10 times, 100 times, 1000 times, 5000 times and 10000 times in turn by a 10-time gradient dilution method. And (3) observing whether the difference of the fluorescence degrees between the sample to be detected and the blank control has statistical significance by using an RT-RPA method at the 30-minute reaction end point, wherein the judgment standard is that p <0.05 is positive and p >0.05 is negative. Through screening, the primer pair RP-F7/RP-R10 and RP-F2/RP-R6 meet the position relation of an inner primer pair and an outer primer pair when the blood cell total RNA sample with the same concentration is detected, the slope value of an amplification curve is large (table 5 and figure 4), and the blood cell total RNA sample diluted by 100 times can be detected at the lowest (figure 5, left). By using the RT-nestRPA method, the RP gene can detect a total RNA sample of blood cells diluted by 5000 times, and the detection sensitivity of the RP gene is obviously improved (figure 5 right).

TABLE 5 preferred primer pairs and Probe combinations for RP genes

The invention relates to a method for detecting RP reference genes by RT-nestRPA (RT-nested RPA), which comprises the following steps:

the method comprises the following steps: carrying out RT-RPA reaction on template RNA by using a pair of external primers of the RP gene to pre-amplify a first segment of the RP gene of a target gene;

step two: using a pair of internal primers of SARS-CoV-2 virus RP gene, using the amplified first segment of target gene as template, and making one RPA reaction under the condition of adding signal probes of different labels of RP gene to amplify second segment of target RP gene. And obtaining the detection result of the internal reference RP gene by detecting probe signals of different labels.

And (3) observing in real time by using a constant-temperature fluorescent amplification instrument, wherein a typical exponential amplification curve can be seen within 15min of on-line detection, the slope of the curve is greater than 20, the difference value of the fluorescence value and the blank/negative control have obvious statistical difference (p is less than 0.05), the result is judged to be a positive result, and the result of the detection of the internal reference RP gene of the sample to be detected is positive.

Example 4 preparation of a sample simulating positive infection by SARS-CoV-2 Virus and RT-nestRPA double Gene detection results

The RPA or RT-RPA detection method usually can only detect one target gene in one reaction, and if the expression condition of the reference gene of a sample to be detected needs to be determined, another reaction tube is needed for detection. Considering the characteristic that SARS-CoV-2 virus is RNA virus, in order to simplify the experimental steps, reduce the detection cost and improve the detection sensitivity, the invention firstly proposes the concept of RT-nestRPA, and innovatively detects the N gene of SARS-CoV-2 virus and the RP gene of human housekeeping gene in the same reaction tube, directly aims at RNA samples, and rapidly detects the N gene and the internal reference RP gene of SARS-CoV-2 virus with high sensitivity, and the monitoring system comprises the RP gene of the internal reference gene to control the quality of the experimental process, thereby avoiding false negative results.

Because SARS-CoV-2 virus has extremely strong infectivity, the RNA quality control of SARS-CoV-2 virus (pseudovirus culture solution, containing N gene, Guangzhou Bangdong Biotechnology Co., Ltd.) is purchased for primer screening and sensitivity test of RT-nestRPA technology. Meanwhile, a healthy human peripheral blood sample is collected (the sample is negative through RT-nestRPA N gene detection and new crown IgG/IgM antibody detection results), blood cells are obtained through high-speed centrifugal separation at 3000rpm, and the two samples are mixed to simulate a nucleic acid composition structure (the sample simultaneously contains SARS-CoV-2 virus RNA and RP gene) conforming to a true SARS-CoV-2 virus infection positive sample. The simulated sample consists of pseudovirus culture solution and healthy human blood cells according to the ratio of 1: 1 volume ratio, and extracting the total RNA of the mixed sample by a column extraction method. Meanwhile, pseudovirus culture solution RNA and human blood cell RNA are respectively extracted for RT-nestRPA double-gene detection.

The RT-nestRPA double-gene detection comprises two steps:

the method comprises the following steps: in the same reaction tube, a pair of external primers of SARS-CoV-2 virus N gene and a pair of external primers of RP gene are used simultaneously to perform RT-RPA reaction on template RNA and pre-amplify the first segment of target gene N gene and RP gene simultaneously.

Step two: in a new reaction tube, a pair of internal primers of SARS-CoV-2 virus N gene and a pair of internal primers of RP gene are used simultaneously, the amplified first segment of target gene is used as template, and under the condition of simultaneously adding signal probes of different labels of N gene and RP gene an RPA reaction is made, and at the same time the second segments of target gene N gene and RP gene are amplified.

And detecting the probe signals of different labels to obtain the detection results of the virus nucleic acid and the reference gene.

The differently labeled signaling probes are probes with different signaling labels, typically probes with different fluorescein labels, such as FAM, HEX fluorescein labeled probes.

In the first step, the RT-RPA reaction system is as follows: the enzyme freeze-dried powder (including reverse transcriptase, RNase inhibitor, recombinase, single-stranded DNA binding protein and polymerase), 25uL buffer solution 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 sample to be tested, and 2.5uL magnesium acetate (280 mM). The reaction started immediately upon addition of magnesium acetate to the reaction system.

In the second step, the RPA reaction system is as follows: lyophilized enzyme powder (including recombinase, single-stranded DNA binding protein, polymerase and exonuclease), 25uL buffer solution VI, 1.0uL N gene forward inner primer (10uM), 1.0uL N gene reverse inner primer (10uM), 1.0uL RP gene forward inner primer (10uM), 1.0uL RP gene reverse inner primer (10uM),0.3uL N gene probe (10uM),0.3uL RP gene probe (10uM), total pre-reaction product (about 37uL), 2.5uL magnesium acetate (280 mM). The reaction started immediately upon addition of magnesium acetate to the reaction system.

The RT-RPA reaction in the first step can be carried out at 37-42 ℃ for 5-20 min.

The reaction temperature is preferably 39 ℃ and the reaction time is preferably 15 min.

The RPA reaction in the second step can be carried out at 37-42 ℃ for 1-30 min.

And (3) observing in real time by using a constant-temperature fluorescent amplification instrument, wherein a typical exponential amplification curve can be seen within 15min of on-line detection, the slope of the curve is greater than 20, and the difference value of the fluorescence value and the blank/negative control has obvious statistical difference (p is less than 0.05), so that a positive result is obtained.

The RT-nestRPA technology is used for detecting the RNA of the simulated sample, the RNA of the pseudovirus and the total RNA of the blood cells, and the result of the detection of the N gene detection channel (FAM fluorescent signal) shows that the RNA of the simulated sample and the RNA of the pseudovirus present a typical amplification curve, while the total RNA of the blood cells does not show a remarkable amplification curve (figure 6, left). The results in the RP gene detection channel (HEX fluorescent signal) showed that the mock sample RNA and total blood cell RNA exhibited typical amplification curves, whereas no significant amplification curve was seen for the pseudoviral RNA (fig. 6 right). The result shows that the RT-nestRPA double-gene detection system can not cause obvious fluorescent signal interference among different channels, and detectable cross reaction can not be generated among different primers.

When the typical exponential amplification curve is generated in the N gene detection channel and the internal reference RP gene detection channel of the sample to be detected simultaneously, the result of the nucleic acid detection of SARS-CoV-2 virus in the sample to be detected is judged to be positive. If the N gene detection channel of the sample to be detected does not have an obvious amplification curve and the internal reference RP gene detection channel has a typical exponential amplification curve, the result of detecting the SARS-CoV-2 virus nucleic acid of the sample to be detected is judged to be negative. If the N gene detection channel of the sample to be detected has an obvious amplification curve and the internal reference RP gene detection channel does not have a typical exponential amplification curve, the detection result of the sample to be detected is judged to be suspicious, and secondary detection is carried out if necessary.

Example 5 RT-nestRPA Co-detection of N Gene and RP Gene in clinical samples

All clinical samples were subjected to nucleic acid extraction (including DNA and RNA) using an automated nucleic acid extractor according to the procedures of the nucleic acid extraction kit (Huayin Biotech co., Shenzhen, China). All nucleic acid samples are detected by real-time quantitative PCR (qPCR) nucleic acid, and whether the samples are positive samples of SARS-CoV-2 infection is judged. During the period from 2 months 2020 to 4 months 2020, 111 samples (nasopharyngeal swabs or sputum) from 84 patients were collected in Shenzhen third people hospital. The results include 15 qPCR test positive samples from 15 patients with confirmed coronary pneumonia, 64 qPCR test negative samples from 37 patients with confirmed coronary pneumonia, and 32 samples from 32 patients with repeat work and healthy physical examination (fig. 7, the numerical values in fig. 7 are the number of cases).

RNA from clinical specimens was co-detected for both N and RP genes using RT-nestRPA as described in example 4. Clinical samples with positive qPCR (N gene and internal reference gene) detection results are detected by the RT-nestRPA method, and 1 of the clinical samples with positive qPCR (N gene and internal reference gene) detection results are continuously diluted by 11 times and 10 times. The serial dilution samples were then tested using the RT-nestRPA dual gene assay. Samples with negative qPCR (N gene) detection results were also tested using the RT-nestRPA double gene method of the invention.

15 nasal swabs or sputum samples from 15 patients with confirmed diagnosis of COVID-19, all positive for detection of SARS-CoV-2N gene using the qPCR method. The detection result of the RT-nestRPA method is consistent with the detection result of the N gene by the qPCR method, and the positive coincidence rate is 100 percent (15/15). Meanwhile, the RT-nestRPA method of the invention is used for detecting the reference genes (RP genes) of 15 samples, and the detection results are positive (Table 6).

TABLE 6 comparison of the results of the detection of the N gene and the reference gene of RT-nestRPA in the qPCR-positive samples

And selecting one positive sample, carrying out continuous 11 times of 10-fold ratio dilution, respectively using the RT-nestRPA and qPCR method to carry out synchronous detection, and comparing the sensitivities of the RT-nestRPA and the qPCR method. The results showed that the SARS-CoV-2 nucleic acid positive sample was still positive after 10 th serial dilution by using the RT-nestRPA method of the present invention, while the qPCR method was negative for the sample after 4 th dilution (Table 7).

TABLE 7 comparison of the results of N gene detection of a positive sample by RT-nestRPA and qPCR methods

64 nasal, anal or sputum samples from 37 COVID-19 confirmed patients were collected and all negative for SARS-CoV-2 nucleic acid using the qPCR method, and 29.69% (19/64) of the samples were found positive using the RT-nestRPA method of the invention (Table 8). The result shows that false negative exists in the result of using qPCR method to detect COVID-19 to diagnose SARS-CoV-2 nucleic acid in patient sample, and the detection sensitivity of using the RT-nestRPA method of the invention is superior to that of qPCR method.

TABLE 8 comparison of the results of detection of N gene and reference gene of RT-nestRPA in qPCR negative samples

In order to observe whether asymptomatic infectors positive for SARS-CoV-2 nucleic acid were present in the general healthy population, 32 throat swab samples of the persons in physical examination were collected, and all of the samples were negative for SARS-CoV-2 nucleic acid detection by the qPCR method. However, 3 samples (9.37%) were found to be positive in the test using the RT-nestRPA method of the present invention (Table 9), and the 3 samples were also positive in the test using the reference gene RP gene (FIG. 8). The result shows that the nucleic acid detection method of the RT-nestRPA has higher sensitivity than the qPCR method, and has important significance for early detection of asymptomatic infectors carrying SARS-CoV-2.

TABLE 9 detection results of RT-nestRPA in the N gene and reference gene of the sample of the repeat physical examination

The nucleic acid detection method of RT-nestRPA of the invention detects N gene of new coronavirus and RP gene of reference gene in 111 cases of clinical samples in total. The detection results of 15 qPCR positive samples by the RT-nestRPA method disclosed by the invention are positive (15/15), the consistency rate with the qPCR detection method is 100%, and the false negative rate is 0%. In addition, the RT-nestRPA method of the invention detects 22 samples (22.91%) with positive result of the new coronavirus N gene in 96 qPCR negative samples (FIG. 9). By combining the results, compared with the qPCR method, the method has higher sensitivity and better application prospect for early detection of new coronavirus infectors.

The above-mentioned embodiments only express the embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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Claims

1. The kit for detecting the pathogen RNA and the animal or human gene RNA is characterized by comprising a primer pair for detecting the pathogen RNA and the animal or human gene RNA by using RT-nested RPA and a primer pair for detecting the internal reference gene RNA by using RT-nested RPA, wherein the primer pair for detecting the pathogen RNA and the animal or human gene RNA and the primer pair for detecting the internal reference gene RNA simultaneously carry out detection reaction in the same reaction tube.

2. The kit for detecting pathogen RNA and animal or human gene RNA according to claim 1, wherein the pathogen RNA comprises RNA of virus, bacteria, chlamydia, mycoplasma, rickettsia, spirochete, fungus, or parasite, and the animal or human gene RNA comprises mRNA of human tumor-related gene and mRNA of genetic disease-related gene.

3. The kit for detecting the RNA of a pathogen, or the RNA of an animal or human gene, according to claim 2, wherein the pathogen is SARS-CoV-2 neocoronavirus.

4. The kit for detecting pathogen RNA, or animal or human gene RNA, according to claim 3, wherein the primer pair for detecting SARS-CoV-2 new coronavirus RNA by RT-nested RPA is a primer pair directed to the nucleotide sequence of SEQ ID NO: 1 or a primer pair designed by the partial sequence of the N gene sequence of the SARS-CoV-2 new coronavirus.

5. The kit for detecting pathogen RNA, animal or human gene RNA according to claim 4, wherein the primer pair for detecting SARS-CoV-2 new coronavirus by RT-nested RPA comprises: SEQ ID NO: 3 and SEQ ID NO: 4, the sequence shown in SEQ ID NO: 5 and SEQ ID NO: 6.

6. The kit for detecting RNA of a pathogen, or RNA of an animal or human gene, according to claim 3, wherein the reference gene is the RP gene of the human housekeeping gene.

7. The kit for detecting RNA of a pathogen and RNA of an animal or human gene as claimed in claim 6, wherein the primer pair for detecting RP gene by nested RPA comprises: SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

8. The kit for detecting pathogen RNA, and animal or human genetic RNA according to claim 3, further comprising a nucleic acid sequence specific for SEQ ID NO: 1 or partial sequence of N gene sequence of SARS-CoV-2 new coronavirus.

9. The kit for detecting pathogen RNA, or animal or human genetic RNA, according to claim 6, further comprising a nucleic acid sequence specific for SEQ ID NO: 7 or a partial sequence thereof.

10. A primer pair for detecting SARS-CoV-2 new coronavirus is a primer pair for detecting SARS-CoV-2 new coronavirus N gene by RT-nested RPA, and comprises an outer primer pair and an inner primer pair.

11. The primer pair for detecting SARS-CoV-2 novel coronavirus according to claim 10, wherein the primer pair is a primer pair directed to SEQ ID NO: 1 or a partial sequence thereof, wherein the outer primer pair has the sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4, and the inner primer pair has a nucleotide sequence shown as SEQ ID NO: 5 and SEQ ID NO: 6 in a sequence listing.

12. A probe for detecting SARS-CoV-2 neo-coronavirus, wherein said probe is specific for SEQ ID NO: 1 or partial sequence of N gene sequence of SARS-CoV-2 new coronavirus.

13. The probe for detecting SARS-CoV-2 neo-coronavirus according to claim 12, wherein the probe has the sequence as shown in SEQ ID NO: 2, or a nucleotide sequence shown in the figure.

14. A kit for detecting SARS-CoV-2 neocoronavirus, comprising the primer pair of claim 10 or 11.

15. The kit for detecting SARS-CoV-2 novel coronavirus according to claim 14, wherein the kit further comprises a primer pair for detecting internal reference gene by RT-nested RPA, and the primer pair for detecting SARS-CoV-2 novel coronavirus N gene and the primer pair for detecting internal reference gene perform detection reaction simultaneously in the same reaction tube.

16. The kit for detecting SARS-CoV-2 neo-coronaviruses according to claim 15, further comprising the probe of claim 12 or 13, and a probe specific for an internal reference gene.

17. The kit for detecting SARS-CoV-2 neo-coronavirus according to claim 15 or 16, wherein the reference gene is a human housekeeping gene RP gene.

18. The kit for detecting SARS-CoV-2 neo-coronavirus according to claim 17, wherein the primer pair for detecting RP gene by RT-nested RPA comprises: SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

19. The kit for detecting SARS-CoV-2 neo-coronavirus according to claim 17, wherein the probe for detecting the RP gene by RT-nested RPA is a probe specifically directed to the nucleotide sequence shown in SEQ ID NO: 7 or a partial sequence thereof, and has a sequence shown in SEQ ID NO: 8 in a nucleotide sequence shown in SEQ ID NO.

20. Use of a kit according to any one of claims 1 to 9 for the manufacture of a product for the detection of RNA from a pathogen.