CN110144422B - Quadruple fluorescent quantitative detection kit for simultaneously detecting four human coronaviruses - Google Patents

Abstract

The invention relates to a quadruple fluorescence quantitative detection kit for simultaneously detecting four human coronaviruses, belonging to the technical field of human coronavirus detection. The human coronavirus is MERS-CoV/HCoV-NL63/HCoV-OC43/HCoV-HKUI; the detection kit specifically comprises: specific primer pairs and probes, positive quality control and negative quality control; each group of specific primer pairs and probes comprise a pair of specific primer pairs and a probe sequence; the positive quality control product is specifically a target sequence synthesized artificially; the negative quality control material is deionized water or sterile water. The kit can realize the simultaneous detection of four human coronaviruses by a single tube, has strong detection specificity and high detection sensitivity, and has quick detection time, thereby avoiding the defects of long time consumption, high detection cost, easy cross contamination and the like caused by one-by-one detection. The similar related product report is not seen in the market at present, and the kit has wide application prospect in the aspects of disease detection, infectious disease prevention and control, clinical treatment guidance and the like.

Description

Quadruple fluorescent quantitative detection kit for simultaneously detecting four human coronaviruses

Technical Field

The invention relates to a quadruple fluorescence quantitative detection kit for simultaneously detecting four human coronaviruses, belonging to the technical field of human coronavirus detection.

Background

Human coronavirus (Human Coronaviruses, hCoVs) infection is one of the important pathogens causing human respiratory diseases, and the detection rate of coronavirus among common respiratory pathogens is about 3% -10%, so that the human coronavirus is an important pathogen for infecting infants, old people and immunocompromised adults, thereby causing clinical symptoms such as cough, fever, laryngitis, bronchitis, pneumonia and the like. Currently, six human coronaviruses are known, namely human coronavirus 229E (Human coronavirus E, HCoV-229E) and HCoV-OC43 found in the sixty of the 19 th century, severe acute respiratory syndrome (Severe acute respiratory syndrome, SARS) coronavirus SARS-CoV found in the Guangdong of China, novel human coronavirus NL63 (HCoV-NL 63) identified in the Netherlands in 2004, novel human coronavirus HKU1 (HCoV-HKU 1) identified in the hong Kong in 2005, and middle east respiratory syndrome (Middle East respiratory syndrome, MERS) coronavirus MERS-CoV found in the middle east region in 2012 month 6.

Epidemiological studies show that four human coronaviruses, HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKUI, are prevalent throughout the world and are distributed globally, and clinical symptoms are mainly manifested as fever, cough, rhinitis, laryngitis, bronchitis, pneumonia and the like, thus causing a certain burden to human life and health. SARS-CoV and MERS-CoV can cause acute respiratory symptoms after infection of human, and severe patients cause respiratory and renal failure and death. SARS-CoV caused about 9000 worldwide infections since the first appearance in Guangdong in China in 2003, about 800 deaths, and mortality rates of nearly 10%. Thanks to the effective disease prevention and control measures worldwide, the SARS-CoV has disappeared and no popular report is found at present. MERS-CoV as a newly emerging sixth human coronavirus, there are 26 countries and regions worldwide in which 2428 cases of confirmed diagnosis, 839 cases of death, and the mortality rate is as high as 34.6%. MERS-CoV is currently mainly popular in the middle east sauter arabian area, and once concentrated outbreaks of popularity in korea occur in 2015, there is also one example of cases imported from korea in our country. The MERS-CoV is not only an infectious disease which is monitored by national disease control emphasis, but also one of important infectious diseases which are prevented from being transmitted by the port of the national border at present.

Early prevention and control are preconditions for preventing infectious disease input from spreading, guaranteeing public health safety and maintaining human life health, and developing a rapid, sensitive and specific molecular detection technical method is a key for early prevention and control, and provides an important basis for guiding clinical and reasonable selection of antiviral drugs. At present, the detection technical methods for human coronaviruses mainly comprise virus separation culture, serological detection method, common PCR method, isothermal amplification method, dissolution curve method and fluorescent quantitative RT-PCR method. The virus separation culture, serological detection method and common PCR method are relatively conventional detection methods, and the methods have the defects of lower detection sensitivity, overlong detection time, high requirements on laboratory hardware, high technical requirements on operators and the like in the experimental process, so that the timeliness of detection and the accuracy of results cannot be ensured. Isothermal amplification and fluorescent quantitative RT-PCR are molecular detection techniques commonly used in most laboratories today. The isothermal amplification method has the outstanding problems that detection equipment and detection reagents are expensive, cross contamination is easy to occur, the detection result is uncertain, and the like, and can only realize single pathogen detection, and can not realize single-tube multiple detection. The method of dissolution profile mainly uses the difference of Tm value of amplified products to distinguish by the position difference of melting profile, which requires that the primer design must have difference of Tm value, and the difference of single base changes Tm value, resulting in unreliability of detection result. The fluorescent quantitative RT-PCR method also faces the problem that single-tube single-fold (up to double or triple) pathogen detection can be realized at present, so that the pathogen screening frequency is increased, the detection result time is greatly prolonged, the operation process is increased, the detection cost is greatly increased, and the rapid diagnosis and early prevention and control of diseases are not facilitated. The rapid and accurate detection of the hCoVs has important significance for monitoring and preventing and controlling the epidemic of the hCoVs, and provides reliable laboratory basis for clinical early diagnosis, reasonable selection of antiviral drug treatment and the like.

Disclosure of Invention

The invention aims to overcome the defects of long time consumption, low detection efficiency, high detection cost, low detection timeliness and the like in the detection process of hCoVs, and provides a quadruple fluorescence quantitative detection kit for simultaneously detecting four human coronaviruses; it can detect four human coronaviruses of MERS-CoV, HCoV-NL63, HCoV-OC43 and HCoV-HKUI simultaneously by a single tube.

According to the technical scheme, the quadruple fluorescence quantitative detection kit for simultaneously detecting four human coronaviruses, wherein the human coronaviruses are MERS-CoV/HCoV-NL63/HCoV-OC43/HCoV-HKUI; the detection kit specifically comprises: specific primer pairs and probes, positive quality control and negative quality control;

each group of specific primer pairs and probes comprise a pair of specific primer pairs and a probe sequence; the positive quality control product is specifically a target sequence synthesized artificially; the negative quality control material is deionized water or sterile water.

Based on the four human coronavirus genomic sequences MERS-CoV, HCoV-NL63, HCoV-OC43 and HCoV-HKUI, the conserved sequences of each coronavirus were searched for, and preferably, specific primer sets and target probes suitable for qRT-PCR were designed, respectively, with the following primer information:

specific primer pairs comprise a primer 1 and a primer 2 aiming at human coronavirus MERS-CoV, wherein the sequence of the primer 1 is shown as SEQ ID No.1, and the sequence of the primer 2 is shown as SEQ ID No.2; the sequence of the probe 1 is shown as SEQ ID No.3;

aiming at the human coronavirus HCoV-NL63, the specific primer pair comprises a primer 3 and a primer 4, wherein the sequence of the primer 3 is shown as SEQ ID No.4, and the sequence of the primer 4 is shown as SEQ ID No.5; the sequence of the probe 2 is shown as SEQ ID No.6;

aiming at the human coronavirus HCoV-OC43, the specific primer pair comprises a primer 5 and a primer 6, the sequence of the primer 5 is shown as SEQ ID No.7, and the sequence of the primer 6 is shown as SEQ ID No.8; the sequence of the probe 3 is shown as SEQ ID No.9;

aiming at the human coronavirus HCoV-HKUI, the specific primer pair comprises a primer 7 and a primer 8, the sequence of the primer 7 is shown as SEQ ID No.10, and the sequence of the primer 8 is shown as SEQ ID No.11; the sequence of the probe 4 is shown as SEQ ID No.12.

Further, the four primer probes are fluorescently labeled with different fluorescent colors.

Further, the fluorescent groups for labeling the 5 'end of the primer probe are FAM, VIC, ROX and Cy5, and the quenching group for labeling the 3' end is MGB.

Further, in group 1, the 5 'end of probe 1 is labeled with FAM fluorescent group, and the 3' end is labeled with MGB fluorescence quenching group; the 5 'end of the probe 2 in the group 2 is marked with a VIC fluorescent group, and the 3' end is marked with an MGB fluorescent quenching group; in the group 3, the 5 'end of the probe 3 is marked with a ROX fluorescent group, and the 3' end is marked with an MGB fluorescent quenching group; the 5 'end of probe 4 in group 4 was labeled with Cy5 fluorescent group and the 3' end was labeled with MGB fluorescence quenching group.

The details are shown in table 1 below:

TABLE 1

Further, the positive quality control product sequence comprises a section of 102bp target fragment covering MERS-CoV specific primers and probes, a section of 109bp target fragment covering HCoV-NL63 specific primers and probes, a section of 122bp target fragment covering HCoV-OC43 specific primers and probes, and a section of 143bp target fragment covering HCoV-HKU1 primers and probes; the quality control material sequence is directly used as a positive quality control material after artificial synthesis, or is constructed on a plasmid vector after artificial synthesis, and clone plasmids are extracted through shaking bacteria to be used as the positive quality control material.

Further, the target sequence of the positive quality control product is specifically shown as SEQ ID No.13.

The method comprises the following steps:

the quadruple fluorescence quantitative detection kit is applied to detection of four human coronaviruses of MERS-CoV, HCoV-NL63, HCoV-OC43 and HCoV-HKUI simultaneously.

The detection method adopting the quadruple fluorescence quantitative detection kit comprises the following steps:

(1) Extraction of sample nucleic acid: taking a sample to be detected, and extracting nucleic acid from the sample to obtain sample nucleic acid;

(2) qRT-PCR detection: preparing a reaction system by adopting MERS-CoV/HCoV-NL63/HCoV-OC43/HCoV-HKUI specific primers and probe sequences, 2-d reaction solution, DNA polymerase and RT reverse transcriptase and the sample nucleic acid extracted in the step 1; taking a positive quality control product and a negative quality control product to respectively prepare a reaction system consistent with sample nucleic acid; qRT-PCR detection is carried out on the three reaction systems;

(3) And (3) analysis and judgment: adjusting a threshold value through the highest point of the fluorescent signal obtained by the negative quality control product in the step (2), and controlling the positive quality control product to have S-shaped amplification curves in all four detection channels; and then comparing the amplification curve obtained by the sample nucleic acid with a positive quality control product, and judging the result.

Further, applicable instrumentation includes, but is not limited to ABI7000/7300/7500/7500Fast/7900HT/StepOned ^TM Etc., instruments from Roche, bio-Rad, QIAGEN, etc.

Preferably, using a Bio-Rad CFX96 instrument as an example, the amplification procedure is set as follows: reacting at 42 ℃ for 10min; reacting for 5min at 95 ℃; the reaction was then cycled 40 times, the cycling procedure being: reacting at 94 ℃ for 10-15 s, reacting at 58-60 ℃ for 30-60 s, and collecting fluorescence.

And (3) detecting and setting: taking a Bio-Rad CFX96 instrument as an example, clicking a "plate" window, entering a "create New" sample information editing interface, respectively selecting FAM, VIC, ROX and Cy5 fluorescent channels in "select Folurophore", and respectively corresponding to detection of MERS-CoV, HCoV-NL63, HCoV-OC43 and HCoV-HKUI.

The analysis and judgment process of the step (3) is specifically as follows:

a. adjusting a threshold value: the threshold setting principle is to take the highest point of the fluorescence signal just exceeding the negative quality control as a threshold line, or to adjust according to the noise condition of the instrument;

b. and (3) quality control: the negative quality control product has no amplification curve, the positive quality control product has S-shaped amplification curves in four detection channels of FAM, VIC, ROX and Cy5, and the experiment is established; otherwise, the experimental result is invalid;

c. and (3) result judgment:

c1, if the detection sample has S-type amplification and Ct or Cq value is less than or equal to 38, judging according to a fluorescent channel corresponding to the detection target;

c2, if S-type amplification exists, and the Ct value of 38< Ct or Cq value is less than 40, judging that the sample is an uncertain sample, and detecting after extracting nucleic acid again;

c3, if the result of the rechecking sample is consistent with that of c2, judging that the sample is weak positive;

c4, if no obvious S-type amplification curve exists, ct or Cq values are reported, and the amplification is nonspecific, and the result is judged as negative.

The invention has the beneficial effects that: the invention can rapidly and simultaneously screen four human coronaviruses MERS-CoV/HCoV-NL63/HCoV-OC43/HCoV-HKUI; the method has the outstanding advantages of high detection sensitivity, strong specificity, capability of realizing multiple rapid detection, simple operation, convenient application and the like, and provides a feasible technical method for rapid pathogen detection in the fields of clinical diagnosis, disease monitoring detection, inspection and quarantine and the like.

Drawings

FIG. 1 is a schematic diagram of the amplification curve of the sample of example 1.

FIG. 2 is a schematic diagram of the amplification curve of the sample of example 2.

FIG. 3 is a schematic diagram of the amplification curve of the sample of example 3.

FIG. 4 is a schematic diagram of the amplification curve of the sample of example 4.

FIG. 5 is a schematic representation of the amplification curves of HCoV-HKU1 samples for the kit of the invention and company A kit.

Detailed Description

In the embodiment of the invention, the kit for simultaneously detecting four human coronaviruses qRT-PCR of MERS-CoV, HCoV-NL63, HCoV-OC43 and HCoV-HKUI comprises an amplification system, and the detection method comprises the following steps:

(1) Sample preparation: the sample nucleic acid can be directly extracted from samples such as nasopharyngeal swab, alveolar lavage liquid, sputum and the like by using a nucleic acid extraction kit, or can be manually extracted, and also can be extracted by using a full-automatic nucleic acid extractor, and the extracted nucleic acid is directly used for the next amplification reaction.

(2) qRT-PCR detection:

the One-step qRT-PCR Kit (TOYOBO) was selected and specifically configured as follows, taking a 25. Mu.L reaction system as an example: 2. Mu.L of a reaction solution of Kjellmaniella; 1. Mu.L of DNA polymerase; 1. Mu.L of RT reverse transcriptase; four sets of specific primer pairs, 8, 0.5 mu L of each primer and 0.5 mu M of final concentration; four probe sequences each 0.375. Mu.L, 0.2. Mu.M final concentration; preparing three tubes of the reaction system, adding 5 mu L of the sample RNA extracted in the step (1), positive quality control products and negative quality control products into each reaction system, and then adopting ultrapure water to fix the volume to 25 mu L;

the amplification procedure set-up is shown in Table 2 using a Bio-Rad CFX96 instrument as an example.

TABLE 2

And (3) detecting and setting: taking a Bio-Rad CFX96 instrument as an example, clicking a "plate" window, entering a "create New" sample information editing interface, respectively selecting FAM, VIC, ROX and Cy5 fluorescent channels in "select Folurophore", and respectively corresponding to detection of MERS-CoV, HCoV-NL63, HCoV-OC43 and HCoV-HKUI.

(3) Result analysis and determination:

a. adjusting a threshold value: the threshold setting principle is to take the highest point of the fluorescence signal just exceeding the negative quality control as a threshold line, or to adjust according to the noise condition of the instrument;

b. and (3) quality control: the negative quality control product has no amplification curve, the positive quality control product has S-shaped amplification curves in four detection channels of FAM, VIC, ROX and Cy5, and the experiment is established; otherwise, the experimental result is invalid;

c. and (3) result judgment:

c1, if the detection sample has S-type amplification and Ct or Cq value is less than or equal to 38, judging according to a fluorescent channel corresponding to the detection target and according to a table 3;

c2, if S-type amplification exists, and the Ct value of 38< Ct or Cq value is less than 40, judging that the sample is an uncertain sample, and detecting after extracting nucleic acid again;

c3, if the result of the rechecking sample is consistent with that of c2, judging that the sample is weak positive;

c4, if no obvious S-type amplification curve exists, ct or Cq values are reported, and the amplification is nonspecific, and the result is judged as negative.

TABLE 3 Table 3

Fluorescent channel	FAM	VIC	ROX	Cy5
					MERS-CoV	Positive (+)	Negative (-)	Negative (-)	Negative (-)
HCoV-NL63	Negative (-)	Positive (+)	Negative (-)	Negative (-)
					HCoV-OC43	Negative (-)	Negative (-)	Positive (+)	Negative (-)
HCoV-HKU1	Negative (-)	Negative (-)	Negative (-)	Positive (+)

Example 1

The sample to be measured is: nasopharyngeal swab.

The results were interpreted according to Table 3, and the specific amplification curves are shown in FIG. 1.

As shown in FIG. 1, the negative quality control product or the blank control product has no amplification curve, the positive quality control product has S-shaped amplification curves in the FAM, VIC, ROX detection channels and the Cy5 detection channels, the sample to be detected only has the S-shaped amplification curve in the FAM fluorescence channel, the Ct (or Cq) value is less than or equal to 38, and the MERS-CoV of the sample to be detected is judged to be positive.

Example 2

The sample to be measured is: nasopharyngeal swab.

The results were interpreted according to Table 3, and the specific amplification curves are shown in FIG. 2.

As shown in FIG. 2, the negative quality control product or the blank control product has no amplification curve, the positive quality control product has S-shaped amplification curves in the FAM, VIC, ROX detection channels and the Cy5 detection channels, the sample to be detected only has the S-shaped amplification curve in the VIC fluorescence channel, the Ct (or Cq) value is less than or equal to 38, and the HCoV-NL63 of the sample to be detected is judged to be positive.

Example 3

The sample to be measured is: nasopharyngeal swab.

The results were interpreted according to Table 3, and the specific amplification curves are shown in FIG. 3.

As shown in FIG. 3, the negative quality control or the blank control has no amplification curve, the positive quality control has S-shaped amplification curves in the FAM, VIC, ROX detection channels and the Cy5 detection channels, the sample to be detected only has the S-shaped amplification curve in the ROX fluorescence channel, the Ct (or Cq) value is less than or equal to 38, and the HCoV-OC43 of the sample to be detected is judged to be positive.

Example 4

The sample to be measured is: nasopharyngeal swab.

The results were interpreted according to Table 3, and the specific amplification curves are shown in FIG. 4.

As shown in FIG. 4, the negative quality control product or the blank control product has no amplification curve, the positive quality control product has S-shaped amplification curves in the FAM, VIC, ROX detection channel and the Cy5 detection channel, the sample to be detected has S-shaped amplification curve only in the Cy5 fluorescence channel, the Ct (or Cq) value is less than or equal to 38, and the HCoV-HKU1 of the sample to be detected is judged to be positive.

Example 5

Other pathogens which have homology with the nucleic acid sequence of the human coronavirus, are easy to cause the same or similar clinical symptoms and are easy to cause normal parasitism or concurrence at the sampling position, such as influenza A virus, influenza B virus, influenza C virus, rhinovirus, respiratory syncytial virus, adenovirus, measles virus, rubella virus and mumps virus are selected as samples to be detected, the detection is carried out by using the kit, the operation is strictly carried out according to the specification of the kit, the detection is carried out on a CFX96 real-time fluorescence quantitative PCR instrument, and the detection results are negative, so that the kit has better detection specificity.

Example 6

The detection of HCoV-HKU1 is carried out by selecting the kit and the A company single detection kit, the result is shown in figure 5, and the HCoV-HKU1 virus is detected by adopting the quadruple qRT-PCR kit and the A company single detection kit, the Ct value of the positive quality control product of the kit is far lower than that of the A company, and the positive HCoV-HKU1 can be detected. The HCoV-HKU1 could not be detected by company A, and the detection sensitivity was lower than that of the kit of the invention.

Example 7

3 batches of the kit are used for carrying out 10 times of repeated detection on the standard substances with three concentrations of positive quality control products, negative quality control products, medium and low quality control products, and the CV range of the in-batch imprecision Ct value is as follows: MERS-CoV 0.68-1.42%, HCoV-NL63 0.72-1.55%, HCoV-OC43 0.77-1.47%, HCoV-HKU1 0.89-1.65%; the CV range of the batch-to-batch imprecision Ct values is: MERS-CoV 1.12-1.19%, HCoV-NL63 1.04-1.40%, HCoV-OC43 1.03-1.52%, HCoV-HKU1 0.95-1.40%; CV values of the Ct values of the precision in the batch and the precision between the batches are less than 5%, and negative controls are negative, which shows that the kit has good precision and the detection results are shown in the following table 4.

TABLE 4 Table 4

The reagent can realize the simultaneous detection of four human coronaviruses, namely MERS-CoV, HCoV-NL63, HCoV-OC43 and HCoV-HKUI, by a single tube, has the advantages of strong detection specificity, high detection sensitivity and quick detection time, and avoids the defects of long time consumption, high detection cost, easiness in cross contamination and the like caused by one-by-one detection. The similar related product report is not seen in the market at present, and the kit has wide application prospect in the aspects of disease detection, infectious disease prevention and control, clinical treatment guidance and the like.

The foregoing has outlined and described the basic principles, features, and advantages of the invention. It will be appreciated by persons skilled in the art that the invention is not limited to the embodiments described above, and that the embodiments and descriptions described in the foregoing are merely illustrative of the principles of the invention, and that various changes and modifications may be made therein without departing from the spirit and scope of the invention, which is defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

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Chinese Center for Disease Control and Prevention

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Claims (6)

1. A quadruple fluorescence quantitative detection kit for simultaneously detecting four human coronaviruses is characterized in that: the human coronavirus is MERS-CoV/HCoV-NL63/HCoV-OC43/HCoV-HKUI; the detection kit specifically comprises: specific primer pairs and probes, positive quality control and negative quality control;

each group of specific primer pairs and probes comprise a pair of specific primer pairs and a probe sequence; the positive quality control product is specifically an artificially synthesized target sequence, and the target sequence of the positive quality control product is specifically shown as SEQ ID No.13; the negative quality control material is deionized water or sterile water;

specific primer pairs comprise a primer 1 and a primer 2 aiming at human coronavirus MERS-CoV, wherein the sequence of the primer 1 is shown as SEQ ID No.1, and the sequence of the primer 2 is shown as SEQ ID No.2; the sequence of the probe 1 is shown as SEQ ID No.3;

aiming at the human coronavirus HCoV-NL63, the specific primer pair comprises a primer 3 and a primer 4, wherein the sequence of the primer 3 is shown as SEQ ID No.4, and the sequence of the primer 4 is shown as SEQ ID No.5; the sequence of the probe 2 is shown as SEQ ID No.6;

aiming at the human coronavirus HCoV-OC43, the specific primer pair comprises a primer 5 and a primer 6, the sequence of the primer 5 is shown as SEQ ID No.7, and the sequence of the primer 6 is shown as SEQ ID No.8; the sequence of the probe 3 is shown as SEQ ID No.9;

aiming at the human coronavirus HCoV-HKUI, the specific primer pair comprises a primer 7 and a primer 8, the sequence of the primer 7 is shown as SEQ ID No.10, and the sequence of the primer 8 is shown as SEQ ID No.11; the sequence of the probe 4 is shown as SEQ ID No.12;

adopting different fluorescent colors to carry out fluorescent labeling on four probe sequences of a probe 1 sequence, a probe 2 sequence, a probe 3 sequence and a probe 4 sequence, wherein fluorescent groups for labeling the 5 'end of the probe sequence are specifically FAM, VIC, ROX and Cy5, and quenching groups for labeling the 3' end are MGB;

the positive quality control product sequence comprises a 102bp target fragment covered by a MERS-CoV specific primer and a probe, a 109bp target fragment covered by an HCoV-NL63 specific primer and a probe, a 122bp target fragment covered by an HCoV-OC43 specific primer and a probe, and a 143bp target fragment covered by an HCoV-HKU1 primer and a probe.

2. The quadruple fluorescent quantitative detection kit for simultaneously detecting four human coronaviruses according to claim 1, wherein: the positive quality control material sequence is directly used as a positive quality control material after artificial synthesis, or is constructed on a plasmid vector after artificial synthesis, and clone plasmids are extracted through shaking to be used as the positive quality control material.

3. Use of the quadruple fluorescent quantitative detection kit according to claim 1 or 2, characterized in that: it is applied to the detection of four human coronaviruses of MERS-CoV, HCoV-NL63, HCoV-OC43 and HCoV-HKUI for non-diagnostic purposes.

4. A method for performing non-diagnostic detection using the quadruple fluorescent quantitative detection kit for simultaneous detection of four human coronaviruses according to claim 1 or 2, characterized by the steps of:

(1) Extraction of sample nucleic acid: taking a sample to be detected, and extracting nucleic acid from the sample to obtain sample nucleic acid;

(2) qRT-PCR detection: preparing a reaction system by adopting MERS-CoV/HCoV-NL63/HCoV-OC43/HCoV-HKUI specific primers and probe sequences, 2-d reaction solution, DNA polymerase and RT reverse transcriptase and the sample nucleic acid extracted in the step 1; taking a positive quality control product and a negative quality control product to respectively prepare a reaction system consistent with sample nucleic acid; qRT-PCR detection is carried out on the three reaction systems;

(3) And (3) analysis and judgment: adjusting a threshold value through the highest point of the fluorescent signal obtained by the negative quality control product in the step (2), and controlling the positive quality control product to have S-shaped amplification curves in all four detection channels; and then comparing the amplification curve obtained by the sample nucleic acid with a positive quality control product, and judging the result.

5. The method for non-diagnostic target detection using a quadruple fluorescent quantitative detection kit of claim 4, wherein the qRT-PCR amplification procedure of step (2) is as follows: reacting at 42 ℃ for 10min; reacting for 5min at 95 ℃; the reaction was then cycled 40 times, the cycling procedure being: reacting at 94 ℃ for 10-30 s, reacting at 58-60 ℃ for 30-60 s, and collecting fluorescence.

6. The method for non-diagnostic target detection using a quadruple fluorescent quantitative detection kit according to claim 4, wherein the analytical determination process in step (3) is specifically as follows:

a. adjusting a threshold value: the threshold setting principle is to take the highest point of the fluorescence signal just exceeding the negative quality control as a threshold line, or to adjust according to the noise condition of the instrument;

b. and (3) quality control: the negative quality control product has no amplification curve, the positive quality control product has S-shaped amplification curves in four detection channels of FAM, VIC, ROX and Cy5, and the experiment is established; otherwise, the experimental result is invalid;

c. and (3) result judgment:

c1, if the detection sample has S-type amplification and Ct or Cq value is less than or equal to 38, judging according to a fluorescent channel corresponding to the detection target;

c2, if S-type amplification exists, and the Ct value of 38< Ct or Cq value is less than 40, judging that the sample is an uncertain sample, and detecting after extracting nucleic acid again;

c3, if the result of the rechecking sample is consistent with that of c2, judging that the sample is weak positive;

c4, if no obvious S-type amplification curve exists, ct or Cq values are reported, and the amplification is nonspecific, and the result is judged as negative.