Fluorescent quantitative PCR (Polymerase Chain Reaction) primer probe group for detecting pangolin-derived hookworms and application of fluorescent quantitative PCR primer probe group

Abstract

The invention discloses a fluorescent quantitative PCR primer probe set for detecting pangolin-derived hookworms and application of the fluorescent quantitative PCR primer probe set. The fluorescent quantitative primer probe group provided by the invention comprises a forward primer with a nucleotide sequence as shown in SEQ ID NO.2, a reverse primer with a nucleotide sequence as shown in SEQ ID NO.3 and a TaqMan probe with a nucleotide sequence as shown in SEQ ID NO.4. The primer probe group is used for performing probe-method fluorescent quantitative PCR (Polymerase Chain Reaction), DNA (Deoxyribonucleic Acid) of pangolin-derived hookworms can be specifically detected, the lowest detection concentration is 3.4 * 10 < 1 > copy number / L, and the primer probe group can be applied to detection of pangolin-derived hookworms. And the sensitivity is 1000 times that of the conventional PCR. The method not only can evaluate the hookworm infection condition of the pangolin, but also can be used for pollution monitoring of exogenous hookworms, and provides technical reserve for research in related fields of epidemiology, pathogenesis and the like of pangolin-derived hookworms.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a fluorescent quantitative PCR primer and probe set for detecting hookworms of pangolin origin and its application. Background Technology

[0002] Hookworms, belonging to the genus *Ancylostoma* in the family Alectostomatidae, are parasitic nematodes. The eggs are oval, thin-shelled, and approximately 50-75 micrometers × 30-40 micrometers in size, typically containing 2-8 egg cells. Hookworms primarily parasitize the intestines of mammals, with hookworms specifically referring to a group of hookworms that primarily parasitize pangolins. After infection, the worms attach to the intestinal mucosa through their buccal capsules, feeding on blood and secreting anticoagulants, leading to intestinal mucosal damage, chronic blood loss, and symptoms such as anemia, malnutrition, and emaciation. Young pangolins are more severely affected by infection, often exhibiting stunted growth and even death due to extreme weakness. Furthermore, hookworm infection weakens the pangolin's immune system, increasing the risk of secondary infections by bacteria, viruses, and other pathogens, which is a significant reason for the failure of wild pangolin rescue efforts. Molecular biological analysis has revealed significant differences between the mitochondrial genome of *Ancylostomasp.*, *Ancylostomasp.*, *Ancylostomasp.*, *Ancylostomasp.*, and *Ancylostomasp.*, providing crucial molecular evidence for designing specific detection targets for *Ancylostomasp.* and effectively avoiding cross-reactions with other common hookworms during detection (Molecular detection of a novel *Ancylostomasp.* by whole mtDNA sequence from *pangolin Manis javanica*. *Parasites & Vectors*, 2022, 15(1): 70). Therefore, developing specific detection methods based on these genomic differences can solve the problem of traditional detection methods failing to accurately distinguish *Ancylostomasp.* from closely related hookworm species, meeting the urgent need for accurate identification of *Ancylostomasp.* in wild pangolin population monitoring, pathogen diagnosis in artificial rescue, and customs quarantine.

[0003] Currently, the detection of hookworm from pangolins mainly relies on traditional fecal smear microscopy and conventional PCR technology. Traditional fecal smear microscopy determines the infection status by identifying the morphology of the eggs, but it has low sensitivity and is prone to missed detection when the number of eggs is small in mild infections. It also cannot distinguish between hookworm from pangolins and hookworms from other mammals (whose eggs have similar morphology). Although conventional PCR technology has improved sensitivity compared to microscopy, it has the disadvantage of non-specific amplification leading to false positive results, and it cannot accurately quantify the intensity of infection, making it difficult to assess the degree of infection and monitor the treatment effect.

[0004] Pangolins are a Class I protected animal in China. Monitoring wild populations and screening for pathogens in artificial rescue operations require rapid, sensitive, specific, and quantitative detection methods to achieve early detection and intervention. Quantitative real-time PCR (qPCR) has become a commonly used molecular biology method for parasite nucleic acid detection. Based on specific primer design, the application of qPCR strategies can effectively solve the problems of false positives and inability to quantify in conventional PCR. Therefore, establishing a qPCR detection method for hookworms derived from pangolins is of great significance for protecting pangolin health, advancing conservation efforts, and strengthening quarantine supervision, and is a pressing technical need that needs to be addressed. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide a fluorescent quantitative PCR primer and probe set for detecting hookworms of pangolin origin and its application.

[0006] The first objective of this invention is to provide a primer-probe set for the specific detection of hookworms from pangolins, the primer-probe set comprising a forward primer, a reverse primer, and a TaqMan probe; the nucleotide sequence of the forward primer is shown in SEQ ID NO.2, the nucleotide sequence of the reverse primer is shown in SEQ ID NO.3, and the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO.4.

[0007] Preferably, the 5' end of the TaqMan probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

[0008] Preferably, the fluorescent group is a FAM fluorescent group, and the quenching group is a TAMRA-N quenching group.

[0009] A second objective of this invention is to provide a kit for detecting hookworms of pangolin origin, comprising the aforementioned primer and probe set and probe-based quantitative PCR reagent.

[0010] Preferably, the kit further includes a plasmid standard of hookworm DNA from pangolins, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0011] Preferably, the concentration of the plasmid standard of hookworm DNA from pangolins is 3.4 × 10⁻⁶. 1 ~3.4×10 8 Copy number / µL.

[0012] Preferably, the concentration of the plasmid standard of hookworm DNA from pangolins is 3.4 × 10⁻⁶. 1 Copy number / µL, 3.4×10 2 Copy number / µL, 3.4×10 3 Copy number / µL, 3.4×104 Copies / µL, 3.4×10 5 Copies / µL, 3.4×10 6 Copies / µL, 3.4×10 7 Copies / µL and 3.4×10 8 Copies / µL.

[0013] The third object of the present invention is to provide a fluorescence quantitative PCR method for detecting hookworms from pangolins for non-diagnostic purposes, comprising the following steps: S1. Collect the test sample from pangolins; S2. Extract the DNA from the test sample; S3. Using the kit described above, with the DNA in step S2 as a template, perform probe-based fluorescence quantitative PCR amplification; S4. Read the amplification curve and Ct value, and judge whether the test sample contains hookworms from pangolins according to the amplification curve and Ct value; The method for judgment is as follows: If there is a Ct value, Ct value ≤ 35, and a specific S-shaped fluorescence amplification curve appears, it is determined that the test sample is positive for hookworms from pangolins; if there is no Ct value and no specific fluorescence amplification curve, it is determined that the test sample is negative for hookworms from pangolins; if there is a Ct value, 35 < Ct value < 40, and a specific fluorescence amplification curve appears, then repeat steps S3 to S4 with the DNA in the same test sample as a template; After repeating steps S3 to S4 once, if there is a Ct value, Ct value ≤ 35, and a specific S-shaped fluorescence amplification curve appears, it is determined that the test sample is positive for hookworms from pangolins; if there is no Ct value and no specific fluorescence amplification curve, it is determined that the test sample is negative for hookworms from pangolins; if there is a Ct value, 35 < Ct value < 40, and a specific fluorescence amplification curve appears, then repeat steps S1 to S4 once or repeat steps S2 to S4 once; After repeating steps S1 to S4 once or repeating steps S2 to S4 once, if there is a Ct value, Ct value < 40, and a specific fluorescence amplification curve appears, it is determined that the test sample is positive for hookworms from pangolins, otherwise it is determined that the test sample is negative for hookworms from pangolins.

[0014] The fourth object of the present invention is to provide a fluorescence quantitative PCR method for detecting the content of hookworms from pangolins for non-diagnostic purposes, comprising the following steps: [[ID=三十二]] [[ID=三十三]]S1. Collect the test sample from pangolins; [[ID=三十四]] [[ID=三十五]]S2. Extract the DNA from the test sample; [[ID=三十六]] S3. Using the kit described in claim 5, respectively taking the plasmid standard of the hookworm DNA from pangolin and the DNA in step S2 as templates, perform fluorescence quantitative PCR amplification by the probe method; S4. Read the amplification curve and Ct value, and judge whether the test sample contains hookworm DNA from pangolin according to the situation of the amplification curve and Ct value; The method of the judgment is as follows: If there is a Ct value, Ct value ≤ 35, and a specific S-shaped fluorescence amplification curve appears, it is determined that the test sample is positive for hookworm DNA from pangolin; if there is no Ct value and no specific fluorescence amplification curve, it is determined that the test sample is negative for hookworm DNA from pangolin; if there is a Ct value, 35 < Ct value < 40, and a specific fluorescence amplification curve appears, repeat steps S3 to S4 once with the DNA in the same test sample as the template; After repeating steps S3 to S4 once, if there is a Ct value, Ct value ≤ 35, and a specific S-shaped fluorescence amplification curve appears, it is determined that the test sample is positive for hookworm DNA from pangolin; if there is no Ct value and no specific fluorescence amplification curve, it is determined that the test sample is negative for hookworm DNA from pangolin; if there is a Ct value, 35 < Ct value < 40, and a specific fluorescence amplification curve appears, repeat steps S1 to S4 once or repeat steps S2 to S4 once; After repeating steps S1 to S4 once or repeating steps S2 to S4 once, if there is a Ct value, Ct value < 40, and a specific fluorescence amplification curve appears, it is determined that the test sample is positive for hookworm DNA from pangolin, otherwise it is determined that the test sample is negative for hookworm DNA from pangolin; S5. Read the Ct value of the plasmid standard of the hookworm DNA from pangolin, take the lg value of the concentration of the plasmid standard of the hookworm DNA from pangolin as the X-axis, and take the Ct value of the plasmid standard of the hookworm DNA from pangolin as the Y-axis to draw a standard curve; S6. Substitute the Ct value of the test sample positive for hookworm DNA from pangolin into the standard curve obtained in step S5 to calculate the concentration of hookworm DNA from pangolin in the test sample positive for hookworm DNA from pangolin.

[0015] Preferably, the test sample from pangolin is an anal swab, feces or intestine from pangolin.

[0016] Preferably, the system for fluorescence quantitative PCR amplification by the probe method is: 2×probe method qPCR universal premixed reaction solution, 0.2 μL of forward primer with a concentration of 10 μM, 0.2 μL of reverse primer mixture with a concentration of 10 μM, 0.4 μL of TaqMan probe with a concentration of 10 mM, 1 μL of DNA template and 3.2 μL of water; The procedure for probe-based real-time PCR amplification is as follows: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing for 30 s, for 40 cycles.

[0017] The beneficial effects of this invention are: 1. A real-time fluorescence quantitative PCR detection method based on a probe of pangolin-derived hookworm was established for the first time. This method can be used to qualitatively or quantitatively detect pangolin-derived hookworm in different clinical samples such as rectal test strips, feces, or intestines.

[0018] 2. Compared with other traditional methods for detecting hookworm from pangolins, such as microscopic examination, ELISA, and conventional PCR, this detection method is more sensitive, more convenient, and significantly improves detection efficiency. It also effectively prevents contamination and is characterized by its speed, high specificity, high sensitivity, and stable reaction. These characteristics are specifically demonstrated in the following ways: (1) Analysis of detection time of the method of the present invention The probe-based quantitative real-time PCR method for detecting hookworm from pangolins takes approximately 1 hour to extract sample DNA. The detection reaction conditions are: 95℃ pre-denaturation for 30 seconds; 95℃ denaturation for 5 seconds, 60℃ annealing for 30 seconds, for 40 cycles; taking approximately 40 minutes. The entire process takes approximately 1 hour and 40 minutes.

[0019] (2) Specificity analysis of the method of the present invention The pangolin-derived hookworm primers and TaqMan probes designed in this invention, after BLAST alignment analysis, exhibit high conservation and specificity. They can not only detect pangolin-derived hookworms but also differentiate them from other pathogens. The primer pairs and probes designed in this invention showed a specific S-shaped fluorescence amplification curve when amplifying only pangolin-derived hookworm DNA. No fluorescence amplification curves were observed when using pangolin-derived Strongyloides stercoralis, hookworm of the dog, and pangolin-derived Cyclostome DNA as templates. The negative control showed no fluorescence amplification curve, indicating the high specificity of the method of this invention.

[0020] (3) Sensitivity and repeatability analysis of the method of the present invention A plasmid standard from pangolins with known copy numbers was serially diluted 10-fold in eight steps, resulting in a concentration of 3.4 × 10⁻⁶ plasmids per μL. 8 Copy number, 3.4 × 10 7 Copy number, 3.4 × 10 6 Copy number, 3.4 × 10 5 Copy number, 3.4 × 10 4 Copy number, 3.4 × 10 3 Copy number, 3.4 × 10 2 Copy number, 3.4 × 10 1Copy number. The established probe-based real-time quantitative PCR method was used for detection. The results showed that the detection limit for hookworm from pangolins using the probe-based real-time quantitative PCR method provided by this invention is 3.4 × 10⁻⁶. 1 The copy number per μL demonstrates the high sensitivity of this invention.

[0021] 3. Compared with other traditional methods for detecting hookworms from pangolins, such as microscopic examination, ELISA, and conventional PCR, this detection method is simple and easy to use. It can also be further developed into a detection kit, with a standardized operation procedure, making it suitable for large-scale promotion and application.

[0022] 4. This detection method can not only evaluate the hookworm infection status of pangolins, but also provide technical reserves for research in related fields such as the epidemiology and pathogenesis of hookworms from pangolins.

[0023] 5. This detection method can also be used for monitoring exogenous hookworm contamination. Attached Figure Description

[0024] Figure 1 This is the sensitivity test result of the probe-based real-time quantitative PCR detection method for hookworms derived from pangolins. Among them, Figure 1 In this context, A represents the amplification curve of probe-based real-time quantitative PCR. Figure 1 In the graph, B represents the results of a routine PCR sensitivity test, 1 is the blank control, and 2 represents the result of a assay with a concentration of 3.4 × 10⁻⁶. 8 Using pangolin-derived hookworm DNA at a copy number / µL as a template, 3 was prepared at 3.4 × 10⁻⁶. 7 Using pangolin-derived hookworm DNA at a copy number / µL as a template, 4 was prepared with 3.4 × 10⁴ copies / µL. 6 Using pangolin-derived hookworm DNA at a copy number / µL as a template, 5 was prepared at 3.4 × 10⁵. 5 Using pangolin-derived hookworm DNA at a copy number / µL as a template, 6 was prepared with 3.4 × 10⁶ copies / µL. 4 Using pangolin-derived hookworm DNA at a copy number / µL as a template, 7 was prepared at 3.4 × 10⁷ copies / µL. 3 Using pangolin-derived hookworm DNA at a copy number / µL as a template, 8 was prepared at 3.4 × 10⁸ copies / µL. 2 Using pangolin-derived hookworm DNA at a copy number / µL as a template, 9 was prepared with 3.4 × 10⁹ copies / µL. 1 Pangolin-derived hookworm DNA at a copy number / µL was used as a template.

[0025] Figure 2 This is the standard curve for the probe-based real-time quantitative PCR detection method of hookworm from pangolins.

[0026] Figure 3 This is the amplification curve of the probe-based real-time quantitative PCR detection method for hookworms from pangolins, representing a specificity test.

[0027] Figure 4 This is the amplification curve of clinical samples detected by a probe-based real-time fluorescence quantitative PCR method for detecting hookworms from pangolins. Detailed Implementation

[0028] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0029] Example 1 1. Materials and Methods 1.1 Materials (1) Sample to be tested The samples to be tested are taken from the feces, rectal test strips, or intestines of Chinese pangolins or Malayan pangolins.

[0030] (2) Extracting DNA from the sample to be tested DNA is extracted from the collected samples to be tested and used as a template.

[0031] (3) Main reagents Nuclease-free water (suitable for electrophoresis, product number W4502) was purchased from the Merck Group in Darmstadt, Germany.

[0032] The universal premixed reaction solution for probe-based qPCR described in this embodiment is Premix Ex Taq (ProbeqPCR) (2X Conc.) from Premix Ex Taq™ (Probe qPCR), product number RR390A, purchased from Baori Biotechnology (Beijing) Co., Ltd.

[0033] In this embodiment, the EZNA® Stool DNA Kit (fecal DNA extraction kit, catalog number D4015, purchased from Omega, USA) was used to extract DNA.

[0034] In this embodiment, 2×Taq Plus Master Mix Ⅱ (Dye Plus), purchased from Nanjing Novizan Biotechnology Co., Ltd., with product number P213-01, was used for routine PCR amplification.

[0035] In this embodiment, the EZNA® Stool DNA Kit (fecal DNA extraction kit, catalog number D4015, purchased from Omega, USA) was used to extract DNA.

[0036] 1.2 Construction of a probe-based real-time quantitative PCR detection method for hookworms derived from pangolins (1) Based on the cytochrome c oxidase I of hookworm from pangolin source ( COX1The primers and probes required for real-time quantitative detection of conserved gene sequence regions are designed to encode the aforementioned... COX1 The nucleotide sequence of the gene is shown in SEQ ID NO.1. The primers and probes are designed as follows: Forward primer: 5'-GGGACTAGTTGAACGGTGTATC-3', SEQ ID NO.2; Reverse primer: 5'-GACCTTAAACCAGCGCAATG-3', SEQ ID NO.3; TaqMan probe: 5'-TAGGTCATCCTGGGAGAAGTGTTGACT-3', SEQ ID NO.4; The 5' end of the TaqMan probe is labeled with a FAM fluorescent group, and the 3' end is labeled with a TAMRA-N quencher group.

[0037] (2) Constructing a probe-based real-time quantitative PCR reaction system The probe-based real-time quantitative PCR reaction system includes 5 μL of 2× probe-based qPCR universal premixed reaction solution, 0.2 μL each of 10 μM forward and reverse primers, 0.4 μL of 10 mM TaqMan probe, 1 μL of DNA template, and 3.2 μL of nuclease-free water.

[0038] The amplification program for probe-based real-time quantitative PCR is as follows: 95℃ pre-denaturation for 30 s, 1 cycle; 95℃ denaturation for 5 s, 60℃ annealing for 30 s, 40 cycles.

[0039] (3) Establishing a standard curve The target fragment, as shown in SEQ ID NO.1, of the hookworm nucleotide sequence from pangolins was constructed into the pUC57 vector, then transformed into *E. coli* (DH5α). After amplification culture, plasmid DNA was extracted, and after confirmation by enzyme digestion, electrophoresis, and sequencing, it was used as a plasmid standard. The plasmid DNA concentration was determined, and the specific plasmid copy number was converted using the copy number conversion formula. Serial dilutions were performed 10-fold to a final concentration of 3.4 × 10⁻⁶. 8 Copy number / μL, 3.4 × 10 7 Copy number / μL, 3.4 × 10 6 Copy number / μL, 3.4 × 10 5 Copy number / μL, 3.4 × 10 4 Copy number / μL, 3.4 × 10 3 Copy number / μL, 3.4 × 10 2 Copy number / μL, 3.4 × 10 1Plasmid standard dilutions of various concentrations were used as templates for probe-based real-time quantitative PCR reactions to obtain fluorescence signals and corresponding Ct values ​​for different concentrations of plasmid standards. A standard curve was plotted with 1g of template concentration as the X-axis and the corresponding Ct value as the Y-axis for quantifying hookworms.

[0040] (4) Interpretation of qualitative results Assuming the quality of the probe-based real-time quantitative PCR detection is accurate, the steps for interpreting the qualitative results are as follows: If the detected sample has a Ct value ≤ 35 and shows a specific S-shaped fluorescence amplification curve, the sample is considered positive for hookworm. If there is no Ct value and no specific fluorescence amplification curve, the sample is considered negative for hookworm. If 35 < Ct value < 40 and a specific fluorescence amplification curve appears, the sample is considered suspicious, and it is recommended to repeat the experiment and test the wells repeatedly.

[0041] If, after repeating the experiment and testing the wells, the detected sample has a Ct value ≤ 35 and exhibits a specific S-shaped fluorescence amplification curve, it is determined that the tested sample is positive for hookworm; if there is no Ct value and no specific fluorescence amplification curve, the corresponding tested sample is determined to be negative for hookworm; if the results are similar (i.e., 35 < Ct value < 40 still appears), it is recommended to resample and test or re-extract sample DNA for testing.

[0042] If, after resampling and testing or re-extracting sample DNA for testing, the Ct value is still between 35 and 40 and a specific S-type fluorescence amplification curve appears, or the Ct value is ≤35 and a specific S-type fluorescence amplification curve appears, the tested sample is determined to be hookworm positive; otherwise, the tested sample is determined to be hookworm negative.

[0043] (5) Interpretation of quantitative results After the qualitative results were interpreted in (4), the Ct value of the pangolin-derived hookworm positive sample was substituted into the standard curve to calculate the copy number of pangolin-derived hookworm and to perform quantitative detection.

[0044] 1.3 Characterization, Validation, and Application of the Probe-Based Real-Time Quantitative PCR Detection Method for Hookworms from Pangolins (1) Establishment of standard curve and determination of sensitivity and repeatability The standard of hookworm plasmid from pangolins was increased from 3.4 × 10⁻⁶. 8 Copies / μL diluted 10-fold to 3.4 × 10⁻⁶ 1 Copies / μL, diluted to a concentration of 3.4 × 10⁻⁶. 8 Copy number / μL, 3.4 × 10 7 Copy number / μL, 3.4 × 10 6 Copy number / μL, 3.4 × 10 5Copy number / μL, 3.4 × 10 4 Copy number / μL, 3.4 × 10 3 Copy number / μL, 3.4 × 10 2 Copy number / μL, 3.4 × 10 1 Eight plasmid standard dilutions with a copy number / μL were used, and each concentration was repeated three times. The detection limit of the probe method constructed above for real-time fluorescence quantitative PCR detection of hookworm from pangolin was determined, and a standard curve was obtained.

[0045] Simultaneously, the above-mentioned plasmid standard dilution was detected using conventional PCR. Specifically, the designed forward primer (nucleotide sequence shown in SEQ ID NO.2) and reverse primer (nucleotide sequence shown in SEQ ID NO.3) were used as amplification primers for conventional PCR. The conventional PCR reaction system was as follows: 12.5 μL 2 × Taq Plus Master Mix II (Dye Plus), 1 μL forward primer, 1 μL reverse primer, 1 μL DNA template, and 9.5 μL nuclease-free water; the conventional PCR reaction program was as follows: 94℃ for 5 min; 94℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s, for 35 cycles; 72℃ for 5 min; and stored at 4℃.

[0046] Table 2. Sensitivity and repeatability test results of the real-time fluorescence quantitative PCR detection method using the hookworm probe method from pangolins. The results are as follows Figure 1 As shown in Figure A, the probe-based real-time quantitative PCR method can detect hookworms from pangolins at eight concentration gradients, and the detection limit for hookworms from pangolins is 3.4 × 10⁻⁶. 1 The result of 1000 copies / μL indicates that the established probe-based real-time quantitative PCR method has a good detection range and excellent sensitivity, which is 1000 times that of conventional PCR detection methods, while the detection limit of conventional PCR detection methods is 3.4 × 10⁻⁶. 3 Copy number / μL ( Figure 1 (B in the middle).

[0047] Furthermore, based on the results obtained from the probe-based real-time quantitative PCR method described above (Table 2), the standard curve is as follows: Figure 2 As shown, the standard curve equation is Y = -3.2688X + 39.079, the correlation coefficient is 0.9984, and the amplification efficiency is 102.265%. Both the correlation coefficient and the amplification efficiency show a good linear relationship between the initial template and the Ct value.

[0048] (2) Specific detection DNA from pangolin-derived Strongyloides stercoralis, Hookworm, and pangolin-derived Cyclostome were used as templates, respectively. Hookworm DNA was used as a positive template. Nuclease-free water was used as a negative control. The fluorescence signal was detected using the probe-based real-time quantitative PCR method described above.

[0049] The results are as follows Figure 3 As shown, using hookworm DNA from pangolins as a positive template, a specific S-shaped fluorescence amplification curve was observed. No fluorescence amplification curves were observed when using DNA from pangolin-derived Strongyloides stercoralis, hookworm of the canine, and pangolin-derived Cyclostome as templates. No fluorescence amplification curve was observed when using nuclease-free water as a negative control. These results demonstrate that probe-based real-time quantitative PCR can specifically detect hookworms from pangolins and shows no cross-reactivity with other parasitic pathogens.

[0050] (3) Validation and application of probe-based real-time quantitative PCR detection method for hookworms from pangolins Forty-one pangolin fecal DNA samples were used for real-time quantitative PCR detection of hookworm from pangolins using a probe method. Each sample was tested in triplicate. Six plasmid standard dilution buffers (3.4 × 10⁻⁶) were used. 8 ~3.4×10 3 A template (copy number / μL) was used as a positive control, and nuclease-free water was used as a negative control. The sample detection system consisted of 10 μL, containing the following components in sequence: 5 μL of 2× probe-based qPCR universal premixed reaction solution, 3.2 μL of nuclease-free water, 0.2 μL each of the forward primer (nucleotide sequence shown in SEQ ID NO.2) and the reverse primer (nucleotide sequence shown in SEQ ID NO.3) (concentrations of 10 μM), 0.4 μL of TaqMan probe (concentration of 10 mM), and 1 μL of DNA template. The detection reaction conditions were: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing for 30 s, for 40 cycles.

[0051] The results are as follows Figure 4 As shown: Among 41 pangolin fecal DNA samples, 5 samples showed specific S-shaped fluorescence amplification curves when used as templates. These curves were obtained using plasmid standard dilutions at 6 concentrations (3.4 × 10⁻⁶). 8 ~3.4×10 3 Using a template of (copy / μL) as a positive control, a specific S-shaped fluorescence amplification curve was observed, while no S-shaped fluorescence amplification curve was observed in the negative control using nuclease-free water as a template. Conventional PCR results showed no positive results in the 41 pangolin fecal DNA samples mentioned above. The results indicate that real-time quantitative PCR has higher sensitivity than conventional PCR, proving the presence of hookworm infection in pangolin feces.

Claims

1. A primer probe set for specifically detecting hookworm of Manis pentadactyla, characterized in that, The primer probe set comprises a forward primer, a reverse primer and a TaqMan probe; the nucleotide sequence of the forward primer is shown as SEQ ID NO. 2, the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 3, and the nucleotide sequence of the TaqMan probe is shown as SEQ ID NO.

4.

2. The primer probe set of claim 1, wherein, The 5' end of the TaqMan probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

3. The primer probe set of claim 2, wherein, The fluorescent group is a FAM fluorescent group, and the quenching group is a TAMRA-N quenching group.

4. A kit for detecting Ancylostoma ceylanicum of origin of Manis, characterized by, The primer probe set and the probe method fluorescent quantitative PCR reagent of any one of claims 1-3 are included.

5. The kit according to claim 4, characterized in that, A plasmid standard of the pangolin-derived hookworm DNA is also included, and the nucleotide sequence of the pangolin-derived hookworm DNA is shown as SEQ ID NO.

1.

6. The kit of claim 5, wherein The concentration of the plasmid standard of the Manis pentadactyla-derived Ancylostoma DNA is 3.4 x 10 1 copies / µL. 8 copies / µL.

7. The kit of claim 6, wherein The concentration of the plasmid standard of Manis pentadactyla-derived Ancylostoma DNA is 3.4 x 10 1 copies / µL, 3.4 x 10 2 copies / µL, 3.4 x 10 3 copies / µL, 3.4 x 10 4 copies / µL, 3.4 x 10 5 copies / µL, 3.4 x 10 6 copies / µL, 3.4 x 10 7 copies / µL, and 3.4 x 10 8 copies / µL.

8. A fluorescent quantitative PCR method for detecting hookworm of Manis pentadactyla for non-disease diagnosis purposes, characterized by, The following steps are included: S1. Collecting a sample to be detected from a pangolin; S2. Extracting DNA in the sample to be detected; S3. Using the kit of claim 4, taking the DNA of step S2 as a template to perform probe method fluorescent quantitative PCR amplification; S4. Reading the amplification curve and Ct value, and judging whether the sample to be detected contains the pangolin-derived hookworm according to the amplification curve and the Ct value; The judging method is as follows: If there is a Ct value, the Ct value is ≤35, and a specific S-shaped fluorescent amplification curve appears, it is determined that the sample to be detected is positive for the pangolin-derived hookworm; if there is no Ct value, and no specific fluorescent amplification curve appears, it is determined that the sample to be detected is negative for the pangolin-derived hookworm; if there is a Ct value, 35 < Ct value < 40, and a specific fluorescent amplification curve appears, the DNA in the same sample to be detected is taken as a template to repeat steps S3-S4 once; After repeating steps S3-S4 once, if there is a Ct value, the Ct value is ≤35, and a specific S-shaped fluorescent amplification curve appears, it is determined that the sample to be detected is positive for the pangolin-derived hookworm; if there is no Ct value, and no specific fluorescent amplification curve appears, it is determined that the sample to be detected is negative for the pangolin-derived hookworm; if there is a Ct value, 35 < Ct value < 40, and a specific fluorescent amplification curve appears, steps S1-S4 are repeated once or steps S2-S4 are repeated once; After repeating steps S1-S4 once or repeating steps S2-S4 once, if there is a Ct value, the Ct value is <40, and a specific fluorescent amplification curve appears, it is determined that the sample to be detected is positive for the pangolin-derived hookworm, otherwise it is determined that the sample to be detected is negative for the pangolin-derived hookworm.

9. A fluorescent quantitative PCR method for detecting the amount of Ancylostoma ceylanicum of the pangolin for a purpose other than disease diagnosis, characterized by, The following steps are included: S1. Collecting a sample to be detected from a pangolin; S2. Extracting DNA in the sample to be detected; S3. Using the kit of claim 5, taking the plasmid standard of the pangolin-derived hookworm DNA and the DNA of step S2 as templates to perform probe method fluorescent quantitative PCR amplification, respectively; S4. Reading the amplification curve and Ct value, and judging whether the sample to be detected contains the pangolin-derived hookworm according to the amplification curve and the Ct value; The judging method is as follows: If Ct value exists, Ct value is less than or equal to 35, and specific S-shaped fluorescence amplification curve appears, the sample is determined to be positive for the pangolin-derived hookworm; if Ct value does not exist, and specific fluorescence amplification curve does not appear, the sample is determined to be negative for the pangolin-derived hookworm; if Ct value exists, 35 < Ct value < 40, and specific S-shaped fluorescence amplification curve appears, the DNA in the same sample is used as a template to repeat steps S3-S4 once; After repeating steps S3-S4 once, if Ct value exists, Ct value is less than or equal to 35, and specific S-shaped fluorescence amplification curve appears, the sample is determined to be positive for the pangolin-derived hookworm; if Ct value does not exist, and specific fluorescence amplification curve does not appear, the sample is determined to be negative for the pangolin-derived hookworm; if Ct value exists, 35 < Ct value < 40, and specific S-shaped fluorescence amplification curve appears, steps S1-S4 are repeated once or steps S2-S4 are repeated once; After repeating steps S1-S4 once or repeating steps S2-S4 once, if Ct value exists, Ct value is less than 40, and specific S-shaped fluorescence amplification curve appears, the sample is determined to be positive for the pangolin-derived hookworm; otherwise, the sample is determined to be negative for the pangolin-derived hookworm; S5. The Ct value of the plasmid standard of the pangolin-derived hookworm DNA is read, and a standard curve is drawn with the lg value of the concentration of the plasmid standard of the pangolin-derived hookworm DNA as the X axis and the Ct value of the plasmid standard of the pangolin-derived hookworm DNA as the Y axis. S6. The Ct value of the sample to be tested that is positive for the pangolin-derived hookworm is substituted into the standard curve obtained in step S5 to calculate the concentration of the pangolin-derived hookworm DNA in the sample to be tested that is positive for the pangolin-derived hookworm.

10. The method of claim 9, wherein, The system of the probe-based fluorescence quantitative PCR amplification is 2x probe-based qPCR universal premix, 0.2 μL of forward primer with a concentration of 10 μM, 0.2 μL of reverse primer mixture with a concentration of 10 μM, 0.4 μL of TaqMan probe with a concentration of 10 m M, 1 μL of DNA template, and 3.2 μL of water; The program of the probe-based fluorescence quantitative PCR amplification is pre-denaturation at 95℃ for 30 s, denaturation at 95℃ for 5 s, annealing at 60℃ for 30 s, and 40 cycles.

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