阅读说明：本技术 一种新冠病毒(2019-nCoV)核酸提取效率的评价方法 (Evaluation method for nucleic acid extraction efficiency of new coronavirus (2019-nCoV) ) 是由 隋志伟 刘思渊 黄文锋 于 2021-06-15 设计创作，主要内容包括：本发明建立了一种评价新冠病毒(2019-nCoV)核酸提取效率的方法,提取前采用流式分析技术结合免疫荧光探针技术精确定量检测2019-nCoV病毒颗粒数,提取后使用数字PCR(dPCR)精确定量检测2019-nCoV病毒核酸拷贝数,该核酸拷贝数与提取前测得的颗粒数的比值即为核酸提取效率。本方法可以识别并定量检测2019-nCoV颗粒,可以准确评价2019-nCoV核酸提取效率,且评价方式直接有效,评价结果可溯源。(The invention establishes a method for evaluating nucleic acid extraction efficiency of a new coronavirus (2019-nCoV), wherein before extraction, a flow analysis technology is adopted to be combined with an immunofluorescence probe technology to accurately and quantitatively detect the number of particles of the new coronavirus (2019-nCoV), after extraction, a digital PCR (dPCR) is used to accurately and quantitatively detect the copy number of nucleic acid of the new coronavirus (2019-nCoV), and the ratio of the copy number of the nucleic acid to the number of particles measured before extraction is the nucleic acid extraction efficiency. The method can identify and quantitatively detect the 2019-nCoV particles, can accurately evaluate the 2019-nCoV nucleic acid extraction efficiency, has a direct and effective evaluation mode, and has a traceable evaluation result.)

1. A method for evaluating nucleic acid extraction efficiency of a novel coronavirus (2019-nCoV), comprising the following steps:

1) dividing one sample containing 2019-nCoV into two parts;

2) taking one sample, and determining the concentration of 2019-nCoV particles;

3) extracting nucleic acid of the 2019-nCoV virus in another sample by using a nucleic acid extraction method to be evaluated;

4) determining the copy number of the nucleic acid extracted in the step 3) by adopting a digital PCR (dPCR) technology;

5) calculating the extraction efficiency E of the nucleic acid extraction method to be evaluated according to the following formula;

in the formula:

e is the nucleic acid extraction efficiency of the method to be evaluated;

c_dPCRthe copy number of the nucleic acid obtained in the step 4) is unit copies/. mu.L;

c_FCMthe concentration of the particles obtained in step 2) was in units of events/. mu.L.

2. The evaluation method according to claim 1, wherein the 2019-nCoV particles are determined in step 2) by the following method:

(1) differentiating 2019-nCoV from non-biological background particles in the sample:

simultaneously labeling the specific coat protein of the 2019-nCoV by using an S protein antibody and an N protein antibody of the 2019-nCoV, which are cross-linked by a chemical group through a fluorescent probe; the S protein antibody uses a red fluorescent probe, and the N protein antibody uses an orange fluorescent probe;

(2) differentiating the 2019-nCoV particles and the 2019-nCoV fragments in the sample:

labeling the genome of the 2019-nCoV nucleic acid with a green nucleic acid fluorescent probe;

(3) counting red, orange and green fluorescent signals generated by 1) and 2) by a flow analyzer to realize the rapid quantitative detection of the 2019-nCoV particles;

in the above (3), the determination method for an event generated by one particle is as follows:

judging the S protein fragment of 2019-nCoV if only red fluorescence is detected;

if only orange fluorescence is detected, judging the protein fragment to be the N protein fragment of 2019-nCoV;

if three kinds of fluorescence of red, orange and green are detected simultaneously, judging the particles to be 2019-nCoV;

if no fluorescence is detected, the particle is judged to be an abiotic foreign particle.

3. The evaluation method according to claim 2, wherein the technical index of the S protein is:

a) can react with 2019-nCoV in the mobile phase;

b) after the cross-linking of the red fluorescent probe, 2019-nCoV can be subjected to fluorescent labeling, and the red fluorescence can be detected by a flow analyzer;

c) has an affinity KD of less than 1 x 10 to the S protein of 2019-nCoV^-10。

4. The evaluation method according to claim 2, wherein the technical index of the N protein is:

a) can react with 2019-nCoV in the mobile phase;

b) after the crosslinking of the orange fluorescent probe, 2019-nCoV can be subjected to fluorescent labeling, and orange fluorescence can be detected by a flow analyzer;

c) the affinity KD of the N protein of 2019-nCoV should be less than 1 x 10^-10。

5. The evaluation method of claim 2, wherein the flow analyzer counts 2019-nCoV particles by gating through a scattered light channel of the flow analyzer and detecting the red, orange and green fluorescent signals through a three fluorescent channel; wherein the ring gate of the forward angle scattered light channel is between 60nm and 150 nm.

6. The method of evaluating of claim 5, wherein the gate is enclosed by: and measuring the 60nm standard microspheres and the 150nm standard microspheres by using a flow analyzer, and performing gate looping according to the signal positions of the microspheres on a histogram of a forward angle scattered light channel, wherein the lower limit is 60nm, and the upper limit is 150 nm.

7. The evaluation method according to claim 2, wherein the fluorescence emission spectrum of the red fluorescent probe is from 601nm to 640 nm; the fluorescence emission spectrum of the orange fluorescent probe is 551 nm-590 nm; the fluorescence emission spectrum of the green fluorescent probe is 501 nm-540 nm.

8. The evaluation method of claim 2, wherein the technical indicators and detection parameters of the flow analyzer are: the fluorescence sensitivity is less than 10MESF, the scattered light sensitivity is less than 30nm, the fluorescence resolution RSD is less than 3%, and the scattered light resolution is less than 3%.

9. A method for determining the concentration of 2019-nCoV particles comprises the following steps:

(1) differentiating 2019-nCoV from non-biological background particles in the sample:

simultaneously labeling the specific coat protein of the 2019-nCoV by using an S protein antibody and an N protein antibody of the 2019-nCoV, which are cross-linked by a chemical group through a fluorescent probe; the S protein antibody uses a red fluorescent probe, and the N protein antibody uses an orange fluorescent probe;

(2) differentiating the 2019-nCoV particles and the 2019-nCoV fragments in the sample:

labeling the genome of the 2019-nCoV nucleic acid with a green nucleic acid fluorescent probe;

(3) counting red, orange and green fluorescent signals generated by 1) and 2) by a flow analyzer to realize the rapid quantitative detection of the 2019-nCoV particles;

in the above (3), the determination method for an event generated by one particle is as follows:

judging the S protein fragment of 2019-nCoV if only red fluorescence is detected;

if only orange fluorescence is detected, judging the protein fragment to be the N protein fragment of 2019-nCoV;

if three kinds of fluorescence of red, orange and green are detected simultaneously, judging the particles to be 2019-nCoV;

if no fluorescence is detected, the particle is judged to be an abiotic foreign particle.

10. The method of determining 2019-nCoV particles of claim 9, wherein the technical indicators of the S protein are:

a) can react with 2019-nCoV in the mobile phase;

b) after the cross-linking of the red fluorescent probe, 2019-nCoV can be subjected to fluorescent labeling, and the red fluorescence can be detected by a flow analyzer;

c) has affinity KD with S protein of 2019-nCoV smaller than1×10^-10；

The technical indexes of the N protein are as follows:

a) can react with 2019-nCoV in the mobile phase;

b) after the crosslinking of the orange fluorescent probe, 2019-nCoV can be subjected to fluorescent labeling, and orange fluorescence can be detected by a flow analyzer;

c) the affinity KD of the N protein of 2019-nCoV should be less than 1 x 10^-10。

The technical field is as follows:

the invention belongs to the field of public health, relates to a novel coronavirus detection method, and particularly relates to an evaluation method for nucleic acid extraction efficiency of a novel coronavirus (2019-nCoV).

Background art:

the real-time fluorescence RT-PCR detection is a main method for detecting the nucleic acid of the new coronavirus at present due to the characteristics of high sensitivity and strong specificity. However, with the development of a great deal of detection work, people find that the positive detection rate of the fluorescence RT-PCR detection is only 30-50%. Among them, the most important reason is that the efficiency of nucleic acid extraction methods differs among nucleic acid detecting reagents. Therefore, it is necessary to evaluate the extraction efficiency of nucleic acid extraction methods of different kits.

The nucleic acid extraction efficiency refers to the ratio of the number of copies of nucleic acid after extraction to that before extraction. However, 2019-nCoV nucleic acid is completely encapsulated inside the virus particle before extraction, and the copy number cannot be directly determined.

At present, the common method for evaluating the extraction efficiency of nucleic acid is to extract the same sample by adopting different extraction methods and respectively measure the copy number of the extracted nucleic acid so as to compare the extraction efficiency of different methods. However, this method can only obtain a relative result, and cannot really calculate the extraction efficiency of the method.

There is also a study of evaluation using 2019-nCoV nucleic acid standards instead of virus particles, namely, performing one extraction operation on the nucleic acid standards by using a method to be evaluated, and indirectly calculating the extraction efficiency by measuring the amount of nucleic acid lost during the extraction. However, the method neglects the influence of virus cracking efficiency on extraction efficiency in actual extraction operation, so that the evaluation result is inaccurate.

Therefore, only by accurately measuring the number of 2019-nCoV particles, the extraction efficiency of the nucleic acid extraction method can be accurately and effectively evaluated!

However, there is currently no method available to accurately quantify 2019-nCoV particles. The mainstream detection technologies at home and abroad, including nucleic acid detection, antibody detection and antigen detection, are to simply detect nucleic acid or surface protein, so that the fact that a sample contains virus particles cannot be proved, and the method cannot accurately detect the number of the virus particles.

In view of the above, in order to accurately and efficiently evaluate nucleic acid extraction efficiency of a novel coronavirus (2019-nCoV), it is necessary to develop a method capable of precisely and quantitatively detecting 2019-nCoV particles for virus particle concentration measurement before nucleic acid extraction.

The invention content is as follows:

the invention aims to provide a method for evaluating the nucleic acid extraction efficiency of a novel coronavirus (2019-nCoV), which firstly needs to be capable of realizing accurate quantitative detection of 2019-nCoV particles.

Therefore, to achieve the above object, the following three technical barriers must be solved.

The technical key points are as follows: the difficulty of accurately identifying the 2019-nCoV particles is solved, and a fluorescence labeling method of the new coronavirus is established.

The difficulties to be overcome are:

at present, the mainstream detection technologies at home and abroad, including nucleic acid detection, antibody detection and antigen detection, are used for simply detecting nucleic acid or surface protein, and the fact that a sample contains virus particles cannot be proved.

It is necessary to recognize both the virus surface-specific protein and the internal nucleic acid in order to verify that the sample contains viral particles. Although the fluorescence labeling of bacteria is well established, the fluorescence labeling of viruses, especially of the new coronaviruses, presents a number of problems to be solved: for example, the nucleic acid of the novel coronaviruses is RNA, while the nucleic acid of the bacteria is DNA. Fluorescent dyes that can be used for bacterial staining generally cannot be used for viral staining.

The solving means is as follows:

first, a suitable antibody specific for 2019-nCoV was developed.

S protein and N protein of 2019-nCoV are selected as targets, and an S protein antibody and an N protein antibody of 2019-nCoV are developed. The S protein antibody is coupled with the red fluorescent probe, and the N protein antibody is coupled with the orange fluorescent probe. While 2019-nCoV was specifically fluorescently labeled with 2 antibodies and analyzed for fluorescence of 2019-nCoV using a flow analyzer.

The technical indexes of the S protein of 2019-nCoV determined by the invention are as follows: a) the antibody can react with 2019-nCoV in a mobile phase; b) after the antibody is crosslinked by a red fluorescent probe, the antibody can carry out fluorescent labeling on 2019-nCoV, and red fluorescence can be detected by a flow analyzer; c) the affinity KD of the antibody and the S protein of 2019-nCoV should be less than 1 x 10^-10。

Antibodies meeting the above requirements may be used in the method of the invention.

The technical indexes of the N protein of 2019-nCoV determined by the invention are as follows: a) the antibody can react with 2019-nCoV in a mobile phase; b) after the antibody is crosslinked by the orange fluorescent probe, 2019-nCoV can be subjected to fluorescent labeling, and orange fluorescence can be detected by a flow analyzer; c) the affinity KD of the antibody and the N protein of 2019-nCoV should be less than 1 x 10^-10。

Antibodies meeting the above requirements may be used in the method of the invention.

Second, a green fluorescent probe was developed that recognizes the 2019-nCoV genome

The invention screens out a green fluorescent probe which has good effect and can identify 2019-nCoV genome from a plurality of nucleic acid probes (example 1), is named Grn-3 (examples 3 and 4) and is suitable for a flow analyzer and is currently stored in China institute of metrology science.

The technical key 2 is as follows: how to realize quantitative detection of 2019-nCoV particles and select proper detection device

The difficulties to be overcome are:

2019-nCoV is a coronavirus, and belongs to the category of microorganisms. The flow analysis technique is a multi-parameter microbiological analysis technique, which can simultaneously analyze the number, size and biological characteristics of microorganisms, and has been successfully used for detecting bacteria. However, flow assay techniques are currently less common for detecting viruses, as detecting viruses can be much more difficult than detecting bacteria.

Since the largest difference between viruses and bacteria is the size difference. Bacteria are typically around 1 μm in size (0.5 μm to 3 μm), while viruses are only around 100nm, much smaller than bacteria. The sensitivity of the flow analyzer is generally difficult to meet the detection requirement, and when new coronavirus with the size of 100nm is detected, many background particles with the size of about 100nm cannot be distinguished by scattered light.

The solving means is as follows:

firstly, flow analyzers of almost all brands in the market are screened and verified, a nanoscale flow analyzer suitable for analyzing tiny particles is screened, and key technical parameters of the flow analyzer are determined.

The invention determines that the technical indexes and detection parameters suitable for the target flow analyzer are as follows: the fluorescence sensitivity is less than 10MESF, the scattered light sensitivity is less than 30nm, the fluorescence resolution RSD is less than 3%, and the scattered light resolution is less than 3%.

Secondly, a flow analysis technology is combined with an immunofluorescence probe technology to develop a method for quantitatively detecting virus particles. The method comprises the following steps: 1) distinguish 2019-nCoV from other background particles in the sample: simultaneously, using a red fluorescent probe to cross-link an S protein antibody of the 2019-nCoV through a chemical group, and using an orange fluorescent probe to cross-link an N protein antibody of the 2019-nCoV through a chemical group, and marking a specific coat protein of the 2019-nCoV; 2) distinguishing 2019-nCoV particles from 2019-nCoV particle fragments in the sample: labeling the genome of the 2019-nCoV nucleic acid with a green nucleic acid fluorescent probe; 3) the red, orange and green fluorescence signals generated by 1) and 2) are simultaneously counted by a flow analyzer, so that the 2019-nCoV particles are rapidly and quantitatively detected.

The technical key is as follows: the difficulty in obtaining a sample of the new coronavirus is great and it is necessary to obtain a real sample from a clinical patient

The difficulties to be overcome are:

2019-nCoV has strong infectivity, belongs to a second class of pathogenic microorganisms, but is managed according to the first class of pathogenic microorganisms. The acquisition of the sample itself is a difficult task when developing the method.

When detecting bacteria, if the number concentration of bacteria in the sample does not reach the detection limit, various methods can be used for culturing and enriching. The detected sample of the virus can not be cultured, and the new coronavirus sample is generally taken from the real sample of the patient. This again makes detection more difficult.

The solving means is as follows:

the project group specially proceeds to Wuhan gold, silver and pool hospital in 2020, and virus real samples of new crown patients are collected.

Therefore, the invention establishes a method for evaluating the nucleic acid extraction efficiency of the new coronavirus (2019-nCoV) for the first time, and the method comprises the following steps:

1) one sample containing 2019-nCoV was mixed well and divided into two portions (one for measuring particle concentration; the other part is used for nucleic acid extraction, and the concentrations of the two parts are the same).

2) Taking one sample, and determining the concentration of 2019-nCoV particles;

3) extracting nucleic acid of the 2019-nCoV virus in another sample by using a nucleic acid extraction method to be evaluated;

4) determining the copy number of the nucleic acid extracted in the step 3) by adopting a digital PCR (dPCR) technology;

5) calculating the extraction efficiency E of the nucleic acid extraction method to be evaluated according to the following formula;

wherein E is the nucleic acid extraction efficiency of the method to be evaluated;

c_dPCRthe copy number of the nucleic acid obtained in the step 4) is unit copies/. mu.L;

c_FCMthe concentration of the particles obtained in step 2) was in units of events/. mu.L.

Wherein, the 2019-nCoV particles are determined in the step 2) by the following method:

(1) differentiating 2019-nCoV from non-biological background particles in the sample:

simultaneously labeling the specific coat protein of the 2019-nCoV by using an S protein antibody and an N protein antibody of the 2019-nCoV, which are cross-linked by a chemical group through a fluorescent probe; the S protein antibody uses a red fluorescent probe, and the N protein antibody uses an orange fluorescent probe;

(2) differentiating the 2019-nCoV particles and the 2019-nCoV fragments in the sample:

labeling the genome of the 2019-nCoV nucleic acid with a green nucleic acid fluorescent probe;

(3) counting red, orange and green fluorescent signals generated by 1) and 2) by a flow analyzer to realize the rapid quantitative detection of the 2019-nCoV particles;

in the above (3), the determination method for an event generated by one particle is as follows:

judging the S protein fragment of 2019-nCoV if only red fluorescence is detected;

if only orange fluorescence is detected, judging the protein fragment to be the N protein fragment of 2019-nCoV;

if three kinds of fluorescence of red, orange and green are detected simultaneously, judging the particles to be 2019-nCoV;

if no fluorescence is detected, the particle is judged to be an abiotic foreign particle.

The technical indexes of the S protein antibody of 2019-nCoV are as follows: 1) the antibody can react with 2019-nCoV in a mobile phase; 2) the antibody can be subjected to fluorescent labeling on 2019-nCoV after being crosslinked by a red fluorescent probe, and the red fluorescence can be detected by a flow analyzer 3) the affinity KD of the antibody and the S protein of the 2019-nCoV is less than 1 x 10^-10。

The technical indexes of the N protein antibody of 2019-nCoV are as follows: 1) the antibody can react with 2019-nCoV in a mobile phase; 2) after the antibody is crosslinked by the orange fluorescent probe, 2019-nCoV can be subjected to fluorescent labeling, and orange fluorescence can be detected by a flow analyzer; 3) the affinity KD of the antibody and the N protein of 2019-nCoV should be less than 1 x 10^-10。

The counting by the flow analyzer is realized by performing a gate enclosing through a scattered light channel of the flow analyzer and detecting the red, orange and green fluorescent signals through three fluorescent channels, so as to count the 2019-nCoV particles; wherein the ring gate of the forward angle scattered light channel is between 60nm and 150 nm. The method of the ring door comprises the following steps: and measuring the 60nm standard microspheres and the 150nm standard microspheres by using a flow analyzer, and performing gate looping according to the signal positions of the microspheres on a histogram of a forward angle scattered light channel, wherein the lower limit is 60nm, and the upper limit is 150 nm.

The fluorescence emission spectrum of the red fluorescent probe is 601 nm-640 nm; the fluorescence emission spectrum of the orange fluorescent probe is 551 nm-590 nm; the fluorescence emission spectrum of the green fluorescent probe is 501 nm-540 nm.

The technical indexes and detection parameters of the flow analyzer are as follows: the fluorescence sensitivity is less than 10MESF, the scattered light sensitivity is less than 30nm, the fluorescence resolution RSD is less than 3%, and the scattered light resolution is less than 3%.

Therefore, by the above method, the evaluation of the nucleic acid extraction efficiency of the novel coronavirus (2019-nCoV) can be achieved.

The invention clearly discloses the method of the invention by a plurality of experiments, which are detailed in the examples. Wherein:

example 1 is screening of S protein antibodies and N protein antibodies.

Example 2 is a specific dual fluorescent label of 2019-nCoV coat.

Example 3 is the screening of 2019-nCoV nucleic acid fluorescent probes.

Example 4 is the specificity evaluation of the 2019-nCoV viral particle measurement method.

Example 5 is a comparison of five brands of nucleic acid extraction reagents.

The following is a specific operation of one practical test of the present invention.

1. 2019-nCoV particle concentration measurement before extraction: and adding a red fluorescent probe cross-linked S protein antibody (Rd-S-Ab) with the final concentration of 1 mu g/mL, an orange fluorescent probe cross-linked N protein antibody (Org-N-Ab) with the final concentration of 1 mu g/mL and a 2019-nCoV nucleic acid green fluorescent probe (Grn) with the concentration of 1 mu g/mL into the sample solution, uniformly mixing, and incubating for 10-30 min in a dark place. Detection was then performed using a flow analyzer with a gate of 50nm to 150nm for the forward angle scattered light channel. The 2019-nCoV particle concentration c is calculated by analyzing the events in the gate in the three fluorescence channels_FCM。

2. Nucleic acid extraction: extracting the nucleic acid of 2019-nCoV from the parallel samples by using a nucleic acid extraction method to be evaluated;

3. 2019-nCoV nucleic acid copy number measurement after extraction: using the dPCR technique, the copy numbers of the N gene, E gene and Orf1ab gene of 2019-nCoV in the extracted sample were measured, and the average of the copy numbers of the three genes was calculated.

4. And (3) calculating the extraction efficiency: the nucleic acid extraction efficiency of the new coronavirus (2019-nCoV) was calculated using the formula.

The invention has the following innovations and advantages:

1. 2019-nCoV particles can be identified and quantitatively detected;

2. can accurately evaluate the extraction efficiency of 2019-nCoV nucleic acid

3. The evaluation mode is direct and effective, and the evaluation result is traceable.

Drawings

FIG. 1 is the fluorescent labeling of the outer shell of the 2019-nCoV particle in example 2. Wherein, the flow detection result of the S protein antibody (Rd-S-Ab) after being singly marked is shown in figure 1; FIG. 1 shows the results of flow assays of protein N antibody (Org-N-Ab) labeled alone; FIG. 1 shows the flow detection results of Rd-S-Ab and Org-N-Ab after double fluorescence labeling.

FIG. 2 shows the results of detection using a flow analyzer after labeling 2019-nCoV nucleic acids with 5 different green fluorescent probes in example 3.

Detailed Description

The invention is further illustrated below with reference to specific examples. It is to be understood that these examples are merely illustrative of the process of the present invention and are not intended to limit the scope of the present invention.

The consumables of the test reagents used in the following examples are all conventional reagents or materials, unless otherwise specified. The experimental method not specified for the specific conditions was carried out under the conventional conditions or the conditions recommended by the manufacturer.

Example 12019-screening of S protein antibody and N protein antibody of nCoV

Materials and methods

1. 5S protein antibodies (accession numbers S-Ab-1 to S-Ab-5) of 2019-nCoV were selected for dilution to the same initial concentration and each 2-fold serial dilution was performed.

2. The S protein of 2019-nCoV was linked to a carboxyl chip using an EDC/NHS reaction. And the carboxyl chip was loaded into an analytical interaction analyzer.

3. For an S protein antibody, 2-fold serial dilutions of the antibody were loaded separately into the molecular interaction apparatus, data collected, and a series of response curves were fitted with TraceDrawer and the affinity KD of the antibody and S protein was calculated.

4. With reference to steps 1 to 3, the affinity of 5 2019-nCoV N protein antibodies (numbered N-Ab-1 to N-Ab-5) for the N protein was determined.

Second, experimental results

The affinity of 5S protein and N protein antibodies of 2019-nCoV was analyzed using an analytical interaction analyzer, and the results are shown in Table 1. The results showed that the affinity of the antibody S-Ab-2 to the S protein was the best, 2.31X 10^-11(ii) a The affinity of the antibody N-Ab-3 to the N protein is preferably 3.99X 10^-11。

Third, conclusion of experiment

The S protein antibody (S-Ab-2) and the N protein antibody (N-Ab-3) of 2019-nCoV with the best affinity are screened out.

TABLE 1 antigen-antibody affinities of S and N proteins

Example 22019 Dual fluorescent labeling of nCoV Shell

Materials and methods

1. The S protein antibody (S-Ab-2) of 2019-nCoV was labeled with red fluorescein Cy5 using a Cy5 antibody coupling kit to give Rd-S-Ab. The N protein antibody (N-Ab-3) was labeled with orange fluorescein PE using a PE antibody coupling kit to give Org-N-Ab.

2. Purified 2019-nCoV particles, a saliva sample from a new coronavirus patient in Wuhan Tantang Hospital.

3. And adding a red fluorescent probe crosslinked S protein antibody (Rd-S-Ab) with the final concentration of 1 mu g/mL and an orange fluorescent probe crosslinked N protein antibody (Org-N-Ab) with the final concentration of 1 mu g/mL into the sample solution, uniformly mixing, and incubating for 30min in the dark.

4. Detection was performed with a flow analyzer. The ring gate of the forward angle scattered light channel is between 50nm and 150 nm. 2019-nCoV particle concentration was calculated by analyzing events within the gates at FL2 and FL3 fluorescence channels.

Second, experimental results

The particles in the gate of the flow analyzer can observe red fluorescence in the histogram of the red fluorescence channel (FL3), and can observe orange fluorescence in the histogram of the orange fluorescence channel. And in the FL2-FL3 scattergrams, particles with both fluorescences simultaneously can be distinguished from background particles. Indicating that 2019-nCoV particles were successfully labeled with red and orange fluorescence (FIG. 1).

Third, conclusion of experiment

The screened and fluorescently-labeled Rd-S-Ab and Org-N-Ab can react with 2019-nCoV particles in a mobile phase. And the bi-fluorescently labeled 2019-nCoV particles can be detected by a flow analyzer.

Example 32019 fluorescent labeling of nCoV nucleic acids

Materials and methods

1. 6 2019-nCoV nucleic acid fluorescent probes (Nos. Grn-1 to Grn-5) were selected to be diluted to the same initial concentration of 1. mu.g/mL.

2. Purified 2019-nCoV particles, a saliva sample from a new coronavirus patient in Wuhan Tantang Hospital. Using a DNA scavenger, the DNA in the sample is cleared.

3. Grn-1 to Grn-5 are respectively added into the sample solution, and the mixture is evenly mixed and incubated for 30min in the dark.

4. Detection was performed with a flow analyzer. The ring gate of the forward angle scattered light channel is between 50nm and 150 nm. The 2019-nCoV particle concentration was calculated by analyzing events within the gate at the FL1 fluorescence channel.

Second, experimental results

Grn-1, Grn-2, Grn-5 failed to make 2019-nCoV particles inside the apron marked green fluorescence. Grn-4 although it was possible to mark 2019-nCoV, it was not possible to distinguish 2019-nCoV from background particles. Only Grn-3 allowed the 2019-nCoV particles within the apron to be labeled green fluorescence and distinguished from background particles (FIG. 2).

Third, conclusion of experiment

The green fluorescent probe which has good screening effect, can identify 2019-nCoV genome and is suitable for the flow analyzer is named as Grn.

Example 4 validation of flow measurement method specificity

Materials and methods

1. Purified 2019-nCoV particles, a saliva sample from a new coronavirus patient in Wuhan Tantang Hospital. Using a DNA scavenger, the DNA in the sample is cleared.

2. The purified vesicular stomatitis virus, influenza A virus, respiratory syncytial virus, hepatitis A virus, hepatitis C virus and enterovirus 71 are from Wuhan virus institute in Chinese academy of sciences.

3. And adding a red fluorescent probe cross-linked S protein antibody (Rd-S-Ab) with the final concentration of 1 mu g/mL, an orange fluorescent probe cross-linked N protein antibody (Org-N-Ab) with the final concentration of 1 mu g/mL and a 2019-nCoV nucleic acid green fluorescent probe (Grn) with the concentration of 1 mu g/mL into the 2019-nCoV sample solution, uniformly mixing, and incubating for 10-30 min in a dark place.

4. Detecting by a flow analyzer: the ring gate of the forward angle scattered light channel is between 50nm and 150 nm. The 2019-nCoV particle concentration was calculated by analyzing events within the gate in the three fluorescence channels.

5. Repeating steps 3 and 4, and measuring the virus sample of the control group.

Second, experimental results

2019-nCoV particles and solutions of different viruses were reacted with Rd-S-Ab, Org-N-Ab and Grn, respectively, and then detected using a flow analyzer to verify the specificity of the method. The results (Table 2) show that only 2019-nCoV particles simultaneously detect red, orange and green fluorescence, and other viruses only detect green fluorescence.

Third, conclusion of experiment

The flow measurement method has good specificity and can accurately identify 2019-nCoV.

TABLE 2 results of specificity experiments for flow methods

Example 5 comparison of five brands of nucleic acid extraction reagents

Materials and methods

1. Purified 2019-nCoV particles, new coronavirus patient saliva from Wuhan gold and silver pond hospitalA liquid sample. Using a DNA scavenger, the DNA in the sample is cleared. The 2019-nCoV particle solution was concentrated to a concentration of about 10⁷one/mL and its precise concentration was measured using flow analysis techniques.

2. The information of five brands of nucleic acid extraction reagents is shown in Table 3. Using 5 kits, nucleic acid extraction was performed on 2019-nCoV samples, respectively.

TABLE 3 basic conditions of five brands of nucleic acid extraction reagents

3. Nucleic acid copy number measurements were performed on 5 extracted samples using dPCR, respectively.

Second, experimental results

The results showed that the concentration of the new coronavirus particles in the sample was 4.2X 10⁷events/mL. The extraction efficiencies of the five brands (a to E) of nucleic acid extraction reagents were: 48.5%, 67.6%, 59.3%, 55.4%, 63.0%.

Third, conclusion of experiment

The method can directly, effectively and accurately evaluate the nucleic acid extraction efficiency of the new coronavirus.