CN111926112A - Primer composition, kit and method for detecting SARS-CoV-2 nucleic acid - Google Patents

Abstract

The invention discloses a primer composition, a kit and a method for detecting SARS-CoV-2 nucleic acid. The primer composition comprises SEQ ID NO: 1-SEQ ID NO: 6, or a primer comprising the sequence shown in SEQ ID NO: 7-SEQ ID NO: 11, or a primer of the sequence shown in figure 11. And (3) filling the primer solution containing the primer composition into a microfluidic chip in advance to assemble the kit. By using the components of the kit, a small-volume rotary microfluidic fluorescence system is formed in the microfluidic chip to detect SARS-CoV-2 nucleic acid. The primer composition has strong specificity and high sensitivity, and the kit and the method have the advantages of high speed and efficiency, simple and convenient operation, strong anti-interference performance, high repeatability and simple and convenient identification, and are suitable for port and field detection.

Description

Primer composition, kit and method for detecting SARS-CoV-2 nucleic acid

Technical Field

The invention relates to the field of nucleic acid detection, in particular to a primer composition, a kit and a method for detecting SARS-CoV-2 nucleic acid.

Background

18 days 3 and 18 months in 2020, Merrieia, USA

The COVID-19 detection kit is approved by the US FDA for the EUA authorization for emergency use and is used for the rapid detection of the novel coronavirus, and the detection is carried out on the test kit

2.0 the detection time is 45min, 3 months and 21 days, the nucleic acid detection reagent of Xpert SARS-CoV-2 is declared to obtain EUA authorization by American Saiping, and the detection time is 30min for the qualitative detection of SARS-CoV-2 virus nucleic acid. On day 3, 28, Yapeh, USA, the FDA awards the product YapeID NOW Emergency Use Authority (EUA) for its molecular-of-care test (POCT) for the detection of the novel coronavirus disease (COVID-19). The test can give positive results within as short as five minutes, and negative results within 13 minutes, and is the instant test with the highest reported speed at present.

The micro-fluidic chip technology is a new subject and technology developed gradually on a micro-electromechanical processing technology-based micro total analysis system (μ TAS) by Manz and Widmer in switzerland in the early 90 th century. The definition of this discipline by professor GM, Whitesides, harvard university is: the microfluidic chip is a technology and science for controlling nano-liter to pico-liter volume fluid in a micron scale structure. One of the advantages of the technology is that the specific surface area of the reaction cavity with small volume and the mass and heat transfer effect of biochemical reaction in the cavity are increased, and the rapid reaction kinetic effect can be effectively realized. There is no report of using micro-fluidic chip technology for SARS-CoV-2 nucleic acid detection.

In summary, the development of molecular biology techniques provides for early diagnosis of pathogens, and the PCR technique is mainly used for clinical diagnosis, and includes various techniques derived based on PCR: including reverse transcription PCR (RT-PCR), multiplex PCR, nested PCR, RAPD technology, real-time fluorescence quantitative PCR, etc. However, the above detection method has problems of long detection period, complex detection system and operation process, difficulty in performing on-site detection, and the like, and requires professional operation. In addition, PCR instruments are expensive and have a long amplification time. Therefore, a detection method which is rapid, simple, convenient, easy to popularize, safe and reliable and suitable for field operation is needed in scientific research and production practice, and especially the rapid detection speed is important for epidemic prevention and control.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a primer composition, a kit and a method for detecting SARS-CoV-2 nucleic acid, which utilize micro-fluidic small-volume effect to realize effective constant-temperature amplification reaction in a reaction cavity and are applied to the rapid and effective analysis of SARS-CoV-2 nucleic acid.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the present invention, there is provided a primer composition for detecting SARS-CoV-2 nucleic acid, comprising SEQ ID NO: 1-SEQ ID NO: 6, or a primer comprising the sequence shown in SEQ ID NO: 7-SEQ ID NO: 11, or a primer of the sequence shown in figure 11.

The second aspect of the invention provides a kit for detecting SARS-CoV-2 nucleic acid, comprising reaction liquid, a microfluidic chip, a positive control, a negative control, an internal standard substance and a chip sealing film;

wherein the microfluidic chip contains a primer solution, and the primer solution comprises the primer composition of the first aspect.

Further, the reaction solution also comprises reverse transcriptase, Bst DNase, dNTPs, 10 multiplied isothermal amplification reaction buffer solution, fluorescent dye and magnesium ion solution.

Further, the positive control contains 10⁵copies/mL of target fragment, the negative control is physiological saline, the internal standard substance is 10⁵copies/mL internal standard fragment of pseudovirus.

Further, the concentration of each primer in the primer composition in the primer solution is 48-52 mu M; the concentration of the Bst DNA polymerase is 8U/. mu.l, the concentration of dNTP is 12mM, the concentration of fluorescent dye is 1 XSSYBR Green I, the concentration of reverse transcriptase is 5U/. mu.l, and the concentration of magnesium ion solution is 8 mM.

Further, the magnesium ion solution is MgSO₄An aqueous solution having a concentration of 8 mM.

Further, each primer in the above primer composition was at a concentration of 50. mu.M.

Furthermore, the volume ratio of the dNTPs, the 10 multiplied isothermal amplification reaction buffer solution and the magnesium ion solution is (7-9): (4-6): 2.

Further preferably, the volume ratio of the dNTPs, the 10 Xisothermal amplification reaction buffer solution and the magnesium ion solution is 8:5: 2.

The third aspect of the present invention provides a method for detecting SARS-CoV-2 nucleic acid using the above-mentioned kit, comprising the steps of:

adding an internal standard substance into a sample to be detected, and extracting nucleic acid of the sample to be detected by adopting a nucleic acid extraction kit;

mixing the nucleic acid of the sample to be detected, the positive control or the negative control and the reaction solution, then carrying out vortex oscillation and uniform mixing, adding all the mixture into a sample adding hole of a microfluidic chip after instantaneous centrifugation, sealing the sample adding hole by using a sealing film, and scraping and pressing the sealing film;

placing the micro-fluidic chip into a micro-fluidic chip detector, and setting reaction conditions for amplification reaction;

and step four, judging the detection result.

Further, the volume of the nucleic acid in the sample, the positive control or the negative control is 5. mu.L, and the volume of the reaction solution is 25. mu.L.

Further, the determination of the detection result adopts the following criteria:

the Ct values of the target and the internal standard are less than or equal to 36, and the result is judged to be positive;

target and internal standard Ct values are indicated as "-" or > 36, and are judged negative.

By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:

the primer composition, the kit and the method for detecting SARS-CoV-2 nucleic acid provided by the invention have the following advantages:

(1) fast and efficient: the whole amplification can be completed within 15-30 min, and the amplification yield can reach 10⁹-10¹⁰A copy;

(2) the operation is simple and convenient: complex instruments, special reagents and complicated steps such as denaturation of double-stranded DNA are not needed in advance, only a real-time fluorescence detector is needed for reaction and detection, and the conditions are mild;

(3) specificity: no cross reaction with other pathogens;

(4) high sensitivity: the minimum detection limit can reach 100 copies;

in conclusion, the primer composition has the advantages of strong specificity and high sensitivity, and the kit and the method have the advantages of rapidness, high efficiency, simplicity and convenience in operation, strong anti-interference performance, high repeatability and convenience in identification, and are suitable for port and field detection.

Drawings

FIG. 1 is a graph showing the amplification curve of the target detection range of SARS-CoV-2ORF1ab of the novel coronavirus in one embodiment of the present invention; wherein, the graph A is 10⁵/10⁴Amplification curves for the copies/. mu.L concentration of pseudovirus samples and negative controls, FIG. B is 10³/10²/10¹Amplification curves for pseudovirus samples at copies/μ L concentration;

FIG. 2 is a graph of the amplification curve of the target detection range of the novel coronavirus SARS-CoV-2N gene in one embodiment of the present invention; wherein, the concentration is 10 in graph A⁵Amplification curves for the copies/. mu.L concentration of pseudovirus samples and negative controls, FIG. B is 10⁴/10³/10²/10¹Amplification curves for pseudovirus samples at copies/μ L concentration;

FIG. 3 shows an embodiment 10 of the present invention¹Amplification profile of novel coronavirus SARS-CoV-2ORF1ab target dilution 1 fold at copies/μ L concentration; wherein, the graph A is eight diluted by 1 timeAmplification curves of multiple wells, panel B is an amplification curve of four multiple wells diluted 1-fold;

FIG. 4 shows an embodiment 10 of the present invention¹Amplification curve diagram of gene target dilution 1-fold for copies/muL concentration novel coronavirus SARS-CoV-2N; wherein, the graph A is an amplification curve of eight duplicate wells diluted by 1 time, and the graph B is an amplification curve of four duplicate wells diluted by 1 time;

FIG. 5 shows an embodiment 10 of the present invention¹Amplification results of novel coronavirus SARS-CoV-2ORF1ab target at copies/μ L concentration; wherein, FIG. A, B, C shows the amplification curves of eight duplicate wells, respectively;

FIG. 6 shows an embodiment 10 of the present invention¹Amplification curve diagram of gene target of novel coronavirus SARS-CoV-2N at copies/muL concentration; wherein, FIG. A, B, C shows the amplification curves of eight duplicate wells;

FIG. 7 is a graph of amplification of a pathogen sample according to an embodiment of the present invention; wherein, the graph A is an amplification curve of influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, parainfluenza virus, chlamydia pneumoniae, mycoplasma pneumoniae and MERSR-CoV, and the graph B is an amplification curve of coronavirus OC43, coronavirus NL63, coronavirus HKU1, coronavirus 229E, streptococcus pneumoniae, norovirus, rotavirus and staphylococcus aureus;

FIG. 8 is a graph of amplification of a pathogen sample according to an embodiment of the present invention; wherein, FIG. 8A is a negative sample (i.e., only one internal standard amplification curve appears); FIG. 8B shows a positive sample (appearance of an internal standard amplification curve, an O gene amplification curve, and an N gene amplification curve);

FIG. 9 is a graph showing the amplification of the ORF1ab target in the concentration reference in the first assay batch according to one embodiment of the present invention; wherein, the pictures A and B are eight duplicate wells of two experiments respectively;

FIG. 10 is a graph of the amplification plot of the ORF1ab target in a first lot of reagent detection threshold concentration reference in one embodiment of the present invention; wherein, the pictures A and B are eight duplicate wells of two experiments respectively;

FIG. 11 is a graph showing amplification curves of the N gene target in a concentration reference in a first batch of reagent assays, according to an embodiment of the present invention; wherein, the pictures A and B are eight duplicate wells of two experiments respectively;

FIG. 12 is a graph showing amplification curves of a first lot of reagents for detecting N gene targets in a threshold concentration reference according to an embodiment of the present invention; wherein, the pictures A and B are eight duplicate wells of two experiments respectively;

FIG. 13 is a graph showing the amplification of dexamethasone acetate, an interference substance, for the target detection of the novel coronavirus SARS-CoV-2ORF1ab in one embodiment of the present invention; wherein panel A is a normal control group and panel B is an interfering substance group;

FIG. 14 is a graph showing the amplification curve of dexamethasone acetate, an interfering substance in the target detection of the SARS-CoV-2N gene of the novel coronavirus, in accordance with an embodiment of the present invention; wherein panel A is a normal control group and panel B is an interfering substance group;

FIG. 15 is a graph showing the amplification of ribavirin as a novel coronavirus SARS-CoV-2ORF1ab target detection interfering substance in one embodiment of the present invention; wherein panel A is a normal control group and panel B is an interfering substance group;

FIG. 16 is a graph showing the amplification curve of the target detection interfering substance ribavirin of the novel coronavirus SARS-CoV-2N gene in one embodiment of the present invention; wherein panel A is a normal control group and panel B is an interfering substance group;

FIG. 17 is a graph showing the amplification curve of the novel coronavirus SARS-CoV-2ORF1ab target for detecting the interfering substance ceftriaxone in one embodiment of the present invention; wherein panel A is a normal control group and panel B is an interfering substance group;

FIG. 18 is a graph showing the amplification curve of the novel coronavirus SARS-CoV-2N gene target for detecting the interfering substance ceftriaxone in one embodiment of the present invention; wherein panel A is a normal control group and panel B is an interfering substance group;

FIG. 19 is a graph showing the amplification of the novel coronavirus SARS-CoV-2ORF1ab target for detecting interfering substance mucin in one embodiment of the present invention; wherein panel A is a normal control group and panel B is an interfering substance group;

FIG. 20 is a graph showing the amplification of mucin as a novel coronavirus SARS-CoV-2N gene target for detection of interfering substances in one embodiment of the present invention; wherein panel A is a normal control group and panel B is an interfering substance group;

FIG. 21 is a graph showing the amplification of hemoglobin for the target detection of interfering substances of the novel coronavirus SARS-CoV-2ORF1ab in one embodiment of the present invention; wherein panel A is a normal control group and panel B is an interfering substance group;

FIG. 22 is a graph showing the amplification curve of hemoglobin in the target assay for the interfering substance of the novel coronavirus SARS-CoV-2N gene in one embodiment of the present invention; wherein, panel A is a normal control group, and panel B is an interfering substance group.

Detailed Description

The invention provides a primer composition, a kit and a method for detecting SARS-CoV-2 nucleic acid. The primer composition with good sensitivity, high specificity, high repeatability and strong anti-interference capability is designed to be combined with the micro-fluidic chip technology to realize the rapid detection of SARS-CoV-2 nucleic acid.

The microfluidic chip is based on a centrifugal fluid driving system, and utilizes centrifugal force to directionally drive a sample to flow so as to enable reaction liquid to enter a reaction tank. In the chip, samples can be uniformly distributed to 4 reaction holes, and synchronous detection of four targets is realized. Specifically, the invention adopts a micro-fluidic chip with pre-embedded specific amplification primers to uniformly mix nucleic acid and reaction liquid, add the mixture into a sample adding hole, seal the sample adding hole, place the sample adding hole into a micro-fluidic chip detector, centrifuge the reagent in the sample adding hole into the reaction hole through a micro-channel by high-speed centrifugation, amplify at constant temperature, read by real-time fluorescence, output signals and present results.

The chip provided by the invention can complete 8 sample detections within 30min (can be upgraded to 16 samples/chips or 32 samples/chips at the later stage), does not consider the sample sending time and the personnel rotation time, can realize 384 samples/devices per day for full load detection, basically meets the requirement of the current SARS-CoV-2 detection quantity, and is very suitable for a quick detection mode of unit batch sample sending and batch detection. Generally speaking, the detection kit has the characteristics of integration (amplification, signal output), miniaturization, multi-project joint detection, easy operation and the like, and completes the detection of the whole virus by combining a constant temperature amplification technology.

The SARS-CoV-2 nucleic acid method using the above-mentioned kit is quick in speed and flexible in flux, and in addition, in the course of detection it can adopt several modes of fluorescence collection system, portable flip type microfluidic amplification reaction fluorescence collection system (can utilize draw-bar box to make assembly to implement portable detection mode of port field) and pull type microfluidic fluorescence collection system which can be in multi-module superposition mode (has higher flux, can implement superposition and synchronous detection of 12 equipment modules, and can implement high-flux detection of 96 sample/equipment).

The present invention will now be described in detail and specifically with reference to the following examples so as to provide a better understanding of the present invention, but the following examples are not intended to limit the scope of the present invention.

Example 1

This example provides a primer composition and kit for detecting SARS-CoV-2 nucleic acid. The information of the designed primer sequence and target sequence for the SARS-CoV-2 conserved fragment is shown in Table 1 and Table 2 below.

TABLE 1 information Table of ORF1ab Gene amplification primer sequences and target sequences

TABLE 2 information Table of primer sequences and target sequences for amplification of the N genes

The kit containing the primer composition comprises the following components (shown in Table 3).

TABLE 1 kit Components

Wherein the primer solution contains 50. mu.M of outer primer 1, 50. mu.M of outer primer 2, 50. mu.M of inner primer 1, 50. mu.M of inner primer 2 and 50. mu.M of loop primer.

Concentration of internal standard 10⁶copies/mL。

The reaction solution contains the following raw materials in concentration: 12mM dNTP, 10 × isothermal amplification reaction buffer, 8mM MgSO₄An aqueous solution. In the reaction solution, 12mM dNTP, 10 Xisothermal amplification reaction buffer and 8mM MgSO₄The volume ratio of the aqueous solution is 8:5: 2.

The DNA polymerase is Bst DNA polymerase, and the concentration of the Bst DNA polymerase is 8U/. mu.l; the concentration of the reverse transcriptase is 5U/mul; the fluorescent dye concentration was 1 × SYBR Green I.

Example 2

This example provides a method for detecting SARS-CoV-2 nucleic acid using the primer composition and kit provided in example 1, comprising the steps of:

1. sample and negative and positive reference substance treatment and extraction

The nucleic acid extraction reagent produced by Shanghai Rapid diagnosis product company Limited is adopted, the specific operation steps are operated according to the instruction of the reagent kit, and 2 mu L of internal standard substance provided by the reagent kit is added into each part during extraction.

2. Reagent preparation

The kit is taken out to room temperature from minus 20 +/-5 ℃, and each reagent is dissolved at room temperature, fully mixed and used after being centrifuged for a short time. N (N ═ number of samples to be tested + negative and positive controls) 1.5mL centrifuge tubes were used, and 25. mu.l of SARS-CoV-2 reaction solution was added to each tube.

3. Sample application

Adding 5 mu L of nucleic acid sample to be detected into a centrifuge tube prepared with SARS-CoV-2 reaction solution, mixing uniformly by vortex oscillation, adding all the mixture into a chip sample adding hole after instantaneous centrifugation, sealing the sample adding hole by a sealing film, scraping and compacting.

4. Nucleic acid amplification

Clicking the device warehouse-in and warehouse-out button, flatly placing the chip on a tray of the device (one side with a label is upward), slightly pressing the chip downwards to clamp the chip, and closing the warehouse door. 4.1 or 4.2 can be selected for subsequent operations.

4.1 reaction conditions are automatically set: after the two-dimensional code of the kit is scanned by the code scanning gun, a transport sequence (suitable for MA3000) is clicked.

4.2 reaction conditions were set manually:

(cracking temperature 65 ℃ C., cracking time omitted; for MA 3000); the amplification temperature is 65 ℃ and 40 minutes; the low-speed rotation is 1600 revolutions per minute, and the low-speed time is 30 seconds (the low-speed times are 3 times, and the method is suitable for MA 3000); rotating at high speed of 4500 rpm for 30 s (high speed times of 3 times, suitable for MA 3000); and operating the program.

5. Analysis of results

The general situation of the threshold line is set to 200 (which can be adjusted according to the actual situation, the set principle is that the threshold line just exceeds the highest point of the atypical S-type amplification curve, and the Ct value shows "-"), and the instrument matching software automatically analyzes the result. Each chip has 8 sample adding holes, each sample adding hole corresponds to 4 detection holes, and the sample adding holes are an ORF1ab gene target detection hole, an N gene target detection hole, a negative detection hole and an internal standard detection hole respectively.

6. Quality control

When the kit is operated for the first time, detection is performed using a negative control and a positive control.

(1) Negative control: the target detection hole has no obvious S-shaped amplification curve or the Ct value is more than 36, the internal standard detection hole is in an S-shaped amplification curve, and the Ct value is less than or equal to 36.

(2) Positive control: the target detection hole and the internal standard detection hole are both in S-shaped amplification curves, the Ct value of the target is less than or equal to 36, and the Ct value of the internal standard is less than or equal to 36.

[ Positive judgment value or reference interval ]

1. And judging the Ct values of the target and the internal standard to be positive if the Ct values are less than or equal to 36.

Ct values indicated as "-" or > 36, judged negative.

[ interpretation of test results ]

On the premise that the experimental quality control is effective, the inspection result is judged according to the following.

1. And (4) internal standard result judgment:

the Ct values of the detection target and the internal standard are less than or equal to 36, and the result is judged to be positive; target and internal standard Ct values are indicated as "-" or > 36, and are judged negative.

2. And (4) judging a target result:

verification example 1

This example uses a sample of SARS-CoV-2 pseudovirus diluted in a gradient to verify the sensitivity of the above products and methods. The specific experimental procedures and results are as follows:

step one, primary screening: with a concentration of 10⁵，10⁴，10³，10²，10¹，10⁰Verification of SARS-CoV-2 pseudovirus sample by Copies/mL, wherein the dilution of SARS-CoV-2 pseudovirus sample is shown in Table 4 below. The results of the tests are shown in Table 5 and FIGS. 1 to 2.

TABLE 4 pseudoviral dilution scheme for ORF1ab target and N Gene target

Note: x represents the target of SARS-CoV-2ORF1ab and N gene target.

TABLE 5 detection results of novel coronavirus SARS-CoV-2

As can be seen from Table 5 and FIGS. 1-2, the pseudoviral RNA templates of the novel coronavirus SARS-CoV-2ORF1ab target and the novel coronavirus SARS-CoV-2N gene target at a concentration of 10 copies/. mu.L were all positive. Therefore, the pseudoviral RNA concentration of the novel coronavirus SARS-CoV-2ORF1ab target and the N gene target was selected to be 10 copies/. mu.L for further experiments to determine the detection limit of the product.

And step two, carrying out 1-fold, 2-fold, 4-fold and 8-fold dilution on the pseudoviral RNA of the ORF1ab target and the N gene target with the concentration of 10 copies/mu L, repeating each gradient for 12 times, and taking the detection rate of 95% positive as the lowest detection limit. The results are shown in Table 6 and FIGS. 3-4.

TABLE 6 measurement results of detection limit of novel coronavirus SARS-CoV-2

As can be seen from Table 6 and FIGS. 3 to 4, the positive rate obtained by diluting 1 time is satisfactory, the positive rate is 100%, and the positive rate obtained by diluting 2 times is less than 95%. Therefore, the concentration of pseudoviral RNA of the target of the novel coronavirus SARS-CoV-2ORF1ab and the N gene target is 10 copies/mu L, which is the lowest detection limit of the kit.

And step three, repeating the pseudoviral RNA of the ORF1ab target and the N gene target at the concentration of 10 copies/mu L for 24 times, and verifying whether the positive rate of the pseudoviral RNA meets 95%. The results are shown in Table 7 and FIGS. 5 to 6.

TABLE 7 validation of the minimum detection limit of the novel coronavirus SARS-CoV-2ORF1ab target

As can be seen from Table 7 and FIGS. 5 to 6, the positive rates of 10 copies/. mu.L for the SARS-CoV-2ORF1ab target and the N gene target of the novel coronavirus were 95% or more, and the results were satisfactory. Therefore, the minimal detection of the novel coronavirus SARS-CoV-2ORF1ab target and the N gene target was limited to 10 copies/. mu.L.

Verification example 2

This example uses different pathogens (as shown in table 8) to perform specificity studies on the products and methods provided in examples 1 and 2, and the results are shown in table 8 and fig. 7-8.

TABLE 8 pathogen sample and its test results

As can be seen from Table 8 and FIGS. 7-8, the detection results of 17 samples all show that the ORF1ab target and the N gene target are negative, and the internal standards are all positive, which is consistent with the expected results, indicating that the primer composition and the kit provided in example 1 have good specificity.

Verification example 3

This example uses different concentrations of the novel coronavirus SARS-CoV-2 pseudovirus (Table 9 below) to perform a reproducibility (precision) study on the primer compositions and kits provided in example 1.

TABLE 9 information Table of novel coronavirus SARS-CoV-2 pseudovirus at different concentrations

The ORF1ab target and the reference product of the N gene target of the novel coronavirus SARS-CoV-2 are detected, the CV values (less than or equal to 5.0 percent) of Ct values of different operators in/between batches and different operators are calculated, and the precision of the product is fully evaluated. The results are shown in tables 10 to 11 and FIGS. 9 to 12.

TABLE 10 precision determination of ORF1ab targets

TABLE 11 results of precision determination of N Gene targets

As can be seen from tables 10-11 and FIGS. 9-12, the positive detection rate of 3 batches of reagents was 100% and Ct CV values were all 10% or less.

Verification example 4

In this embodiment, the anti-interference performance of the product provided in embodiment 1 is determined by performing anti-interference research on common interference substances (dexamethasone acetate, ribavirin, ceftriaxone, mucin, and hemoglobin).

Interference is one of the important causes of errors in clinical laboratory measurements, and both qualitative and quantitative methods can be affected by interfering substances. In some cases, the error in the results due to the interference may affect the clinical decision of the doctor and may adversely affect both the doctor and the patient. The interfering substances and their concentrations used in this example are shown in Table 12.

TABLE 12 interfering substances and their concentrations

Name (R)	Final concentration of interferents
		Dexamethasone acetate	1mg/mL
Ribavirin	0.05mg/mL
		Ceftriaxone	20mg/mL
Mucins	2mg/mL
		Hemoglobin	2mg/mL

First, research of anti-dexamethasone acetate interference ability

Dexamethasone acetate at corresponding high-value concentrations is added into clinical samples respectively and then detected, and the detection results are shown in table 13 and figures 13-14.

TABLE 13 test results after addition of the interfering substance dexamethasone acetate

As can be seen from Table 13 and FIGS. 13 to 14, the detection result of the reagent is consistent with the positive detection result of the normal control group sample, and the Ct value of the sample is slightly delayed, which indicates that the interferent slightly inhibits the amplification, but the detection result is not changed, which indicates that dexamethasone acetate interferes with the kit in example 1, but the inhibition effect is low, and the judgment of the result is not affected.

Second, study of anti-ribavirin interference ability

Ribavirin with corresponding high concentration is added into clinical samples respectively and then the clinical samples are detected, and the detection results are shown in table 14 and figures 15-16.

TABLE 14 test results after addition of the interfering substance ribavirin

As can be seen from Table 14 and FIGS. 15 to 16, the detection results of ribavirin group are consistent with the positive detection results of the normal control group samples, and the slight delay of Ct value of the samples indicates that the amplification is slightly inhibited by the interferent, but the detection results are not changed, which indicates that ribavirin interferes with the kit in example 1, but the inhibition effect is low, and the judgment of the results is not affected.

Research on anti-ceftriaxone interference capacity

After the clinical samples are respectively added with the ceftriaxone at the corresponding high-value concentration, the detection results are shown in table 15 and figures 17-18.

TABLE 15 test results after addition of interfering substance ceftriaxone

As can be seen from table 15 and fig. 17 to 18, the detection result of the ceftriaxone preparation is consistent with the positive detection result of the normal control sample, and after the Ct value of some samples is slightly delayed, it indicates that the interferent slightly inhibits the amplification, but the detection result is not changed, which indicates that the ceftriaxone preparation interferes with the kit in example 1, but the inhibition effect is low, and the judgment of the result is not affected.

Fourth, study of anti-mucin interference ability

Mucin at corresponding high-value concentration is added into clinical samples respectively and then detection is carried out, and the detection results are shown in table 16 and figures 19-20.

TABLE 16 test results after addition of interfering substance mucin

As can be seen from table 16 and fig. 19 to 20, the detection result of the mucin group is consistent with the positive detection result of the normal control group sample, and the Ct value thereof is slightly delayed, which indicates that the interferent slightly inhibits the amplification, but does not change the detection result, which indicates that the mucin interferes with the kit, but the inhibition effect is low, and has no influence on the judgment of the result.

Fifth, research of anti-hemoglobin interference capability

Hemoglobin with corresponding high-value concentration is added into clinical samples respectively and then detected, and the detection results are shown in table 17 and figures 21-22.

TABLE 17 results of measurement after addition of interfering substance hemoglobin

As can be seen from table 17 and fig. 21 to 22, the detection result of the hemoglobin group is consistent with the positive detection result of the normal control group sample, and the Ct value of the sample is slightly delayed, which indicates that the interfering substance slightly inhibits the amplification, but the detection result is not changed, which indicates that the hemoglobin interferes with the kit, but the inhibition effect is low, and the judgment of the result is not affected.

In conclusion, the clinical samples respectively added with dexamethasone acetate, ribavirin, ceftriaxone, mucin and hemoglobin extract tests show that: the detection result of the reagent is consistent with the positive detection result of a normal control group sample, and the Ct value of the sample is slightly delayed, which indicates that the interference substance slightly inhibits the amplification, but the detection result is not changed, which indicates that the interference substance interferes with the kit, but the inhibition effect is low, and the judgment of the result is not influenced.

The embodiments of the present invention have been described in detail, but the embodiments are only examples, and the present invention is not limited to the above-described embodiments. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

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Tianjin international travel health care center (Tianjin customs port outpatient department)

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tgtgatcaac tccgcgaacc catgcttcag tcagctgatg cacaatcgtt tttaaacggg 120

tttgcggtgt aagtgcagcc cgtcttacac cgtgcggcac aggcactagt actgatgtcg 180

tatacagggc ttttgacatc tacaatgata aagtagctgg ttttgctaaa ttcctaaaaa 240

ctaattgttg tcgcttccaa gaaaaggacg aagatgacaa tttaattgat tcttactttg 300

tagttaagag acacactttc tctaactac 329

<210> 13

<211> 340

<212> DNA

<213> Artificial Sequence

<400> 13

gcggcagtca agcctcttct cgttcctcat cacgtagtcg caacagttca agaaattcaa 60

ctccaggcag cagtagggga acttctcctg ctagaatggc tggcaatggc ggtgatgctg 120

ctcttgcttt gctgctgctt gacagattga accagcttga gagcaaaatg tctggtaaag 180

gccaacaaca acaaggccaa actgtcacta agaaatctgc tgctgaggct tctaagaagc 240

ctcggcaaaa acgtactgcc actaaagcat acaatgtaac acaagctttc ggcagacgtg 300

gtccagaaca aacccaagga aattttgggg accaggaact 340

Claims (9)

1. A primer composition for detecting SARS-CoV-2 nucleic acid, comprising SEQ ID NO: 1-SEQ ID NO: 6, or a primer comprising the sequence shown in SEQ ID NO: 7-SEQ ID NO: 11, or a primer of the sequence shown in figure 11.

2. A kit for detecting SARS-CoV-2 nucleic acid is characterized by comprising reaction liquid, a microfluidic chip, a positive control, a negative control, an internal standard substance and a chip sealing film;

the microfluidic chip contains a primer solution, and the primer solution comprises the primer composition of claim 1.

3. The kit for detecting SARS-CoV-2 nucleic acid as claimed in claim 2, wherein the reaction solution comprises reverse transcriptase, Bst DNA polymerase, dNTPs, 10 × isothermal amplification reaction buffer, fluorescent dye and magnesium ion solution.

4. The kit for detecting SARS-CoV-2 nucleic acid according to claim 2, wherein the positive control comprises 10⁵copies/mL of target fragment, normal saline as negative control, and 10-contained internal standard as internal standard⁵copies/mL internal standard fragment of pseudovirus.

5. The kit for detecting SARS-CoV-2 nucleic acid according to claim 2, wherein the concentration of each primer in the primer composition in the primer solution is 48 to 52. mu.M; the concentration of the Bst DNA polymerase is 8U/mu l, and the concentration of dNTP is 12 mM; the fluorescent dye concentration was 1 XSSYBR Green I, the reverse transcriptase concentration was 5U/. mu.l, and the magnesium ion solution concentration was 8 mM.

6. A kit for detecting SARS-CoV-2 nucleic acid as claimed in claim 1, wherein the volume ratio of the dNTPs, 10X isothermal amplification reaction buffer and magnesium ion solution is (7-9): (4-6): 2.

7. A method for detecting SARS-CoV-2 nucleic acid using the kit of any one of claims 2 to 6, comprising the steps of:

adding an internal standard substance into a sample to be detected, and extracting nucleic acid of the sample to be detected by adopting a nucleic acid extraction kit;

mixing the nucleic acid of the sample to be detected, the positive control or the negative control and the reaction solution, then carrying out vortex oscillation and uniform mixing, adding all the mixture into a sample adding hole of the microfluidic chip after instantaneous centrifugation, sealing the sample adding hole by using a sealing film, and scraping and pressing the sealing film;

placing the micro-fluidic chip into a micro-fluidic chip detector, and setting reaction conditions for amplification reaction;

and step four, judging the detection result.

8. The method of claim 7, wherein the volume of the nucleic acid, positive control or negative control of the sample to be tested is 5 μ L, and the volume of the reaction solution is 25 μ L.

9. The method for detecting SARS-CoV-2 nucleic acid as claimed in claim 7, wherein the determination of the detection result uses the following criteria:

the Ct values of the target and the internal standard are less than or equal to 36, and the result is judged to be positive;

target and internal standard Ct values are indicated as "-" or > 36, and are judged negative.

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