Double-channel real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection primer, probe, kit and method for Endumvirus

Abstract

The invention belongs to the technical field of virus detection, and particularly relates to a double-channel real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection primer, a probe, a detection kit, a detection method and application thereof. The detection sensitivity is high, and the lowest detection limit can reach 10 copies / mL; the detection time period is short, nucleic acid detection can be completed within 45 minutes, and the kit is suitable for clinical and bedside rapid detection and diagnosis; the detection specificity is high, and cross amplification with other 80 pathogens is avoided; the detection accuracy is 100%; virus quantitative analysis can be carried out, and the quantitative linear range is good; the detection repeatability is good, and the precision is high; uDG enzyme is added into a detection system, so that pollution can be prevented; a reference gene is also added into the detection system, so that the quality of the whole extraction and amplification process of the sample can be monitored according to the detection result of the reference gene, and the existence of the RNA of the Enduma virus in samples such as whole blood, serum, plasma, cerebrospinal fluid, urine, alveolar lavage fluid, throat swab and excrement can be detected.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus detection, and in particular relates to primers and probes, a kit and a method for real-time fluorescence quantitative PCR detection of Ndum virus. Background Art

[0002] Ndumu virus is a member of the Togaviridae family. Togaviridae ) Alphavirus ( Alphavirus ) Ndum alphavirus species ( Alphavirus ndumuvirus ) is a virus with a single-stranded positive-sense RNA genome consisting of a 5' untranslated region, an open reading frame, and a 3' untranslated region. It encodes non-structural proteins (such as RNA replicase) and structural proteins (capsid protein, envelope glycoproteins E1 and E2). The envelope glycoproteins can mediate the binding of the virus to host cell receptors and membrane fusion. Ndum virus was first isolated from the mosquito Mansoni in South Africa in 1959. Ndum virus can infect rodents and may be transmitted to mammals such as ungulates through mosquito vectors. It is a pathogen with potential zoonotic risk. Currently, there are no reports on dual-channel real-time fluorescence quantitative PCR detection kits and methods for Ndum virus. Summary of the Invention

[0003] Based on the full genome sequence information of the Ndum virus, the present invention provides a real-time fluorescence quantitative PCR detection primer and probe for Ndum virus, a detection kit, a detection method and its application, aiming to meet the needs of rapid, sensitive and accurate diagnosis.

[0004] The present invention is achieved in that: One of the purposes of the present invention is to provide a real-time fluorescence quantitative PCR detection primer and probe for Ndum virus, including Ndum virus specific primers as shown in SEQ ID NO.1-2 and a probe as shown in SEQ ID NO.3.

[0005] The second object of the present invention is to provide a real-time fluorescence quantitative PCR detection kit for Ndum virus, comprising the real-time fluorescence quantitative PCR detection primers and probes, as well as internal standard gene RNase P specific primers as shown in SEQ ID NO.4-5, and its probe as shown in SEQ ID NO.6.

[0006] Furthermore, it also includes: 5× reaction buffer: a solution containing Tris·Cl, KCl, (NH4)2SO4, DTT, MgCl2, dNTP, dUTP, random hexamer primers, Oligo dT, etc. Positive control: pseudovirus containing the target fragment of Ndum virus amplification; Internal standard solution: a pseudovirus solution containing the amplified target fragment of Rnase P; Negative control: ultrapure sterile water.

[0007] Furthermore, it also includes a positive standard for quantitative detection, and the positive standards are all pseudoviruses containing the amplified target fragment of Ndumu virus with concentration gradients.

[0008] Preferably, the positive standards include Standard 1 with a concentration of 1×10 copies / mL; Standard 2 with a concentration of 1×10 4 copies / mL; Standard 3 with a concentration of 1×10 5 copies / mL; Standard 4 with a concentration of 1×10 6 copies / mL.

[0009] The third object of the present invention is to provide a method for real-time fluorescence quantitative PCR detection of Ndumu virus, including: using sample RNA as a template, preparing an amplification reaction system to perform real-time fluorescence PCR amplification to obtain an amplification curve, analyzing the amplification curve, and making a judgment; the amplification reaction system includes the real-time fluorescence quantitative PCR detection primer pair and probe for Ndumu virus described above.

[0010] Furthermore, the amplification reaction system includes: sample RNA template, the specific detection primer for Ndumu virus as claimed in claim 1 and the probe, the specific detection primer for Rnase P as claimed in claim 2 and the probe, the enzyme mixture as claimed in claim 3, 5×PCR reaction buffer, and internal standard solution. The amplification program is: 50°C for 2 min; 95°C for 1 min; 95°C for 8 sec, 60°C for 40 sec (collect fluorescence signal), 45 cycles.

[0011] Furthermore, the principle for analyzing and judging the amplification curve is: When the Ct value in the FAM fluorescence channel ≤ 40 and there is or no amplification curve in the HEX fluorescence channel, it is judged that the sample is positive for Ndumu virus; When 40 < Ct ≤ 45 in the FAM fluorescence channel and there is or no amplification curve in the HEX fluorescence channel, repeat the experiment once. If the Ct value is still within this range or less than or equal to 40, it is judged that the sample is positive for Ndumu virus, otherwise it is judged that the sample is negative for Ndumu virus; When there is no amplification curve in the FAM fluorescence channel and the Ct value in the HEX channel ≤ 45, it is judged that the sample is negative for Ndumu virus; When there is no amplification curve in the FAM fluorescence channel and there is also no amplification curve in the HEX channel, it is judged that the result of this experiment is abnormal, and it is necessary to re-collect the sample and / or re-extract the RNA of the specimen for detection and amplification.

[0012] The fourth object of the present invention is to provide the use of the primers and probes for real-time fluorescence quantitative PCR detection of Ndum virus in the preparation of a kit for detecting Ndum virus.

[0013] In summary, the advantages and positive effects of the present invention are: 1. The present invention provides primer pairs, probes, kits, and methods for real-time fluorescent PCR detection of Endum virus, which are capable of rapid, accurate, and sensitive detection of Endum virus with strong specificity and high sensitivity, up to 10 copies / mL. Simultaneously with qualitative and quantitative viral analysis, the method offers good linearity and repeatability, with high precision. The method has a short detection cycle, capable of completing detection within 30 minutes, making it suitable for rapid clinical and bedside diagnosis, significantly reducing diagnostic time.

[0014] 2. The present invention systematically screened multiple pre-designed Endum virus primer and probe combinations to obtain a set of Endum virus detection primers and probes with the highest sensitivity, the most regular amplification curve, and the highest amplification efficiency. Subsequently, the dosage and concentration of each component in the PCR reaction system were optimized to determine the optimal reaction system concentration. By continuously adjusting the PCR reaction conditions, a highly efficient and time-efficient reaction condition was obtained. After comprehensive consideration, this method achieved an Endum virus detection sensitivity of 10 copies / mL, requiring only 45 minutes to complete the detection, while also demonstrating excellent stability, repeatability, and precision. Maintaining high detection sensitivity while ensuring stability, repeatability, precision, and rapid detection is the primary challenge of this technology. This study employed a single-tube dual fluorescence channel to simultaneously detect the presence of Endum virus and the internal reference gene RNase P. This method can be used to detect Endum virus RNA in a variety of samples, including whole blood, serum, plasma, cerebrospinal fluid, urine, bronchoalveolar lavage fluid, throat swabs, and feces. In view of the possible presence of reverse transcription or PCR inhibitors in the sample, it is easy to cause the quantitative results of viral nucleic acid to be unprepared or even "false negative". To address this difficulty and technical problem, the present invention designs an internal reference gene, which can monitor the quality of the entire process of sample extraction and amplification, monitor whether the RNA is successfully extracted and whether the subsequent reverse transcription and PCR are carried out smoothly, and monitor whether manual operation errors occur. Fluorescent quantitative PCR products easily form aerosols, causing the next test result to be contaminated and generating a "false positive" report. To address this difficulty and technical problem, the present invention adds UDG enzyme and dUTP, which can degrade and eliminate previous PCR products by heating at 50°C before a new amplification reaction, thereby greatly eliminating the possibility of contamination. The present invention uses a high-efficiency reverse transcriptase that can complete reverse transcription at 50°C for 2 minutes. At the same time, it uses a fast Taq enzyme that can complete PCR within 30 minutes, thereby ensuring that the test can be completed within 45 minutes, which is 2 hours shorter than the general fluorescent quantitative PCR test, meeting the needs of rapid and accurate detection and diagnosis.

[0015] 3. The present invention provides primer pairs, probes, kits and detection methods for real-time fluorescent PCR detection of Ndum virus, which not only shortens the operation time and reduces contamination, but also reduces the cost of sample diagnosis, and has potential application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the amplification curve of the Ndum virus standard; Figure 2 is the standard curve of Ndum virus standard concentration; Figure 3 is the quantitative linear range analysis of the present invention; Figure 4This is the fluorescence PCR amplification curve of Ndum virus of different clinical simulation positive specimens; Figure 5 This is the fluorescence PCR amplification curve of the internal reference gene RNase P in different clinical simulation positive specimens. DETAILED DESCRIPTION

[0017] Example 1 Development of a Real-time Fluorescence Quantitative PCR Detection Kit 1. In this example, fluorescent probe PCR amplification technology was used to select the Endum virus capsid protein gene, envelope glycoprotein E1 gene, E2 gene, and RNA replicase gene sequences as detection target genes. Multiple primer and probe test sets were designed using MEGA11 software and synthesized by the company. PCR amplification tests were then performed on a nucleic acid amplification analyzer for these different primer and probe test sets. The sensitivity, amplification curve regularity, peak onset time, amplification efficiency, time required to reach the plateau phase, and specificity were analyzed and compared to screen out a set of primer pairs and probes for the viral E1 gene with the highest sensitivity, the most regular amplification curve, the earliest peak onset time, the highest amplification efficiency, and the best specificity. The sequence information is shown in SEQ ID NOs. 1-3 in Table 1, where SEQ ID NOs. 1 and 2 are primer pairs, and SEQ ID NO. 3 is a probe. The internal standard is a primer and probe designed for the human housekeeping gene RNase P. The sequence information is shown in SEQ ID NO. 4-6 in Table 1, where SEQ ID NO. 4 and SEQ ID NO. 5 are a primer pair, and SEQ ID NO. 6 is a probe.

[0018] After confirming the optimal primer and probe sequences, we systematically optimized the reaction system. Through experimental exploration, we determined the optimal reaction concentrations for the Ndum virus primers and probe, as well as the RNase P primer and probe. We also determined the optimal dosages of the various enzymes in the enzyme mixture and the optimal concentrations of each component in the reaction buffer.

[0019] In addition, we also conducted experimental exploration and optimization of the PCR reaction conditions and determined the PCR reaction conditions with short time consumption and high amplification efficiency.

[0020] Table 1

[0021] The kit also includes positive controls: Ndum virus standards 1 to 4; positive control: pseudovirus solution containing the Ndum virus amplification sequence; negative control: ultrapure sterile water; internal standard solution: pseudovirus solution containing the RNase P amplified target fragment; 5× PCR reaction buffer and enzyme mixture. The preparation method of the 5× PCR reaction buffer and enzyme mixture is as follows: Preparation of 5× PCR reaction buffer: Tris·Cl 20mM KCl 80mM (NH4)2SO480mM DTT 1.5mM MgCl2 12mM dNTP 10mM dUTP 2.5mM Random hexamer primers 0.5 μM Oligo dT 0.25μM After mixing, store in a refrigerator at -20℃.

[0022] Enzyme mixture preparation: Tris·Cl 20mM KCl 80mM DTT 1mM EDTA 0.1mM Nonidet P-40 0.5% Tween 20 0.5% Glycerin 50% RNase inhibitor 20U / μl UDG enzyme 2U / μl High-efficiency reverse transcriptase 200 U / μl Taq Fast enzyme 2.5 U / μl After mixing, store in a refrigerator at -20℃.

[0023] 2. Real-time fluorescence PCR detection method for Ndum virus (1) Extraction of viral nucleic acid An internal standard solution was added to the collected sample at a ratio of 1:1000, and then extracted using a commercial viral RNA extraction kit to obtain an RNA solution.

[0024] (2) Real-time fluorescence quantitative PCR amplification (25 μl system per aliquot) Take 5 μl of RNA as a template and add 20 μl of PCR reaction solution (the preparation system of fluorescent quantitative PCR reaction solution is shown in Table 2) to the eight-tube strip for fluorescent quantitative PCR amplification.

[0025] Table 2 Fluorescence quantitative PCR reaction solution preparation system

[0026] (3)The real-time fluorescence quantitative PCR reaction procedure is as follows: 50°C for 2 min; 95°C for 1 min; 95°C for 8 sec, 60°C for 40 sec (collect fluorescence signal), for 45 cycles.

[0027] (4)Obtain the real-time fluorescence quantitative PCR amplification results, analyze the amplification curve, and make a judgment. The judgment rules are as follows: When the Ct value in the FAM fluorescence channel is ≤ 40 and there is or is no amplification curve in the HEX fluorescence channel, the sample is judged to be positive for Endum virus; When 40 < Ct ≤ 45 in the FAM fluorescence channel and there is or is no amplification curve in the HEX fluorescence channel, repeat the experiment once. If the Ct value is still within this range or ≤ 40, the sample is judged to be positive for Endum virus; otherwise, the sample is judged to be negative for Endum virus. When there is no amplification curve in the FAM fluorescence channel and the Ct value in the HEX channel is ≤ 45, the sample is judged to be negative for Endum virus; When there is no amplification curve in the FAM fluorescence channel and there is also no amplification curve in the HEX channel, it is judged that the experimental result is abnormal, and it is necessary to re-collect the sample or re-extract the specimen RNA and re-amplify.

[0028] 3. Experimental results The amplification curve of the Endum virus standard is as Figure 1 shown. The standard is a pseudovirus containing the Endum virus amplification sequence. The concentration of standard 1 is 1×10 3 copies / mL; the concentration of standard 2 is 1×10 4 copies / mL; the concentration of standard 3 is 1×10 5 copies / mL; the concentration of standard 4 is 1×10 6 copies / mL; Figure 2 is the standard concentration curve of the Endum virus standard. The equation of the standard concentration curve is: y = -3.426x + 48.57, where x is the log value of the concentration and y is the Ct value. As can be seen from the Figure 2 left side of the standard curve, the amplification efficiency (efficiency) of each round of PCR is 1.959 on average, only differing from the theoretical amplification efficiency of 2 by 2.05%, indicating that the detection efficiency of the present invention is high. The error value (error) given on the left side of the standard curve is 0.0167, which is much smaller than the upper limit value of 0.2, indicating that the standard curve generated by this method fits well and the error is small.

[0029] Performance determination of the real-time fluorescence PCR kit for Endum virus in Example 2 1. Accuracy verification The gold standard for viral nucleic acid detection is genome sequencing. Due to the lack of positive samples, we added pseudovirus containing the target fragment of the Ndum virus amplification to clinical blood samples to obtain five simulated positive blood samples. Then, we took five negative blood samples and compared them with the method of this kit using sequencing to analyze the accuracy of this kit. The results are shown in Table 3 below. All five sequencing-positive samples were detected, and none of the five sequencing-negative samples were detected. The positive and negative coincidence rates were both 100%, indicating that the accuracy of this kit is 100% (10 / 10).

[0030] Table 3 Accuracy test results

[0031] 2. Specificity Verification The specificity of the kit was assessed by testing other pathogens. In this example, 80 positive specimens or nucleic acids from blood, respiratory, zoonotic, and other common pathogens, including viruses, bacteria, fungi, and parasites, were selected. The results are shown in Table 4. Testing revealed no amplification of these 80 pathogens, demonstrating that the real-time fluorescence quantitative PCR method for detecting Ndum virus provided by this invention has a specificity of 100%.

[0032] Table 4 Specificity analysis of the present invention

[0033] 3. Sensitivity detection Sensitivity, also known as the minimum detection limit, refers to the statistical probability of detecting the target nucleic acid in the same sample at the lowest dilution gradient after a positive sample has been subjected to a gradient dilution, with a probability of >95%. Samples used for sensitivity assessment should be tested no fewer than 20 times at each concentration level to be assessed, with at least 19 positive amplification signals present as acceptable. Here, we diluted a positive standard pseudovirus of the Ndum virus at specific copy number ratios, then divided each dilution into 20 samples. Testing was performed using the method of this invention. The minimum detection limit was defined as the number of positive copies detected at 19 or more. The results are shown in Table 5. The detection rate was verified to be 100% at a concentration of 10 copies / mL; below this concentration, the detection rate was less than 95%. This demonstrates that the sensitivity of the real-time fluorescence PCR for Ndum virus provided by the present invention is 10 copies / mL.

[0034] Table 5 Sensitivity analysis of the present invention

[0035] 4. Accuracy testing Accuracy refers to the consistency of the results of multiple tests on the same positive sample. A Ct value coefficient of variation (CV) of less than 5% is considered good accuracy. In this example, four positive pseudovirus samples of the same concentration were tested three times in each concentration gradient. The results are shown in Table 6 below. 8 Within the copy / mL range, the intra-batch CV of this invention is <5%, and the inter-batch CV is <5%, with good precision.

[0036] Table 6 Accuracy analysis of the present invention

[0037] 5. Linear range analysis This example analyzes the 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 and 1×10 8 The linear range of the accuracy of the quantitative results of this invention is within the range of copies / ml, and the results are as follows Figure 3 As shown, the real-time fluorescence quantitative PCR results of Ndum virus provided by this invention are as follows: 2 ~1×10 7 The linear range is good within the copy / ml range, indicating that the linear range of the present invention is good.

[0038] Example 3 Clinical Testing Since there were no real clinical positive samples, we added pseudovirus containing the target fragment of Ndum virus amplification into human whole blood, serum, plasma, cerebrospinal fluid, urine, bronchoalveolar lavage fluid, throat swab, sputum and stool specimens to simulate positive clinical samples. These samples were tested using the above method. The results are as follows: Figure 4 As shown in Figure 2, these samples all have amplification curves, and the virus positive control and virus negative control are normal. At the same time, the internal reference gene RNase P amplification curves of these samples are normal, as shown in Figure 2. Figure 5 The results show that the extraction and amplification processes in this experiment were normal, and the positive and negative results were accurate. This proves that this method is applicable to the detection of whole blood, serum, plasma, cerebrospinal fluid, urine, bronchoalveolar lavage fluid, throat swabs, sputum, and stool specimens from patients suspected of Ndum virus infection.

[0039] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0040] Example 4 Comparison of performance parameters of the kit of the present invention and other PCR detection kits We found two fluorescent probe PCR detection kits for Ndum virus available online, namely Fluorescent Probe PCR Detection Kit A (Cat. No.: HBDY1566400) and Fluorescent Probe PCR Detection Kit B (Cat. No.: BES23390GDK). After purchasing, we conducted a comprehensive comparison between these two fluorescent PCR kits and the kit of the present invention. The comparison results are shown in Table 7 below. Among them, in terms of sensitivity, the sensitivity of the test kit of the present invention is 10 copies / ml, and the sensitivity of fluorescent probe PCR test kits A and B is 1000 copies / mL; in terms of detection time, the test kit of the present invention takes 45 minutes, and the other two fluorescent probe PCR test kits take about 2.5 hours; the test kit of the present invention adds UNG enzyme, which has anti-pollution performance, and the other two fluorescent probe PCR test kits do not add anti-pollution UNG enzyme; the test kit of the present invention has internal positive quality control measures, which can monitor the entire experimental process from RNA extraction to constant temperature amplification to prevent false negatives, while the other two fluorescent probe PCR test kits do not have internal positive quality control; the test kit of the present invention can detect whole blood, serum, plasma, urine, cerebrospinal fluid, alveolar lavage fluid, throat swabs, sputum and stool, while the other two fluorescent probe PCR test kits can only detect serum and plasma. It can be seen that the sensitivity of the test kit of the present invention is significantly higher than that of the two fluorescent probe PCR test kits, the detection time is much less than that of the two fluorescent probe PCR test kits, the types of test specimens are more than those of the two fluorescent probe PCR test kits, and it has the advantages of anti-pollution and anti-false negative that the two fluorescent probe PCR detection kits do not have.

[0041] Table 7 Performance comparison of the present invention and other PCR detection kits

[0042] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0043] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0044] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A real-time fluorescence quantitative PCR detection primer and probe for Ndum virus, characterized in that: It includes primers specific to Endom virus as shown in SEQ ID NO.1-2 and its probe as shown in SEQ ID NO.

3.

2. A real-time fluorescence quantitative PCR detection kit for Endom virus, comprising the real-time fluorescence quantitative PCR detection primers and their probes as described in claim 1; and primers specific to the internal standard gene Rnase P as shown in SEQ ID NO.4-5 and its probe as shown in SEQ ID NO 6.

3. A real-time fluorescent quantitative PCR detection kit for Ndum virus according to claim 2, characterized in that, It further includes: Enzyme mixture: A mixed solution containing highly efficient reverse transcriptase, fast Taq enzyme, UDG enzyme, RNA enzyme inhibitor, etc. 5× reaction buffer: A solution containing Tris·Cl, KCl, (NH4)2SO4, DTT, MgCl2, dNTP, dUTP, random hexamer primers, Oligo dT, etc. Positive control: Pseudovirus containing the amplified target fragment of Endom virus. Internal standard solution: Pseudovirus solution containing the amplified target fragment of Rnase P. Negative control: Ultra-pure sterile water.

4. A real-time fluorescent quantitative PCR detection kit for Ndum virus according to claim 2, characterized in that It further includes a positive standard for quantitative detection, and the positive standards are all pseudoviruses containing Endom virus amplified target fragments with concentration gradients.

5. A real-time fluorescent quantitative PCR detection kit for Ndum virus according to claim 4, characterized in that: The positive standard includes standard 1, with a concentration of 1×10 3 copies / mL; Standard 2, concentration is 1×10 4 copies / mL; Standard 3, concentration is 1×10 5 copies / mL; standard 4, concentration is 1×10 6 copies / mL.

6. A real-time fluorescence quantitative PCR detection method for Ndum virus, characterized in that: The method includes: Using sample RNA as a template, preparing an amplification reaction system for real-time fluorescence PCR amplification to obtain an amplification curve, analyzing the amplification curve, and making a judgment; the amplification reaction system includes the real-time fluorescence quantitative PCR detection primer pair and probe for Endom virus as described in claim 1.

7. The method for detecting Ndum virus by real-time fluorescent quantitative PCR according to claim 6, wherein: The amplification reaction system includes: sample RNA template, the Endom virus-specific detection primers and probe as described in claim 1, the Rnase P-specific detection primers and probe as described in claim 2, the enzyme mixture as described in claim 3, 5×PCR reaction buffer, and internal standard solution.

8. The method for detecting Ndum virus by real-time fluorescent quantitative PCR according to claim 6, wherein: The amplification program is: 50°C for 2 min; 95°C for 1 min; 95°C for 8 sec, 60°C for 40 sec (collecting fluorescence signals), 45 cycles.

9. The method for detecting Ndum virus by real-time fluorescence quantitative PCR according to claim 6, wherein: The principle for analyzing and judging the amplification curve is: When Ct in the FAM fluorescence channel ≤ 40 and there is or is no amplification curve in the HEX fluorescence channel, the sample is judged to be positive for Endom virus; When 40 < Ct ≤ 45 in the FAM fluorescence channel and there is or is no amplification curve in the HEX fluorescence channel, repeat the experiment once. If Ct is still within this range or less than or equal to 40, the sample is judged to be positive for Endom virus, otherwise the sample is judged to be negative for Endom virus; When there is no amplification curve in the FAM fluorescence channel and Ct in the HEX channel ≤ 45, the sample is judged to be negative for Endom virus; When there is no amplification curve in the FAM fluorescence channel and there is also no amplification curve in the HEX channel, it is judged that the result of this experiment is abnormal, and it is necessary to re-collect the sample and / or re-extract the specimen RNA for detection and amplification.

10. Use of the real-time fluorescence quantitative PCR detection primers for Endom virus and their probes as described in claim 1 in the preparation of a kit for detecting Endom virus.

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