Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method and kit for nematode filamentosa

Abstract

The invention discloses a real-time fluorescent quantitative PCR (polymerase chain reaction) detection method and a kit for nematode filamentosa, relates to the technical field of bioengineering, and is technically characterized in that the kit comprises a primer and a TaqMan probe which are used for performing real-time fluorescent quantitative PCR detection on the nematode filamentosa by a probe method, the gene is designed according to a gene conserved region of an internal transcriptional spacer 2 (ITS2) of the nematode filamentosa; according to the invention, specific primers and probes are designed on the basis of a sequence conserved region of the nematode filamentosa, and a probe-method real-time fluorescent quantitative PCR detection method which is simple to operate, high in sensitivity, strong in specificity, good in stability and wide in dynamic range is established by constructing a plasmid standard substance and drawing a standard curve; the method can be used for qualitatively, quantitatively, quickly and accurately detecting the nematode filamentosa in different clinical samples, and has important significance for guaranteeing animal quality and human health. The method has great practical significance in the fields of control of the quality of animal-derived biological products for human and inspection and quarantine of import and export animals.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a real-time fluorescence quantitative PCR detection method and a kit for Dictyocauda filariata. Background Art

[0002] Lungworm disease (hoose) is one of the major diseases that threatens global human health, animal quality and public health safety. Dictyocaulus filaria (Rudolphi, 1809) is one of the important pathogens that causes lungworm disease. The parasite lives in the trachea and bronchioles. The host's response to infection leads to bronchitis, eosinophilic pneumonia and respiratory bronchiolar obstruction, which can cause dyspnea, cough, fever, loss of appetite, poor growth and development, and even death.

[0003] Accurate diagnosis of lungworm disease through reliable detection technology is a prerequisite for formulating targeted prevention and control measures, and is of great significance for the effective control of lungworm disease. At present, the diagnosis of lungworm disease mostly relies on microscopy, which is often used as the "gold standard" for diagnosing lungworm disease (Yaghoub Firouzivand. A field outbreak of Dictyocaulus filaria insheep in the northwest of Iran. J Altern Vet Med, 2023, 6(18): 1097-2002.). However, the microscopic examination method is prone to miss cases with unclear morphological characteristics of parasites, a small number of parasites in the early stage of infection, and subclinical symptoms. The laboratory diagnosis of lungworm disease mainly relies on serological and molecular biological detection methods. Serological tests can still detect antibodies specific to Dictyocaulus filariae weeks or months after successful treatment of lungworm disease, and there is also a risk of false positive results due to cross-reactions with antibodies caused by other pathogens (Parfitt JW, Sinclair IJ. Crossresistance to Dictyocaulus viviparus produced by Dictyocaulus filaria infections in calves. Res Vet Sci, 1967, 8 (1): 6-13.). Therefore, reliable detection technology is needed to diagnose lungworm disease in a timely and accurate manner to prevent its widespread spread. Due to the challenges associated with serological testing, molecular testing has become the preferred diagnostic test method for detecting certain pathogenic infections. For parasitological diagnosis, molecular testing has a high specificity because it can detect parasite-specific nucleic acids, which better solves the "bottleneck" problem of traditional microscopic parasite examination methods that are prone to miss early infection cases. Summary of the invention

[0004] The purpose of the present invention is to solve the above problems and provide a real-time fluorescence quantitative PCR detection method and a kit for Dictyocauda filariata.

[0005] In order to achieve the above object, the technical scheme of the present invention is as follows: a real-time fluorescent quantitative PCR detection kit for Dictyocauda filamentosa comprises primers and TaqMan probes, and the nucleotide sequences of the primers and TaqMan probes are as follows:

[0006] Forward primer: 5′-AACCTGAGCCTGCAGAAACATTAT-3′;

[0007] Reverse primer: 5′-CCACAGGTTTTGCAAACGCTATA-3′;

[0008] TaqMan probe: 5′-AAAGTTGCGAAGCAG-3′.

[0009] Furthermore, the 5' end of the TaqMan probe is labeled with a FAM fluorescent group, and the 3' end is labeled with a MGB-NFQ quenching group.

[0010] According to the above-mentioned probe method real-time fluorescence quantitative PCR detection kit, the present invention also provides a real-time fluorescence quantitative PCR detection method for Dictyocaudus filarius, comprising the following steps:

[0011] S1. Obtain a sample of Dictyochondria filariata to be tested, and extract DNA from the sample to be tested;

[0012] S2. Prepare the probe method real-time fluorescence quantitative PCR reaction system: take 10 μL TaqMan Mix, 8 μL nuclease-free pure water, 1 μL primer and TaqMan probe Mix, and 1 μL template DNA;

[0013] S3, performing probe method real-time fluorescence quantitative PCR reaction: adding the prepared 20 μL reaction system into the probe method real-time fluorescence quantitative PCR reaction tube for reaction, and collecting the fluorescence signal in real time;

[0014] S4. Establish a standard curve: Take 1 μL of the plasmid standard after 10-fold serial dilution as a template, perform the same probe method real-time fluorescence quantitative PCR reaction as the sample, collect the fluorescence signal in real time, and draw a standard curve;

[0015] S5. Data analysis: Substitute the Ct value measured by the test sample into the standard curve to obtain the concentration of the sample to be tested; perform qualitative and quantitative detection on the sample to be tested based on the measured Ct value.

[0016] Preferably, in step S1, the Dictyocaudus filarius sample is taken from one or more of whole blood, rectal swab, serum, brain, heart, lung, liver, gallbladder, kidney, intestine, and bladder.

[0017] Preferably, in step S2, the forward primer in the primer and TaqMan probe Mix is ​​used at a concentration of 440 nM, the reverse primer is used at a concentration of 440 nM, and the TaqMan probe is used at a concentration of 120 nM.

[0018] Preferably, in step S3, the amplification program of the probe method real-time fluorescence quantitative PCR is: 50°C for 2 min, 1 cycle; 95°C for 10 min, 1 cycle; 95°C for 15 s, 60°C for 1 min, 40 cycles.

[0019] Preferably, in step S3, establishing a standard curve specifically comprises the following steps:

[0020] S301. Prepare a plasmid standard containing the target fragment and perform a 10-fold serial dilution to 11 concentrations, starting at 1.5×10 10 Copies / μL to 1.5×10 0 copies / μL;

[0021] S302, take 1 μL of the plasmid standard after 10-fold serial dilution as a template, and perform the same real-time fluorescence quantitative PCR reaction as the sample;

[0022] S303, collecting the fluorescence signals corresponding to each dilution concentration in real time, and drawing a standard curve.

[0023] Preferably, the qualitative result is interpreted as follows: when the quality of probe-based real-time fluorescence quantitative PCR detection is under control, if the Ct value of the sample being tested is ≤35 and a specific S-type fluorescence amplification curve appears, the corresponding object being detected is determined to be positive; if there is no Ct value and no specific fluorescence amplification curve, the corresponding object being detected is determined to be negative; if 35<Ct value<40 and a specific fluorescence amplification curve appears, it is determined to be an uncertain sample, and the nucleic acid needs to be re-extracted for re-testing. If there is still an S-type fluorescence amplification curve and the Ct value is <40 after the re-testing, the corresponding object being detected is determined to be positive, otherwise it is determined to be negative.

[0024] Preferably, the quantitative result interpretation method is as follows: construct the target fragment of Dictyocaurus filamentosa onto the pMD18-T vector, transform the recombinant plasmid into JM109 Escherichia coli competent cells, extract plasmid DNA after amplification and culture, use it as a plasmid standard after restriction digestion, electrophoresis and sequencing to identify the correctness, measure the concentration of plasmid DNA, convert it into a specific plasmid copy number through a copy number conversion formula, perform 10-fold serial dilution, use the plasmid standard of each dilution concentration as a template to perform probe method real-time fluorescence quantitative PCR reaction, draw a standard curve with the lg value of the template concentration as the X axis and the corresponding Ct value as the Y axis. Substitute the Ct value of the sample to be tested into the standard curve, calculate the copy number of Dictyocaurus filamentosa for quantitative detection.

[0025] Preferably, the detection limit of the detection method for Dictyocaudus filarius is 1.5 copies / μL.

[0026] The sequences involved in the scheme of the present invention are:

[0027] Forward primer: 5′-AACCTGAGCCTGCAGAAACATTAT-3′;

[0028] Reverse primer: 5′-CCACAGGTTTTGCAAACGCTATA-3′;

[0029] TaqMan probe: 5′-AAAGTTGCGAAGCAG-3′.

[0030] The 5' end of the TaqMan probe is labeled with a FAM fluorescent group, and the 3' end is labeled with a MGB-NFQ quencher group.

[0031] Compared with the prior art, this solution has the following beneficial effects:

[0032] 1. For the first time, a real-time fluorescence quantitative PCR detection method for Dictyocaudus filarius was established. This detection method can be used to qualitatively and / or quantitatively detect Dictyocaudus filarius in different clinical samples such as whole blood, rectal swabs, serum, brain, heart, lung, liver, gallbladder, kidney, intestine, and bladder.

[0033] 2. Compared with other traditional detection methods for Dictyochondria filariata, such as microscopy, ELISA, and conventional PCR, this detection method has higher sensitivity, is convenient for sampling, greatly improves detection efficiency, and effectively prevents contamination.

[0034] 3. Compared with other traditional detection methods for Dictyochondria filariata, such as microscopy, ELISA, and conventional PCR, this detection method has the characteristics of simple and easy operation procedures, and can be further made into a detection kit with standardized operation, which is suitable for large-scale promotion and application.

[0035] 4. This detection method can not only evaluate the infection status of Dictyocaurus filarius in animals, but also provide technical reserves for the research in related fields such as the epidemiology and pathogenic mechanism of Dictyocaurus filarius.

[0036] 5. This detection method can also be used to monitor the contamination of exogenous Dictyochondria filariformis in animal-derived biological products for human use, providing an effective tool for the safety inspection of biological products. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is the amplification curve of the real-time fluorescence quantitative PCR sensitivity test of the Dictyocaudus filarius probe method in the embodiment of the present invention;

[0038] Figure 2 is a specific test amplification curve of the real-time fluorescence quantitative PCR method of the Dictyocaurus filamentosa probe in the embodiment of the present invention;

[0039] Figure 3 is the real-time fluorescence quantitative PCR standard curve of the Dictyocaudus filarius probe method in the embodiment of the present invention;

[0040] Figure 4 This is the amplification curve of clinical samples detected by real-time optical quantitative PCR using the Dictyocaudus filarius probe method in the embodiment of the present invention. DETAILED DESCRIPTION

[0041] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0042] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0043] Embodiment 1:

[0044] 1 Materials and methods

[0045] 1.1 Materials

[0046] 1.1.1 Specimens of Dictyocauda filariformis to be tested

[0047] The samples for detecting Dictyocaudus filarius of the present invention are taken from different clinical samples such as whole blood, rectal swab, serum, brain, heart, lung, liver, gallbladder, kidney, intestine and bladder.

[0048] 1.1.2 DNA samples to be tested

[0049] DNA is extracted from the collected samples.

[0050] 1.1.3 Main reagents

[0051] Nuclease-free water (Cat. No. P1193) was purchased from Promega Corporation, USA.

[0052] TaqMan Mix (Applied Biosystem TM TaqMan TM Qiankun Platinum Multiplex Premix (Cat. No. A55163) was purchased from Applied Biosystem (ABI), USA.

[0053] DNeasy Blood&Tissue Kit (total DNA extraction kit, catalog number 69504) was purchased from Qiagen, Germany.

[0054] 1.2 Methods

[0055] The detection method provided by the present invention is a probe-based real-time fluorescence quantitative PCR detection method using the primers and TaqMan probes of the present invention. The primers and TaqMan probes provided by the present invention are designed based on the conservative region of the internal transcribed spacer 2 (ITS2) gene of the filamentous nematode.

[0056] 1.2.1 Design of primers and probes

[0057] Forward primer: 5′-AACCTGAGCCTGCAGAAACATTAT-3′;

[0058] Reverse primer: 5′-CCACAGGTTTTGCAAACGCTATA-3′;

[0059] TaqMan probe: 5′-AAAGTTGCGAAGCAG-3′.

[0060] The 5' end of the TaqMan probe is labeled with a FAM fluorescent group, and the 3' end is labeled with a MGB-NFQ quencher group.

[0061] 1.2.2 Construction of probe-based real-time fluorescence quantitative PCR reaction system

[0062] Probe method real-time fluorescence quantitative PCR reaction system: including: 10μL TaqMan Mix, 8μL nuclease-free pure water, 1μL primer and TaqMan probe Mix, 1μL template DNA.

[0063] The amplification program of the probe method real-time fluorescence quantitative PCR was as follows: 50°C for 2 min, 1 cycle, 95°C for 10 min, 1 cycle; 95°C for 15 s, 60°C for 1 min, 40 cycles.

[0064] 1.2.3 Establishing a standard curve

[0065] Use the plasmid DNA containing the target fragment as the standard and perform 10-fold serial dilutions as follows: (1) 1.5×10 10 copies / μL; (2) 1.5×10 9 copies / μL; ⑶1.5×10 8 copies / μL; (4) 1.5×10 7 copies / μL; (5) 1.5×10 6 copies / μL; ⑹1.5×10 5 copies / μL;⑺1.5×10 4 copies / μL;⑻1.5×10 3 copies / μL; ⑼1.5×10 2 copies / μL;⑽1.5×10 1 copies / μL;⑾1.5×10 0 The plasmid standard was used as a template for real-time fluorescence quantitative PCR detection, and the fluorescence signals corresponding to the plasmid standard of each dilution concentration were collected in real time. After the probe method real-time fluorescence quantitative PCR reaction was completed, the template concentration lg value was plotted on the X-axis and the corresponding Ct value was plotted on the Y-axis to draw a standard curve for quantitative detection of Dictyocauda filariata.

[0066] 1.2.4 Interpretation of qualitative results

[0067] When the quality of probe-based real-time fluorescence quantitative PCR detection is under control, if the Ct value of the sample being tested is ≤35 and a specific S-type fluorescence amplification curve appears, the corresponding sample being tested is judged to be positive; if there is no Ct value and no specific fluorescence amplification curve, the corresponding sample being tested is judged to be negative; if 35<Ct value<40 and a specific fluorescence amplification curve appears, it is judged to be an uncertain sample and the nucleic acid needs to be re-extracted for re-testing. If there is still an S-type fluorescence amplification curve and the Ct value is <40, the corresponding sample being tested is judged to be positive, otherwise it is judged to be negative.

[0068] 1.2.5 Interpretation of quantitative results

[0069] The target fragment of Dictyocaurus filamentosa was constructed into the pMD18-T vector, and the recombinant plasmid was transformed into JM109 competent E. coli cells. After amplification and culture, plasmid DNA was extracted. After restriction digestion, electrophoresis and sequencing were confirmed, it was used as a plasmid standard. The concentration of plasmid DNA was measured and converted into a specific plasmid copy number using the copy number conversion formula. A 10-fold series of dilutions was performed, and the plasmid standard of each dilution concentration was used as a template for probe method real-time fluorescence quantitative PCR reaction. The standard curve was drawn with the lg value of the template concentration as the X-axis and the corresponding Ct value as the Y-axis. The Ct value of the sample to be tested was substituted into the standard curve, and the copy number of Dictyocaurus filamentosa was calculated for quantitative detection.

[0070] 1.2.6 Experimental Conclusion

[0071] like Figure 1 As shown, the plasmid standard of Dictyocaurus filamentosa was increased from 1.5×10 10 Copies / μL were serially diluted 10-fold to 1.5×10 0 The detection limit of the probe-based real-time fluorescence quantitative PCR was 1.5 copies / μL, which has excellent sensitivity.

[0072] like Figure 2 As shown in the figure, the probe method real-time fluorescence quantitative PCR detected the fluorescence signal, and the DNA of Dictyocaurus filamentosa was used as the positive template for detection, and a specific S-shaped fluorescence amplification curve appeared. No fluorescence amplification curve was observed when the DNA of Strongyloides stercoralis, Tubules occultus, Tubules muris, and Tetrapterygium spondyloptera was used as the template for detection. No fluorescence amplification curve was observed when nuclease-free pure water was used as the negative control. The probe method real-time fluorescence quantitative PCR specifically detected Dictyocaurus filamentosa, and there was no cross reaction with other parasitic pathogens.

[0073] like Figure 3 As shown in the figure, the plasmid standard of Dictyocaurus filamentosa was serially diluted 10 times to establish the standard curve. Eleven dilution concentrations of plasmid standard (1.5×10 10 ~1.5×10 0 The standard curve of probe-based real-time fluorescence quantitative PCR was established by using 1000 copies / μL) as the template. The standard curve equation was y=-3.231x+38.077, the correlation coefficient was 0.992, and the amplification efficiency was 103.926%. Both the correlation coefficient and the amplification efficiency showed that the initial template had a good linear relationship with the Ct value. The probe-based real-time fluorescence quantitative PCR detected 11 concentration gradients of Dictyocaurus filamentosa, with a wide dynamic range.

[0074] like Figure 4As shown, 600 samples of whole blood, rectal swabs, serum, brain, heart, lung, liver, gallbladder, kidney, intestine, and bladder DNA from Hu sheep and 132 rabbits were used as detection objects, and real-time fluorescence quantitative PCR detection of Dictyochondria filamentosa probe method was performed. Each sample was repeated in parallel 3 times. The clinical sample detection system is 20μL, and the following components are added in sequence: 10μLTaqMan Mix, 8μL nuclease-free pure water, 1μL primer and TaqMan probe Mix, 1μL template DNA. The detection reaction conditions are: 50℃2min, 1 cycle 95℃10min, 1 cycle; 95℃15s, 60℃1min, 40 cycles. The results are shown in Figure 4 As shown in the figure, the whole blood DNA of 7 Hu sheep was used as template to produce a specific S-shaped fluorescence amplification curve. 7 ~1.5×10 3 A specific S-shaped fluorescence amplification curve was observed using 1000 copies / μL) as a template as a positive control. No S-shaped fluorescence amplification curve was observed using nuclease-free pure water as a negative control. The conventional PCR method we established previously did not detect it. The results showed that the sensitivity of real-time fluorescence quantitative PCR is higher than that of conventional PCR. It was proved that there was indeed infection with filamentous nematodes in Hu sheep, but the infection rate was extremely low and the worm load was very low.

[0075] The rapidity, specificity, sensitivity and stability of the present invention are embodied in the following ways:

[0076] 1) Analysis of the detection time of the method of the present invention

[0077] The probe-based fluorescent quantitative PCR method for detecting the filamentous nematode takes about 0.5 h to extract sample DNA; the detection reaction conditions are: 50°C for 2 min, 1 cycle, 95°C for 10 min, 1 cycle; 95°C for 15 s, 60°C for 1 min, 40 cycles, which takes about 1.5 h, and the whole process takes 2 h.

[0078] 2) Specificity analysis of the method of the present invention

[0079] The primer pair and TaqMan probe designed by the present invention are highly conservative and specific after BLAST comparison analysis, and can not only detect the filamentous digitocaurus in the present invention, but also distinguish it from other related parasitic pathogens. Using the filamentous digitocaurus DNA as a positive sample, using nuclease-free pure water as a negative control, and using Strongyloides stercoralis, Tubules cryptica, Tubules muris, and Tetrapterygium spondylorhizium DNA as templates, probe method real-time fluorescence quantitative PCR detection is performed to evaluate the specificity of the method.

[0080] The primer pair and probe designed by the present invention for the filamentous digitocauda nematode only amplify the filamentous digitocauda nematode DNA, and a specific S-shaped fluorescence amplification curve is observed. No fluorescence amplification curve is observed for the DNA of Strongyloides stercoralis, Tubulesoma cryptica, Tubulesoma muris, and Tetrapterygium spondyloptera, and no fluorescence amplification curve is observed for the negative control, indicating that the method of the present invention has strong specificity.

[0081] 3) Sensitivity analysis of the method of the present invention

[0082] The plasmid standard of Dictyocaurus filamentosa with known copy number was diluted 10-fold to 11 concentrations, starting from 1.5×10 10 ~1.5×10 0 The established probe-based real-time fluorescence quantitative PCR method was used for detection. The fluorescence amplification curve is shown in Figure 1 The test results show that under the current detection method and conditions, the detection limit of Dictyocaudus filarius is 1.5 copies / μL, as shown in Table 1. This shows that the method of the present invention has high sensitivity.

[0083] Table 1 Sensitivity test results of real-time fluorescence quantitative PCR method using Dictyocaudus filarius probe

[0084]

[0085] 4) Stability analysis of the method of the present invention

[0086] TaqMan Mix, primers and TaqMan probe Mix, plasmid standards, and nuclease-free pure water were repeatedly frozen and thawed 15 times. 1 μL of plasmid standards (1.5×10 7 ~1.5×10 3 1000 copies / μL) as template, and the probe method real-time fluorescence quantitative PCR reaction was carried out under the same conditions. In the same experiment, 3 replicates were made for each dilution of plasmid standard to analyze the intra-batch differences. Then, using the same plasmid standard as template, an independent repeat experiment was carried out every 3 days for 15 consecutive days. In the same experiment, 3 replicates were made for each dilution of plasmid standard to analyze the inter-batch differences. The coefficient of variation (CV) of the Ct value was statistically calculated to verify the repeatability and stability of the method.

[0087] The analysis results are shown in Table 2: the intra-batch coefficient of variation is 0.11% to 0.58%, the inter-batch coefficient of variation is 0.33% to 2.19%, and the intra-batch and inter-batch coefficients of variation are both less than 3%, indicating that the method of the present invention has good repeatability and stability.

[0088] Table 2 Results of repeatability and stability test of real-time fluorescence quantitative PCR detection method of Dictyochondria filariata probe

[0089]

[0090] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A real-time fluorescence quantitative PCR detection kit for Dictyocauda filariata, characterized in that: The kit contains a primer pair and a probe for detecting Dictyochondria filariata, and the nucleotide sequences of the primers and TaqMan probe are as follows: Forward primer: 5′-AACCTGAGCCTGCAGAAACATTAT-3′; Reverse primer: 5′-CCACAGGTTTTGCAAACGCTATA-3′; TaqMan probe: 5′-AAAGTTGCGAAGCAG-3′.

2. The real-time fluorescent quantitative PCR detection kit for Dictyocauda filariata according to claim 1, characterized in that: The 5' end of the TaqMan probe is labeled with a FAM fluorescent group, and the 3' end is labeled with a MGB-NFQ quenching group.

3. A real-time fluorescence quantitative PCR detection method for Dictyocauda filariata, characterized in that: The following steps are involved: S1. Obtain a sample of Dictyochondria filariata to be tested, and extract DNA from the sample to be tested; S2. Prepare the probe method real-time fluorescence quantitative PCR reaction system: take 10 μL TaqMan Mix, 8 μL nuclease-free pure water, 1 μL primer and TaqMan probe Mix, and 1 μL template DNA; S3, performing probe method real-time fluorescence quantitative PCR reaction: adding the prepared 20 μL reaction system into the probe method real-time fluorescence quantitative PCR reaction tube for reaction, and collecting the fluorescence signal in real time; S4. Establish a standard curve: Take 1 μL of the plasmid standard after 10-fold serial dilution as a template, perform the same probe method real-time fluorescence quantitative PCR reaction as the sample, collect the fluorescence signal in real time, and draw a standard curve; S5. Data analysis: Substitute the Ct value measured by the test sample into the standard curve to obtain the concentration of the sample to be tested; perform qualitative and quantitative detection on the sample to be tested based on the measured Ct value.

4. The real-time fluorescence quantitative PCR detection method of Dictyocauda filariata according to claim 3, characterized in that: In step S1, the Dictyocaudus filarius sample is taken from one or more of whole blood, rectal swab, serum, brain, heart, lung, liver, gallbladder, kidney, intestine, and bladder.

5. The real-time fluorescence quantitative PCR detection method of Dictyocauda filariata according to claim 3, characterized in that: In step S2, the forward primer concentration in the primer and TaqMan probe Mix is ​​440 nM, the reverse primer concentration is 440 nM, and the TaqMan probe concentration is 120 nM.

6. The real-time fluorescence quantitative PCR detection method of Dictyocauda filariata according to claim 3, characterized in that: In step S3, the amplification program of the probe method real-time fluorescence quantitative PCR is: 50°C for 2 min, 1 cycle; 95°C for 10 min, 1 cycle; 95°C for 15 s, 60°C for 1 min, 40 cycles.

7. The real-time fluorescence quantitative PCR detection method of Dictyocauda filariata according to claim 3, characterized in that: In step S3, the quantitative detection includes the establishment of a standard curve, and the specific steps are as follows: S301. Prepare a plasmid standard containing the target fragment and perform a 10-fold serial dilution. There are 11 dilution concentrations, 1.5×10 10 Copies / μL to 1.5×10 0 copies / μL; S302, using 1 μL of 11 dilution concentrations of plasmid standard after 10-fold serial dilution as positive template and nuclease-free pure water as negative control, perform the same probe method real-time fluorescence quantitative PCR reaction as the sample; S303, collecting the fluorescence signals corresponding to the plasmid standard products at each dilution concentration in real time, and after the probe method real-time fluorescence quantitative PCR reaction is completed, drawing a standard curve with the lg value of the template concentration as the X-axis and the corresponding Ct value as the Y-axis; S304, the standard curve equation is y=-3.231x+38.077, the correlation coefficient is 0.992, and the amplification efficiency is 103.926%; S305 , substituting the Ct value of the sample to be tested into the standard curve to obtain the concentration of the sample to be tested.

8. The real-time fluorescent quantitative PCR detection kit for Dictyocauda filariata according to claim 1, characterized in that: The qualitative result interpretation method is as follows: under the condition that the detection quality of the probe method real-time fluorescence quantitative PCR is under control, if the Ct value of the tested sample is ≤35 and a specific S-type fluorescence amplification curve appears, the corresponding tested sample is judged to be positive; if there is no Ct value and no specific fluorescence amplification curve, the corresponding tested sample is judged to be negative; if 35<Ct value<40 and a specific fluorescence amplification curve appears, it is judged to be an uncertain sample, and the nucleic acid needs to be re-extracted for re-testing. If there is still an S-type fluorescence amplification curve and the Ct value is <40 after the re-testing, the corresponding tested sample is judged to be positive, otherwise it is judged to be negative.

9. The real-time fluorescent quantitative PCR detection kit for Dictyocauda filariata according to claim 1, characterized in that: The quantitative result interpretation method is as follows: construct the target fragment of the filamentous dactyloid nematode into the pMD18-T vector, transform the recombinant plasmid into JM109 Escherichia coli competent cells, extract the plasmid DNA after amplification and identification, use it as a plasmid standard after enzyme digestion electrophoresis sequencing is correct, determine the concentration of the plasmid DNA, convert it into a specific plasmid copy number through a copy number conversion formula, perform a 10-fold serial dilution, use the plasmid standard of each dilution concentration as a template to perform a probe method real-time fluorescence quantitative PCR reaction, and draw a standard curve with the lg value of the template concentration as the X axis and the corresponding Ct value as the Y axis. Substitute the Ct value of the sample to be tested into the standard curve, calculate the copy number of the filamentous dactyloid nematode for quantitative detection.

Images