CN111074006B

CN111074006B - Salivirus virus double-channel real-time fluorescence PCR detection primer pair, probe, kit, method and application

Info

Publication number: CN111074006B
Application number: CN202010059987.XA
Authority: CN
Inventors: 刘映乐; 刘为勇
Original assignee: Wuhan University WHU
Current assignee: Zhejiang Anweiluo Diagnostic Technology Co ltd
Priority date: 2020-01-19
Filing date: 2020-01-19
Publication date: 2021-08-03
Anticipated expiration: 2040-01-19
Also published as: CN111074006A

Abstract

The invention discloses a double-channel real-time fluorescence PCR detection primer pair, a probe, a kit, a method and application of Salivirus virus. The invention adopts a pair of amplification primer sequences specific to the Salivirus virus, and quantitatively detects the RNA of the Salivirus virus by a fluorescent probe. Meanwhile, human endogenous RNase P gene is used as an internal reference gene, and an RNase P specific primer and a probe are used for detecting the RNA of the internal reference gene in the sample. The invention adopts a single-tube double-fluorescence channel to simultaneously detect the existence of Salivirus virus and reference gene RNase P, and can detect the existence of Salivirus virus RNA in a specimen. The virus can be qualitatively analyzed and quantitatively analyzed at the same time, and the quantitative linear range is good; the specificity is high, the accuracy is high, the repeatability is good, the precision is high, and the quality monitoring can be carried out on the whole process of extracting and amplifying the sample through the detection result of the internal reference gene.

Description

Salivirus virus double-channel real-time fluorescence PCR detection primer pair, probe, kit, method and application

Technical Field

The invention relates to the technical field of virus detection, in particular to a double-channel real-time fluorescence PCR detection primer pair, a probe, a kit, a method and application of Salivirus virus.

Background

The Salivirus virus is the only member belonging to the genus Salivirus belonging to the family picornaviridae, and is a novel human-induced disease RNA virus discovered in 2009. The Salivirus virus is currently found to cause gastroenteritis in humans. There are currently two types of Salivirus, Salivirus 1 and Salivirus 2. There are currently no reports on detection kits and methods for nucleic acids from Salivirus.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a double-channel real-time fluorescent PCR detection primer pair, a probe, a kit, a method and application of Salivirus virus, which can detect Salivirus 1 type and Salivirus2 type viruses quickly, accurately and sensitively at the same time and have strong specificity and high sensitivity.

In order to achieve the purpose, the scheme of the invention is as follows:

one of the purposes of the invention is to provide a double-channel real-time fluorescence PCR detection primer pair and a probe of Salivirus virus, which comprise a Salivirus virus specific primer pair and a probe, and an internal reference gene RNase P specific primer pair and a probe;

the Salivirus virus specific primer pair comprises: the Salivirus virus specific upstream primer sequence is shown as SEQ ID NO.1, the Salivirus virus specific downstream primer sequence is shown as SEQ ID NO.2, the Salivirus virus specific probe sequence is shown as SEQ ID NO.3, the 5 'end of the Salivirus virus specific probe sequence is connected with a fluorescence reporter group FAM, and the 3' end of the Salivirus virus specific probe sequence is connected with a fluorescence quencher group BQ 1.

The primer pair specific to the internal reference gene RNase P comprises: the sequence of the RNase P gene specific upstream primer is shown as SEQ ID NO.4, and the sequence of the RNase P gene specific downstream primer is shown as SEQ ID NO. 5; the sequence of the RNase P gene specific probe is shown as SEQ ID NO.6, the 5 'end of the sequence of the RNase P gene specific probe is connected with a fluorescent reporter group HEX, and the 3' end of the sequence of the RNase P gene specific probe is connected with a fluorescent quenching group BQ 1.

The invention also aims to provide a double-channel real-time fluorescent PCR detection kit for the Salivirus virus, which comprises a double-channel real-time fluorescent PCR detection primer pair and a probe.

Preferably, the kit further comprises: positive control: a Salivirus virus standard; internal standard solution: a virus-like particle solution containing an RNase P sequence; negative control: RNase Free H₂O。

The invention also aims to provide the double-channel real-time fluorescent PCR primer pair, the probe and the application of the kit in simultaneously detecting Salivirus 1 type viruses and Salivirus2 type viruses. The kit provided by the invention can simultaneously detect Salivirus 1 and Salivirus2 viruses, but does not classify the Salivirus 1 and Salivirus2 viruses; a positive detection of a Salivirus virus in the sample indicates that the sample contains at least one of a Salivirus 1 virus or a Salivirus2 virus.

The fourth purpose of the invention is to provide a double-channel real-time fluorescent PCR detection method of the Salivirus virus, which utilizes the double-channel real-time fluorescent PCR detection primer pair and the probe of the Salivirus virus to carry out double-channel real-time fluorescent PCR amplification.

Specifically, the method comprises the following steps:

step 1, extracting RNA from a sample as a template;

step 2, preparing an amplification reaction system to perform double-channel real-time fluorescent PCR amplification to obtain an amplification curve, wherein the amplification reaction system comprises the template, the double-channel real-time fluorescent PCR detection primer pair and the probe of the Salivirus virus of claim 1;

and 3, analyzing the amplification curve and judging.

Preferably, the step 1 can be performed by a conventional RNA extraction method or a commercially available RNA extraction kit.

Preferably, the amplification reaction system in step 2 specifically comprises: the Salivirus virus of claim 1, a pair of primers and a probe for dual channel real-time fluorescent PCR detection, 5 XPCR Buffer and Enzyme Mix; the amplification procedure was: 30min at 40 ℃; 5min at 95 ℃; 94 ℃ 15sec, 58 ℃ 30sec, 45 cycles.

Preferably, the specific judgment rule in step 3 is:

when Ct in the FAM fluorescent channel is less than or equal to 40 and the HEX fluorescent channel has or does not have an amplification curve, judging that the sample is positive to Salivirus virus;

when Ct in the FAM fluorescent channel is more than 40 and less than or equal to 45, and when an HEX fluorescent channel has or does not have an amplification curve, repeating the experiment once, if the Ct of the FAM fluorescent channel is still within the range or less than or equal to 40, judging that the sample is Salivirus virus positive, otherwise, judging that the sample is Salivirus virus negative;

when the FAM fluorescence channel has no amplification curve and the Ct in the HEX fluorescence channel is less than or equal to 45, judging that the sample is negative to the Salivirus virus;

when the FAM fluorescence channel has no amplification curve and the HEX channel also has no amplification curve, the experiment is judged to be abnormal, and the RNA of the sample needs to be extracted again and amplified again.

The invention has the following advantages and beneficial effects:

1. the invention provides a double-channel real-time fluorescent PCR detection primer pair, a probe, a kit and a detection method for Salivirus viruses, which can detect Salivirus 1 type viruses and Salivirus2 type viruses quickly, accurately and sensitively at the same time, and have strong specificity and high sensitivity (up to 45 copies). Not only shortens the operation time and reduces the pollution, but also reduces the cost of sample diagnosis, and has potential application value.

2. The invention provides a double-channel real-time fluorescent PCR detection primer pair, a probe, a kit and a detection method of Salivirus virus, on the basis of the analysis of Salivirus virus nucleic acid sequences, a pair of specific primers and a specific fluorescent probe are designed by selecting the same or similar sections of Salivirus 1 type and Salivirus2 type conserved gene sequences, and Salivirus 1 type and Salivirus2 type viruses can be simultaneously detected by using a real-time fluorescent quantitative PCR technology. For those skilled in the art, how to use a pair of primer pairs and a probe to simultaneously detect the Salivirus 1 type and the Salivirus2 type viruses, and make the specificity strong and the sensitivity high become the biggest difficulty of the application, the inventors obtain the primer pairs, the probe, the kit and the detection method of the application through a large number of innovative tests, and the difficulties and the technical problems are solved.

3. The invention provides a double-channel real-time fluorescence PCR detection primer pair, a probe, a kit and a detection method of Salivirus virus, and is also provided with an internal reference, when reverse transcription or PCR inhibition exists in cerebrospinal fluid, whole blood, plasma or nasopharyngeal swab, the quantitative result of virus nucleic acid is easily prepared or even false negative appears, aiming at the difficulty and the technical problem, the invention designs the internal reference gene, can carry out quality monitoring on the whole process of specimen extraction and amplification, and can monitor whether RNA is successfully extracted and whether subsequent reverse transcription and PCR are carried out successfully, and can monitor whether manual operation errors occur.

Drawings

FIG. 1 is a Salivirus virus standard amplification curve;

FIG. 2 is a Salivirus virus standard concentration standard curve;

FIG. 3 is a fluorescent quantitative PCR amplification curve for 4 samples of Salivirus virus positive specimens;

FIG. 4 is a 15-sample reference gene RNase P amplification curve;

FIG. 5 is a linear range analysis of the dual channel real-time fluorescent PCR detection kit for Salivirus virus provided by the present invention;

FIG. 6 is an amplification curve of 5 Salivirus virus positive specimens provided in example 3;

FIG. 7 is the amplification curve of the 18 specimen internal reference genes RNase P provided in example 3.

Detailed Description

Example 1 Salivirus virus double-channel real-time fluorescence PCR kit and detection method

1. Composition of the kit

The kit comprises a pair of specific primers (F and R) of the Salivirus virus and a specific Fluorescent Probe (FP); an internal standard gene RNase P pair of specific primers (F and R) and a specific fluorescent probe; positive control, negative control, internal standard solution, 5 × PCR Buffer and Enzyme Mix;

(ii) a pair of primers and fluorescent probe specific to Salivirus virus (shown in Table 1)

② a pair of specific primers and fluorescent probe of RNase P (as shown in Table 1)

TABLE 1

Primer name	Primer sequences
		Salivirus upstream primer	5'-cgcctcacccccaccgccccagcc-3' (shown as SEQ ID NO: 1)
Salivirus downstream primer	5'-gagatcaccaagaccctggcggcgg-3' (shown as SEQ ID NO: 2)
		Salivirus fluorescent probe	5'-FAM-caccctcttcctctccgcgggagctgccct-BQ1-3' (shown as SEQ ID)ID NO.5 shows
RnaseP upstream primer	5'-agatttggacctgcgagcg-3' (shown as SEQ ID NO: 4)
		RnaseP downstream primer	5'-gagcggctgtctccacaagt-3' (shown as SEQ ID NO: 3)
RnaseP fluorescent probe	5'-HEX-ttctgacctgaaggctctgcgcg-BQ1-3' (shown in SEQ ID NO: 6)

Note that: because both SEQ ID NO 5 and SEQ ID NO 6 have fluorescent groups, the sequence table software cannot identify and read, and are therefore only indicated in the description.

③ Positive contrast

A Salivirus virus standard;

negative control

RNase Free H₂O；

Fifthly, internal standard solution

RNase P sequence-containing virus-like particle solution

Preparing 5 times PCR Buffer:

mixing, and storing at-20 deg.C;

preparing Enzyme Mix:

mixing, and storing at-20 deg.C in refrigerator.

2. Double-channel real-time fluorescence PCR detection method for Salivirus virus

(1) Extraction of viral nucleic acids

Firstly, adding absolute ethyl alcohol into

buffer solutions

1 and 2, and respectively adding 25ml of absolute ethyl alcohol and 30ml of absolute ethyl alcohol; adding 30 mu g/ml carrier RNA into the rinsing solution;

② putting 30 mul protease into a 1.5ml centrifuge tube;

③ taking 200 mul of specimen (such as cerebrospinal fluid, nasopharyngeal swab, etc.) and adding into the tube, adding 5 mul of internal standard solution, and mixing well;

adding 200 mul of rinsing liquid (containing 30 mu g/ml carrier RNA) into each tube respectively, uniformly mixing and oscillating for 30s, and incubating for 10min at 70 ℃;

adding 250 μ l of anhydrous ethanol, mixing, oscillating for 30s, and cracking at room temperature for 5 min;

sixthly, adding the lysate into a centrifugal column, centrifuging at 8000rpm for 1min, and discarding the centrifugate in a collecting pipe; the filter column is still put back on the collecting pipe, all the residual mixed liquid in the third step is sucked into the filter column, and the centrifugate is discarded after centrifugation;

seventhly, adding 500 mu l of buffer solution 1 at 12000rpm into the filter column, centrifuging for 1min, and discarding the centrifugate in the collection tube;

eighthly, another clean collection tube of 2ml is taken, the filter column after centrifugation is moved to a new collection tube, 500 mul of buffer solution 2 is added into the filter column, 12000rpm is carried out, and centrifugation is carried out for 1 min. Repeating the step one;

ninthly, moving the filter column into a clean collecting pipe, centrifuging at 12000rpm for 3min, and then placing the filter column at 37 ℃ for 15min to dry the filter membrane;

r.A.the filter column was placed on a 1.5ml Eppendorf tube, and 50. mu.l of RNase-free H was added to the filter column₂O, standing for 2min at room temperature; centrifuging at 12000rpm for 2min, and collecting centrifugate as extracted nucleic acid;

nucleic acid was extracted from 15 suspected samples of herpes fluid of Salivirus infected patients by the above method.

(2) Real-time fluorescent quantitative PCR amplification (25. mu.l each system)

Mu.l of RNA was used as a template, and 20. mu.l of PCR reaction solution (the system for preparing the fluorescent quantitative PCR reaction solution is shown in Table 2) was added to the octaplex tube to perform the fluorescent quantitative PCR amplification.

TABLE 2

(3) The real-time fluorescent quantitative PCR reaction program is as follows:

15min at 40 ℃; 5min at 95 ℃; 94 ℃ for 15sec, 60 ℃ for 30sec, 45 cycles.

Collecting fluorescence signals from step (c) 60 ℃.

The invention uses LightCycle 480II fluorescence quantitative PCR instrument to detect.

(4) And obtaining a real-time fluorescent quantitative PCR amplification result, analyzing an amplification curve, and judging according to a judgment principle. The specific judgment rule is as follows:

when the Ct in the FAM fluorescence channel is less than or equal to 40, judging that the sample is positive to the Salivirus virus;

when the Ct of 40< Ct is less than or equal to 45 in the FAM fluorescence channel, repeating the experiment once, if the Ct is still within the range or less than 40, judging that the sample is positive to the Salivirus virus, otherwise, judging that the sample is negative to the Salivirus virus;

when the FAM fluorescence channel has no amplification curve and Ct in the HEX channel is less than or equal to 45, judging that the sample is negative to Salivirus virus;

3. Results of the experiment

FIG. 1 is an amplification curve of a Salivirus virus standard, and FIG. 2 is a concentration standard curve of the Salivirus virus standard, wherein the standard concentration curve equation is as follows: y-3.37 x +48.41, where x is the log of the concentration and y is the Ct value.

The detection result is that 15 samples of herpes fluid of a suspected patient infected by the Salivirus virus are detected, and 4 positive samples of the Salivirus virus are detected;

FIG. 3 shows the fluorescent quantitative PCR amplification curves of the 4 Salivirus positive specimens, the C according to the 4 positive results_tThe values were combined with the standard curve equation and automatically analyzed by Roche LightCycler480 analysis software to obtain the virus concentrations of the 4 Salivirus virus positive specimens, and the specific results are shown in Table 3.

TABLE 3-4 virus concentrations in Salivirus positive specimens

Sample numbering	Ct value	Salivirus virus concentration (copy/mL)
			Sample 1	25.67	4.46×10⁵
Sample 2	29.18	4.02×10⁴
			Sample 3	31.36	6.99×10³
Sample No.4	22.37	4.12×10⁷

FIG. 4 is a 15 specimen internal reference gene RNase P amplification curve; as can be seen from FIG. 4, the amplification curve of the internal reference gene RNase P of the 15 specimens is normal, which indicates that the extraction and amplification processes of the experiment are normal, and the positive and negative results are accurate.

Example 2 Performance determination of the Dual channel real-time fluorescent PCR kit for Salivirus Virus

1. Accuracy verification

The gold standard for virus nucleic acid detection is genome sequencing, and the detection result of the kit is compared with the virus genome sequencing to analyze the accuracy of the detection result. 20 samples determined as salivirus by genome sequencing are selected, wherein 10 samples of

salivirus type

1 and 10 samples of salivrius type 2 are selected, and the results after detection by using the kit provided by the invention are shown in the following table 4. As can be seen from the results, 20 positive specimens were all detected, indicating that the accuracy of the dual-channel real-time fluorescent PCR of the Salivirus virus provided by the invention is 100%.

TABLE 4 accuracy analysis of the invention

Viral type	Number of examples	Positive in sequencing	The method is positive	Rate of accuracy
					Salivirus type
1	10	10	10	100％
					Type
2 salivrius	10	10	10	100％

2. Specificity verification

The specificity of the kit was assessed by detecting other pathogens and we selected 20 common pathogen positive specimens with the results shown in table 5 below. Through detection, the invention has no amplification on 20 common pathogen positive specimens, and shows that the specificity of the double-channel real-time fluorescent PCR of the Salivirus virus provided by the invention is 100%.

TABLE 5 specificity analysis of the invention

Positive pathogens	Whether or not there is amplification		Whether or not there is amplification
				Adenoviral vectors	Is free of	Human T cell virus	Is free of
Cytomegalovirus	Is free of	Human herpesvirus type 6	Is free of
				Influenza A virus	Is free of	Kaposi sarcoma virus	Is free of
Influenza B virus	Is free of	Staphylococcus aureus	Is free of
				Hepatitis B virus	Is free of	Escherichia coli	Is free of
Hepatitis C virus	Is free of	Acinetobacter baumannii	Is free of
				EB virus	Is free of	Pseudomonas aeruginosa	Is free of
Human herpes simplex virus type 1	Is free of	Candida albicans	Is free of
				Human herpes simplex virus type 2	Is free of	Pneumocystis yeri	Is free of
HIV-1	Is free of	Aspergillus	Is free of

3. Sensitivity detection

The sensitivity, i.e., the lowest detection limit, is the probability statistically > 95% that a target nucleic acid will be detected in the same sample at the lowest dilution gradient after the positive sample is diluted with the gradient. The number of detections of the sample for sensitivity assessment at each concentration level to be assessed should be not less than 20, and at least 19 positive amplification signals are qualified. After the positive standard plasmid of the Salivirus virus is diluted according to a certain copy number multiple ratio, each dilution is averagely divided into 20 samples, the detection is carried out by the method, the copy number of the positive plasmid which appears for 19 times or more is the lowest detection limit, and the result is shown in Table 6. The detection rate is 100% when the concentration is 20 copies/ml, and the detection rate is less than 95% when the concentration is lower than the concentration. Therefore, the sensitivity of the double-channel real-time fluorescent PCR of the Salivirus virus provided by the invention is 20 copies/mL.

TABLE 6 sensitivity analysis of the invention

4. Accuracy detection

The accuracy refers to the consistency of the judgment of results of multiple detections of the same positive sample, and the accuracy is determined to be good when the Coefficient of Variation (CV) is less than 5. We divided in each concentration gradient30 positive specimens were tested 3 times, and the results are shown in Table 7 below. Verified to be 20-1 × 10⁷In the context of copying, the intra-batch CV of this invention<5%, good precision, 20-1 × 10⁶In the context of copying, the intra-batch CV of this invention<5%, good precision.

TABLE 7 accuracy analysis of the invention

5. Linear range analysis

We analyzed the results at 10, 20, 50, 1 × 10²、1×10³、1×10⁴、1×10⁵、1×10⁶、1×10⁷And 1X 10⁸The results of the linear range of the present invention in copy/ml range are shown in FIG. 5, and the linear range of the real-time fluorescent quantitative PCR of the Salivirus virus provided by the present invention is 20 to 1X 10⁷Copy/ml shows a good linear range.

Example 3 clinical assays

18 samples of throat swabs of another suspected patient infected with the salivirus virus were tested by the above method, wherein the detection result of the salivirus virus was positive for 5 cases, the fluorescence quantitative PCR amplification curve of the virus is shown in FIG. 6, and C is obtained according to the positive results of the 5 cases_tThe values were combined with the amplification curve and automatically analyzed by Roche LightCycler480 analysis software to obtain the virus concentrations of the 5 specimen positive for salinivirus virus, and the specific results are shown in Table 8. Meanwhile, the amplification curve of the internal reference gene RNase P of the 18 throat swab specimens is normal, as shown in FIG. 7, which shows that the extraction and amplification process of the experiment is normal, and the positive and negative results are accurate.

TABLE 8-5 virus concentrations in specimen positive for salivirus

The invention is not to be considered as limited to the particular embodiments shown, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> Wuhan university

Double-channel real-time fluorescent PCR detection primer pair, probe, kit, method and application of Salivirus virus

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 24

<212> DNA

<213> Salivirus upstream primer (Salivirus)

<400> 1

cgcctcaccc ccaccgcccc agcc 24

<210> 2

<211> 25

<212> DNA

<213> Salivirus downstream primer (Salivirus)

<400> 2

gagatcacca agaccctggc ggcgg 25

<210> 3

<211> 20

<212> DNA

<213> RNase P downstream primer (RNase P)

<400> 3

gagcggctgt ctccacaagt 20

<210> 4

<211> 19

<212> DNA

<213> RNase P upstream primer (RNase P)

<400> 4

agatttggac ctgcgagcg 19

Claims

1. A double-channel real-time fluorescence PCR detection primer pair and a probe of Salivirus virus are characterized in that: the primer comprises the following two groups of primer pairs and probes:

(1) salivirus virus specific primer pair and probe:

the sequence of the Salivirus virus specific upstream primer is shown as SEQ ID NO. 1;

the sequence of the Salivirus virus specific downstream primer is shown as SEQ ID NO. 2;

the sequence of the Salivirus virus specific probe is shown as SEQ ID NO. 5; the 5 'end of the sequence of the Salivirus virus specific probe is connected with a fluorescence reporter group FAM, and the 3' end of the sequence is connected with a fluorescence quenching group BQ 1; and the combination of (a) and (b),

(2) an internal reference gene RNase P specific primer pair and a probe:

the sequence of the RNase P gene specific upstream primer is shown as SEQ ID NO. 4;

the sequence of the RNase P gene specific downstream primer is shown as SEQ ID NO. 3;

the sequence of the RNase P gene specific probe is shown as SEQ ID NO. 6; the 5 'end of the sequence of the RNase P gene specific probe is connected with a fluorescence reporter group HEX, and the 3' end of the sequence is connected with a fluorescence quenching group BQ 1.

2. A double-channel real-time fluorescence PCR detection kit for Salivirus virus is characterized in that: the kit comprises the Salivirus virus double-channel real-time fluorescent quantitative PCR detection primer pair and the probe of claim 1.

3. The Salivirus virus dual-channel real-time fluorescent PCR detection kit of claim 2, wherein: the kit further comprises:

positive control: a Salivirus virus standard;

internal standard solution: a virus-like particle solution containing an RNase P sequence;

negative control: RNase Free H₂O。

4. Use of the Salivirus virus double channel real-time fluorescent PCR detection primer pair and the probe of claim 1 in preparation of a reagent for simultaneously detecting Salivirus type 1 and Salivirus type 2 viruses.

5. Use of the Salivirus virus double channel real-time fluorescent PCR assay kit of claim 2 or 3 for preparing a reagent for simultaneously detecting Salivirus type 1 and Salivirus type 2 viruses.

6. The use of the Salivirus virus dual-channel real-time fluorescent PCR detection primer set and the probe of claim 4 for preparing a reagent for simultaneously detecting Salivirus type 1 and Salivirus type 2 viruses, wherein the reagent comprises: the Salivirus virus double-channel real-time fluorescent PCR detection method is to carry out double-channel real-time fluorescent PCR amplification by using the Salivirus virus double-channel real-time fluorescent PCR detection primer pair and the probe of claim 1, and comprises the following steps:

(1) extracting RNA from a sample as a template;

(2) preparing an amplification reaction system to perform double-channel real-time fluorescent PCR amplification to obtain an amplification curve, wherein the amplification reaction system comprises the template in the step (1), the double-channel real-time fluorescent PCR detection primer pair and the probe of the Salivirus virus of claim 1;

(3) and analyzing the amplification curve and making a judgment.

7. The use of the Salivirus virus dual-channel real-time fluorescent PCR detection primer set and the probe of claim 6 for preparing a reagent for simultaneously detecting Salivirus type 1 and Salivirus type 2 viruses, wherein the reagent comprises: the amplification reaction system in the step (2) specifically comprises: the Salivirus virus of claim 1, a pair of primers and a probe for dual channel real-time fluorescent PCR detection, 5 XPCR Buffer and Enzyme Mix; the amplification procedure was: 15min at 40 ℃; 5min at 95 ℃; 94 ℃ for 15sec, 60 ℃ for 30sec, 45 cycles.

8. The use of the primer set and probe for the dual channel real-time fluorescent PCR detection of Salivirus virus of claim 6 or 7 for the preparation of a reagent for the simultaneous detection of Salivirus type 1 and Salivirus type 2 viruses, wherein: the specific judgment rule in the step (3) is as follows: