Real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection method for delvorii paragonimus

Abstract

The invention relates to the technical field of molecular biology, in particular to a real-time fluorescent quantitative PCR (polymerase chain reaction) detection method for delvorei paragonimus, aiming at the problems of low sensitivity of traditional microscopic examination and difficulty in distinguishing sibling species in morphology, a specific primer pair of PD-F / PD-R and MH5F / MH5R targeted ITS genes is designed, and a recombinant plasmid standard substance pMD18-T-ITS-1 is constructed; establishing an absolute quantification system based on copy number calculation; the lowest detection limit reaches a single digit copy level, the sensitivity is greatly improved compared with that of common PCR, and cross reaction with six common parasites is avoided; an intra-group / inter-group repeatability variation coefficient lt; and 3.5%. The assorted kit contains the primers, a premix solution and a standard substance, can accurately detect the insect body load in snails and host samples, and provides technical support for rapid identification and prevention and control of Devorii pseudolariagonia.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a real-time fluorescence quantitative PCR detection method for Pseudohypogonimus devorei. Background Art

[0002] Pseudocatatropis dvoryadkini, also known as Pseudocatatropis dvoryadkini, was first discovered in the southern region of the Russian Far East by Dvoryadkin in 1989. It belongs to the phylum Platyhelminthes, class Trematoda, order Echinostomida, family Notocotylidea, and genus Genus Catatropis. Pseudocatatropis vietnamese is the first trematode in the genus Genus Catatropis, using snails as its primary host. Previously, the only known trematode in the family Notocotylidea was Notocotylus magniovatus, whose life cycle is mediated by the mollusk Ceritioidea. The life history of other Hypogonid trematodes has been studied using the gill-bearing snails Bithyniidae Gray and Hydrobiidae Stimpson, as well as the lung-bearing planulae of the family Rafinesque and Chillinidae Dall, as first intermediate hosts. Among most Hypogonid trematodes, only N. indicus, C. pricei, C. harwoodi, C. chinensis, C. misrai, C. poecyclorhynchai, and C. pakistanensis have a genital pore located anterior to the intestinal bifurcation. Based on the number of papillae per side, Hypogonid vietnamensis closely resembles C. harwoodi (7–9) and C. pakistanensisi (9–10), but Hypogonid vietnamensis differs from these two species in other structures and life history. Morphological findings at different developmental stages and genetic evolutionary analysis indicate that C. vietnamensis is a new species of Hypogonid trematode. In terms of evolutionary relationship, P. dvoryadkini is closely related to Notocotylusmalhamensis.

[0003] The first intermediate host of Pseudohypogonimus devorei is the mollusk Helicorbis sujfunensis. It primarily parasitizes its digestive glands and, under laboratory conditions, can infect Anas platyrhynchadom, primarily in the intestines. Morphologically, Pseudohypogonimus devorei is very similar to Pseudocatatropis joyeuxi, with similar morphological characteristics of cercariae, metacercariae, and adults. Pseudocatatropis joyeuxi is found in Europe and primarily uses three species of snails—Anisus leucostomus, Gyraulus albus, and Segmentina nitida—as intermediate hosts in its life cycle. Both trematodes have short, eyeless, and unpigmented cercariae throughout their life cycles and do not leave their intermediate hosts. Although the trematodes appear similar at different developmental stages, geographical variations in the hosts involved in their life cycles result in distinct geographic strains. The developmental process of Pseudohypogonid flukes of Devori consists of the adult, redia, cercariae, and metacercariae stages. Adults have a flat, elongated body with a concave abdomen, tapered anteriorly and rounded posteriorly. The central surface of the anterior half of the body is covered with scaly spines. The central papillae coalesce into a continuous ridge that extends from the internal seminal vesicle to the posterior end of the ovary. Six to seven papillae are present on each side. The oral sucker is proximal, the esophagus is short, and the cecum extends laterally to the uterus. The testes are symmetrically located at the posterior end of the body. The genital pore is located medianally, just posterior to the intestinal bifurcation, and the excretory opening is vesicular. Rediae are slender, yellow-brown in color, measuring 1.232–1.309 mm in length by 0.200–0.308 mm. Cercariae are slender, oval, and dotted with gray pigment spots, measuring 0.231–0.258 mm in length by 0.104–0.116 mm. The body is filled with numerous glands. The oral sucker measures 0.035–0.042 mm x 0.039–0.046 mm, and the excretory opening is vesicular. The tail is short, with a diameter of only 0.02–0.03 mm. Metacercariae are transparent, spherical cysts with a diameter of 0.119–0.135 mm and a wall thickness of 0.008–0.019 mm.

[0004] Since its discovery in 1989, few studies have been reported on Pseudomonas devorei, both domestically and internationally. Further research is needed to determine whether it is a zoonotic parasite or poses a zoonotic risk. Currently, detection methods for Pseudomonas devorei rely primarily on microscopic observation of larvae to determine infection. This method has disadvantages such as low sensitivity and a high risk of missed detection. Furthermore, Pseudomonas devorei shares similar morphological structures with other trematodes during the larval stage, making it difficult to distinguish based on morphology alone. Existing detection methods lack high sensitivity or specificity, and there are no reports on the use of molecular biology techniques for its detection. Therefore, establishing a highly sensitive and specific detection and identification method is of great value for the detection of Pseudomonas devorei. Summary of the Invention

[0005] The purpose of the present invention is to provide a real-time fluorescence quantitative PCR detection method for Pseudomonas devoirii. By designing specific primers targeting the ITS gene and combining it with recombinant plasmid standards for absolute quantification, highly sensitive and specific detection is achieved. The sensitivity is increased by 1000 times compared with conventional PCR, solving the problems of missed detection and misjudgment based on morphology.

[0006] In order to achieve the above technical objectives and the above technical effects, the present invention is implemented through the following technical solutions: A real-time fluorescence quantitative PCR detection method for Pseudomonas devoirii comprises the following steps: S1: Select primer pair MH5F / MH5R; S2: Preparation of standard: After extracting Pseudomonas devoirii DNA, PCR amplification was performed based on primers PD-F / PD-R, and the gel-recovered product was ligated to pMD TM 18-T vector and transformed into DH5α competent cells, and the recombinant plasmid was extracted and the concentration was determined by UV nucleic acid and protein analyzer; S3: Set up the reaction system: Each reaction contains 10 μL of 2× Super Real Pre Mix Plus, 0.6 μL of forward primer, 0.6 μL of reverse primer, 2 μL of template DNA, and 6.8 μL of ddH2O; S4: Perform the following reaction program: incubate at 95°C for 15 minutes, followed by 40 cycles of denaturation at 95°C for 10 seconds and annealing / extension at 60–66°C for 30 seconds.

[0007] Furthermore, the sequences of the primer pair PD-F / PD-R are shown in SEQ ID NO. 1 and 2. Furthermore, the sequences of the primer pair MH5F / MH5R are shown in SEQ ID NO. 3 and 4.

[0008] Furthermore, the positive plasmids that were sequenced correctly were used as standards, and their concentration and purity were determined using an ultraviolet nucleic acid and protein analyzer. The DNA copy number was calculated according to the formula: copy number (copy / µL) = Atschoff constant × plasmid concentration (ng / µL × 10 9 ) / DNA length (bp)×660, where the Attenborough constant is 6.02×10 23 The standard was diluted 10-fold with RNase-free water, and the solution was pipetted several times to mix the solution thoroughly. The samples were diluted 10 times and stored at -20℃ for later use.

[0009] Furthermore, in the standard copy number calculation formula, the DNA length is fixed at 595 bp, and during gradient dilution, 10 μL of plasmid solution is added to 90 μL of ddH2O and mixed.

[0010] In another aspect, the present invention provides a detection kit comprising: The nucleotide sequences of the primer pair (MH5F / MH5R) defined above are shown in Table 4 ; 2×Super Real Pre Mix Plus premix; A recombinant plasmid standard containing the ITS gene of Pseudomonas devoiryi.

[0011] Beneficial effects of the present invention: Since its discovery in 1989, few studies have been reported on Pseudomonas devorei. Whether it is a zoonotic parasite or poses a zoonotic risk requires further investigation. Currently, detection methods for Pseudomonas devorei primarily rely on microscopic observation of larvae to determine infection, a method with drawbacks such as low sensitivity and a high risk of missed detections. Furthermore, traditional methods rely on morphological features such as the location of the genital pore and the number of papillae for identification. However, Pseudomonas devorei larvae are highly similar in morphology to closely related species, making identification difficult. Existing detection methods lack high sensitivity and specificity, and there are no reports on the application of molecular biology techniques to its detection. Therefore, developing new, highly sensitive and specific detection methods is of great significance for the detection and identification of Pseudomonas devorei. This study targets the ribosomal ITS gene, which exhibits significant sequence variation among species but is highly conserved within them. Specific primers (MH5F / MH5R) are designed and combined with a dual validation mechanism of conventional PCR and real-time fluorescence quantitative PCR to ensure detection specificity. The 126 bp fragment amplified by primers MH5F / MH5R showed a single sharp peak in the melting curve and had no cross-reaction with six common trematodes / tapeworms, confirming that it can accurately distinguish Pseudomonas devoirii.

[0012] This method utilizes real-time fluorescence quantitative PCR technology, dynamically monitoring changes in fluorescence signals during amplification, combined with short-segment primer design and premix optimization to significantly enhance detection sensitivity. The method's ability to capture fluorescence signals during the exponential amplification phase enables detection of single-digit copy levels of templates. Gradient dilution experiments confirm that its minimum detection limit is three orders of magnitude lower than that of conventional PCR, effectively eliminating the problem of missed detection of trace amounts of parasite nucleic acid in snail or host intestinal samples.

[0013] The present invention constructs a recombinant plasmid standard containing the ITS gene, pMD18-T-ITS-1. Ultraviolet spectrophotometry is used to precisely determine plasmid concentration, and a copy number calculation formula is derived based on Avogadro's constant, achieving absolute quantification. After tenfold gradient dilution of the standard, the amplification curve exhibits excellent linearity, covering a concentration range of nine orders of magnitude. Repeated experiments within and between groups show a coefficient of variation below the critical value, demonstrating its stability and reliability. The Ct value can be directly converted to parasite nucleic acid copy number, providing a quantitative basis for investigating infection rates in intermediate hosts of snails and studying parasitic load in definitive hosts, with applications in transmission dynamics risk warning and public health safety monitoring.

[0014] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 Schematic diagram of the PCR amplification results of the primer pair PD-F / PD-R of Pseudomonas devoirii; M: DL2000 DNA Marker; 1-7: test samples; -: negative control; Figure 2 Schematic diagram of the phylogenetic tree based on the ITS gene sequence of Pseudomonas devorii. Note: ▲ in the figure represents the Pseudomonas devorii sequence discovered in this study. Figure 3 Schematic diagram of gel recovery results of amplification of the ITS gene by the primer pair PD-F / PD-R of Pseudomonas devoiryi; M: DL2000 DNA Marker; 1-3: samples; -: negative control; Figure 4 Schematic diagram of PCR identification of the recombinant plasmid using the PD-F / PD-R primer pair; M: DL2000 DNA Marker; 1-3: recombinant plasmid; —: negative control; Figure 5 Schematic diagram of the validation results of real-time fluorescence quantitative PCR primers MH4F / MH4R and MH5F / MH5R; M: DL2000 DNA Marker; 1-2: MH4F / MH4R test results of negative samples; 3-4: MH5F / MH5R test results of negative samples; 5-6: MH4F / MH4R test results of Pseudomonas devorei positive samples; 7-8: MH5F / MH5R test results of Pseudomonas devorei positive samples; Figure 6 Schematic diagram of amplification and melting curves of primers MH4F / MH4R and MH5F / MH5R; Figure 7 Schematic diagram of the sensitivity test results of conventional PCR using MH5F / MH5R primers; M: DL2000 DNA Marker; 1-10: 9.74×10 9 copies / µL, 9.74×10 8 copies / µL, 9.74×10 7 copies / µL, 9.74×10 6 copies / µL, 9.74×10 5 copies / µL, 9.74×10 4 copies / µL, 9.74×10 3 copies / µL, 9.74×10 2 copies / µL, 9.74×10 1 copies / µL, 9.74×10 0 Recombinant plasmid copies / µL; —: negative control; Figure 8 Schematic diagram of the specific amplification curve of real-time fluorescence quantitative PCR with primers MH5F / MH5R; 1; 9.74×10 6 Copies / µL standard sample; 2-7: Anterior and posterior trematodes, Anterior and posterior disc flukes, Leaf flukes, Fasciola zingiberensis, Schistosoma japonicum, and Taenia multiceps; 8: negative control; Figure 9 Schematic diagram of the specificity test results of common PCR with MH5F / MH5R primers; 1:9.74×10 6 Copies / µL standard sample; 2-7: Anterior and posterior trematodes, Anterior and posterior disc flukes, Leaf flukes, Fasciola zingiberensis, Schistosoma japonicum, and Taenia multiceps; 8: negative control. DETAILED DESCRIPTION

[0017] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with 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 making any creative efforts shall fall within the scope of protection of the present invention.

[0018] Example 1 The real-time fluorescence quantitative PCR method for detecting Pseudomonas devoirii described in this embodiment comprises the following steps: S1: Select primer pair MH5F / MH5R; S2: Preparation of standard: After extracting Pseudomonas devoirii DNA, PCR amplification was performed based on primers PD-F / PD-R, and the gel-recovered product was ligated to pMD TM 18-T vector and transformed into DH5α competent cells, and the recombinant plasmid was extracted and the concentration was determined by UV nucleic acid and protein analyzer; S3: Set up the reaction system: Each reaction contains 10 μL of 2× Super Real Pre Mix Plus, 0.6 μL of forward primer, 0.6 μL of reverse primer, 2 μL of template DNA, and 6.8 μL of ddH2O; S4: Perform the following reaction program: incubate at 95°C for 15 minutes, followed by 40 cycles of denaturation at 95°C for 10 seconds and annealing / extension at 60–66°C for 30 seconds.

[0019] In this embodiment, the PD-F sequence and PD-R sequence are shown in Table 1.

[0020] In this embodiment, the MH5F sequence and MH5R sequence are shown in Table 4.

[0021] In this example, the positive plasmid that was sequenced correctly was used as a standard. The concentration and purity of the standard were determined using an ultraviolet nucleic acid and protein analyzer. The DNA copy number was calculated according to the formula: Copy number (copy / µL) = Atschoff constant × plasmid concentration (ng / µL × 10 9 ) / DNA length (bp)×660, where the Attenborough constant is 6.02×10 23 The standard was diluted 10-fold with RNase-free water, and the solution was pipetted several times to mix the solution thoroughly. The samples were diluted 10 times and stored at -20℃ for later use.

[0022] In this example, in the standard copy number calculation formula, the DNA length is fixed at 595 bp, and during gradient dilution, 10 μL of plasmid solution is added to 90 μL of ddH2O and mixed.

[0023] In another aspect, the present invention provides a detection kit comprising: The nucleotide sequences of the primer pair MH5F / MH5R defined above are shown in Table 4 ; 2×Super Real Pre Mix Plus premix; A recombinant plasmid standard containing the ITS gene of Pseudomonas devoiryi.

[0024] Example 2 In this example, PCR was used to amplify the ITS gene of Pseudohyponigma devoryii.

[0025] DNA extraction Snail tissue DNA was extracted according to the instructions of the blood / cell / tissue genomic DNA extraction kit produced by Beijing Tiangen Biochemical Technology Co., Ltd. The detailed steps are as follows: (1) Sample processing: Take approximately 50 mg of snail muscle tissue or golden apple snail lung sac tissue, mince it and place it in a 2 mL sterile centrifuge tube. Add 200 μL of buffer GA and 20 μL of proteinase K, and vortex to mix thoroughly. Place the centrifuge tube containing the tissue in a 56°C water bath for overnight digestion, inverting the tube several times until the tissue sample is completely digested. (2) Add 200 μL of buffer GB, vortex to mix thoroughly, place in a 70°C water bath for 10 minutes, and centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0026] (3) Add 200 μL of anhydrous ethanol and vortex to mix for 15 seconds. Flocculent precipitation may appear at this time. Centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0027] (4) First place the adsorption column CB3 into the collection tube, then transfer the solution and flocculent precipitate obtained in step (3) into the adsorption column, centrifuge at 12000 rpm for 45 seconds, and discard the waste liquid in the collection tube.

[0028] (5) Add 500 μL of buffer GD (add anhydrous ethanol before use) to the adsorption column, centrifuge at 12,000 rpm for 45 seconds, and discard the waste liquid in the collection tube.

[0029] (6) Add 600 μL of rinse solution PW (add anhydrous ethanol before use) to the adsorption column CB3, centrifuge at 12,000 rpm for 30 s, and discard the waste liquid in the collection tube.

[0030] (7) Repeat step (6).

[0031] (8) Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 min, discard the waste liquid, and place the adsorption column CB3 at room temperature for 3 to 5 min to allow the residual rinse liquid in the adsorption column to evaporate and dry.

[0032] (9) Transfer the adsorption column CB3 to a clean centrifuge tube and drop 50 μL of elution buffer TE (preheated) onto the middle of the adsorption membrane. Allow to stand at room temperature for 2–5 min. Centrifuge at 12,000 rpm for 2 min. Collect the solution into a centrifuge tube and repeat the elution twice. Finally, collect 100 μL of DNA solution and store it at -20°C for later use.

[0033] PCR amplification primers and amplification conditions The PCR amplification primers for Pseudomonas devorei are shown in Table 1 , and the PCR reaction system and reaction procedure for Pseudomonas devorei are shown in Table 2 .

[0034] Table 1 PCR amplification primers for Pseudogyna devoryii Table 2 PCR reaction system and procedure for Pseudomonas devoirii Agarose gel electrophoresis and sequencing of positive products 5 µL of the PCR product of Pseudohypogonimus devoirii was loaded onto a well of a 1.5% agarose gel. Using a DL2000 marker, the electrophoresis was performed at 120 V for 30 minutes. After electrophoresis, the gel was removed and placed on a UV gel imaging analyzer for observation and photography. PCR products close to the target fragment were sent to Shanghai Sangon Biotechnology for bidirectional sequencing.

[0035] Sequence alignment to identify insect species and genetic evolution analysis (1) Download the sequencing results and splice them using the SeqMan software in the DNA sequence analysis software DNAStar, or directly download the splicing results and perform online comparison using the NCBI database Genbank-Blast to determine the insect species and similarity.

[0036] (2) The reference sequences of insect species were retrieved from the NCBI database, the reference sequences were downloaded and sorted, and the phylogenetic tree was constructed using the neighbor-joining method (NJ) in the MAGEX software. The reliability analysis of the phylogenetic tree was estimated by using the bootstrap test and repeated 1000 times. The analysis was considered significant if it was greater than 95%.

[0037] Data statistics and analysis SPSS20.0 and SAS9.1 software were used to calculate P value, odds ratio (OR) and 95% confidence interval (95% CI) to analyze the influence of different sampling time, location, species, sampling environment and other factors on the infection rate of freshwater snails. When P < 0.05, the difference was significant, when P < 0.01, the difference was extremely significant, which had statistical significance. OR value: OR > 1, indicating that the factor was a risk factor; OR < 1, indicating that the factor was a protective factor; 95% CI: lower limit greater than 1, indicating that the factor was a risk factor, and upper limit less than 1, indicating that the factor was a protective factor.

[0038] Preparation of plasmid standard Extraction of DNA and amplification of target fragment Referring to the above extraction steps and Table 2, a gel containing the target fragment was obtained.

[0039] Gel recovery and verification of target band The gel containing the target fragment was quickly cut off with a clean scalpel in the ultraviolet transmission instrument, the gel block was weighed, and the DP219 type agarose gel DNA recovery kit was used to recover and purify the PCR product. The specific steps were operated according to the kit instruction manual. After the PCR product was purified, 5 μL of the purified product was used for agarose gel electrophoresis to verify the recovery effect.

[0040] Connection of pMD18-T vector and gel recovery product According to the operation steps of the cloning vector instruction manual, the components in Table 3 were added to the sterilized 1.5 mL PCR tube, gently mixed, connected at 16°C overnight, and stored at 4°C for standby.

[0041] Table 3 Connection reaction system Transformation of connection product The connection product was transformed according to the following steps: (1) The DH5α competent cells were ice-bathed for 30 min, and then 10 μL of the connection product was added to 100 μL of the DH5α competent cells, which were gently mixed with a pipette gun, and ice-bathed for 30 min.

[0042] (2) The centrifuge tube was placed in a 42°C water bath pot and heat shocked for 90 s.

[0043] (3) After heat shock, the centrifuge tube was quickly transferred to the ice box and water bathed for 3 min~5 min.

[0044] (4) In the clean bench, 900 μL of LB (without Amp ＋) culture medium was added to the centrifuge tube and cultured in a 37°C water bath constant temperature shaker (150 rpm / min) for 1 h.

[0045] (5) Centrifuge the bacterial solution at 4000 rpm / min for 2 min to allow the bacteria to settle to the bottom of the centrifuge tube. Discard part of the supernatant and mix the remaining supernatant with the bacteria. Take 100 μL of the bacterial solution and spread it evenly on the plate containing 100 mg / mL Amp. ＋ The cells were plated on LB agar plates and incubated upside down in a 37°C incubator for 12 h to 16 h.

[0046] (6) Pick a single colony with a smooth surface and inoculate it into 5 mL of LB medium (containing Amp+). Place it in a 37°C water bath in a constant temperature shaker (140 rpm / min) and culture for 12 h to 16 h. When the OD value reaches about 1.0, remove the bacterial solution and extract the plasmid.

[0047] Extraction and identification of recombinant plasmids Plasmids were extracted according to the Tiangen High-Purity Plasmid Extraction Kit instructions. PCR amplification was performed using the extracted plasmid as a template (conditions refer to Table 2). 5 µL of the amplified product was subjected to agarose gel electrophoresis. The PCR product was sent to Shanghai Sangon Biotechnology for bidirectional sequencing. Sequencing results were verified by online comparison with the NCBI database Genbank-Blast.

[0048] Recombinant plasmid concentration determination and copy number calculation The positive plasmids that were sequenced correctly were used as standards. The concentration and purity of the plasmids were determined using an ultraviolet nucleic acid and protein analyzer. The DNA copy number was calculated according to the formula: copy number (copy / µL) = Atschoff constant × plasmid concentration (ng / µL × 10 9 ) / DNA length (bp)×660, where the Attenborough constant is 6.02×10 23 Dilute the standard sample with RNase-free water in a 10-fold gradient (take 10 µL of plasmid and add it to 90 µL of ddH2O). Pipette several times to mix the solution thoroughly. Dilute the sample in 10 gradients and store at -20℃ until ready for use.

[0049] Establishment of real-time fluorescence quantitative PCR method Design of primers for real-time fluorescence quantitative PCR Two pairs of primers specific for the ITS gene of Pseudomonas devoirii were designed using Primer 5.0 software, referring to the relevant gene sequences published in GenBank: MH4F / MH4R and MH5F / MH5R. The primer sequences are shown in Table 4. The primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0050] Table 4 Real-time fluorescent quantitative PCR amplification primers PCR amplification results of D. dvoryakini In this example, 4502 freshwater snails were collected from Kunming, Yuxi, Dali, Baoshan, Dehong, Lincang, Pu'er and Xishuangbanna in Yunnan Province, respectively. D. dvoryakini was identified by PCR amplification of ITS gene. Eight samples were amplified as D. dvoryakini positive at the ITS gene site, with an overall infection rate of 0.178% (8 / 4502). The target fragment was about 595 bp (see Figure 1 ).

[0051] D. dvoryakini species identification and phylogenetic tree construction In this example, sequence analysis was performed based on ITS gene. The eight positive samples were sent to Shanghai Shengong Bioengineering Co., Ltd. for sequencing. The sequencing results were compared and analyzed with the GenBank database. The sequence similarity of the eight positive samples with the gene sequences (P. dvoryadkini) with accession numbers MH750022.1, MH750023.1 and MH750024.1 was 98.83%~98.98%.

[0052] The sequences of the eight positive samples amplified based on ITS gene were uploaded to the Genbank database, and the gene accession numbers were OM022103, OM022104, OM022105, OM022106, OM022107, OM022108, OM022109 and OM022110, respectively. The reference sequences of the species were retrieved from the NCBI database, and the nucleotide sequences with the highest homology were downloaded and sorted. The genetic development phylogenetic tree was constructed using the Neighbor-joining method (NJ) in MEGAX, and the Bootstrap method was used for verification with a repetition number of 1000. The results showed that the eight D. dvoryakini strains identified based on ITS gene in this example were clustered into the same branch with the Russian strains, part of the Paramphistomum strains and the Amphistomum isolated strains, indicating that the isolated strains obtained in this example were relatively close in genetic relationship (see Figure 2 ).

[0053] Preparation results of plasmid standard Amplification and gel recovery of target fragments After the ITS gene fragments were recovered by agarose gel DNA recovery kit, 1.5% agarose gel electrophoresis was performed, and the results were observed under the ultraviolet gel imager. A specific band appeared at 500 bp~750 bp with DL2000 Maker as the standard (see Figure 3), which was consistent with the expected size, and was preliminarily determined as an ITS gene fragment.

[0054] Construction and identification of recombinant plasmid The ITS gene fragment was cloned into the pMD18-T vector to construct a recombinant plasmid, which was identified by PCR. The target band with a length of 595 bp was successfully amplified, which was similar to the expected fragment. The recombinant plasmid was named pMD18-T-ITS-1. The results showed that the sequencing results of pMD18-T-ITS-1 had 98.82% homology with the gene sequences of MH750022.1, MH750023.1, and MH750024.1 (P. dvoryadkini) published on Genbank. The recombinant plasmid was identified by sequencing, and the target gene fragment was correctly inserted into the recombinant plasmid (see Figure 4 ).

[0055] Determination of recombinant plasmid concentration and calculation of copy number The concentration and purity of the extracted positive plasmid were determined by ultraviolet nucleic acid protein analyzer. The concentration of the plasmid standard was 66 ng / µL, and the OD 260 / OD 280 was 1.94, which was suitable for the purity. The concentration of the plasmid standard was brought into the copy number calculation formula: copy number (copies / µL) = Avogadro's constant × plasmid concentration (ng / µL × 10 9 ) / DNA length (bp) × 660, and the copy number of the plasmid standard was 9.74 × 10 10 copies / µL.

[0056] Verification of real-time fluorescent quantitative PCR primers by ordinary PCR The designed real-time fluorescent quantitative PCR primers were verified by ordinary PCR and real-time fluorescent quantitative PCR using the plasmid standard as the template. The results showed that in ordinary PCR, only primers MH4F / MH4R and MH5F / MH5R amplified the target band (2 repeated holes) (see Figure 5 ). Then, primers MH4F / MH4R and MH5F / MH5R were amplified by real-time fluorescent quantitative PCR, and the amplification curve and melting curve showed that the two primers could specifically amplify, and the melting curve peak was single and sharp (see Figure 6 ). However, under the condition of MH5F / MH5R primers, the amplification reaction of the plasmid standard could show relatively small cycle number (Ct value) and relatively high fluorescence signal value. Therefore, primer MH5F / MH5R was selected as the subsequent real-time fluorescent quantitative PCR amplification primer.

[0057] Sensitivity test of real-time fluorescent quantitative PCR The plasmid standard pMD18-T-ITS-2 was diluted by 10 times gradient, and the concentration was 9.74 × 109 copies / µL to 9.74 x 10 0 copies / µL as standard, the ordinary PCR amplification results showed that: the standard concentration of 9.74 x 10 3 copies / µL, there has been a fuzzy band (see Figure 7 ); real-time fluorescent quantitative PCR amplification results showed that: the standard concentration of 9.74 x 10 0 copies / µL (Ct value of 32.67), there is still amplification curve, indicating that the minimum detection concentration of this experiment is 9.74 x 10 0 copies / µL. Comparison of the two methods, the sensitivity of the real-time fluorescent quantitative method established this time is 1000 times of ordinary PCR (see Table 5).

[0058] Table 5 Comparison of the sensitivity of ordinary PCR and real-time fluorescent quantitative PCR Specificity test of real-time fluorescent quantitative PCR Japanese blood flukes, leaf flukes, front and rear flukes, front and rear flukes, ginger flukes and multi-headed tape worms were used as control species for fluorescent quantitative PCR specificity test, and the concentration of 10 6 copies / µL plasmid standard as positive template to verify its specificity. The results showed that the Ct value of the control sample was less than 32.67 or no specific amplification appeared (see Figure 8 ), (see Table 6 for specificity test results), ordinary PCR amplification results showed that the plasmid standard amplified a bright single band (see Figure 9 ), indicating that the method has certain specificity.

[0059] Table 6 Specificity test results Repeatability test of fluorescent quantitative PCR 9.74 x 10 9 copies / µL~9.74 x 10 6 copies / µL of the standard for inter-group repeated experiments and intra-group repeated experiments. The detected Ct value and coefficient of variation (CV%) results are shown in Table 7 and Table 8, the coefficient of variation of intra-group and inter-group is less than 3.5%, indicating that the fluorescent quantitative PCR detection method established in this embodiment has good repeatability and stability.

[0060] Table 7 Results of inter-group repeatability test Table 8 Results of intra-group repeatability test The present invention has established a fluorescence quantitative PCR detection method with high sensitivity, strong specificity and good repeatability, and has been used to detect Pseudohyponigma devoirii. 6 When the standard plasmid with 10 copies / µL was used as template, the optimal final concentration of the primers was 8 µM, and the optimal annealing temperature was 60.0°C, the real-time fluorescence quantitative PCR amplification curve showed a small Ct value and a small standard deviation of the Ct value. The melting curve had only a single peak, and the negative curve had no peak. The plasmid standard pMD18-T-ITS-1 was diluted 10-fold in series, and at 9.74×10 9 copies / µL-9.74×10 2 The standard curve showed good linearity at a concentration of 100 copies / µL. Its amplification efficiency was 92.3%, the correlation coefficient was 0.987, the slope was -3.522, the intercept was 40.883, and the linear relationship expression was y = -3.522x + 40.883. The real-time fluorescence quantitative method established this time (with a minimum detection limit of 9.74×10 0 copies / µL), is a common PCR (the minimum detection limit is 9.74×10 3 The specificity was 1000 times higher than that of Schistosoma japonicum, Schistosoma japonicum, Schistosoma japonicum, Schistosoma japonicum, Fasciola zingiberensis, and Taenia multiceps. 9 copies / µL~9.74×10 6 The experiments were repeated within and between groups using four different concentrations of standard samples with different copies / µL. The results showed that the coefficient of variation of the Ct values ​​between groups did not exceed 1.03%, and the coefficient of variation of the Ct values ​​within the group did not exceed 3.36%. The above data show that the real-time fluorescence quantitative PCR method established by the present invention has high amplification efficiency, good sensitivity, specificity, repeatability and stability.

[0061] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the present invention to the specific embodiments described. Obviously, many modifications and variations are possible based on the content of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A real-time fluorescence quantitative PCR detection method for Pseudomonas devorei, characterized in that: The following steps are involved: S1: selected primer pair MH5F / MH5R; S2: Preparation of standard: After extracting Pseudohypogonimus devoirii DNA, PCR amplification of PD-F / PD-R was performed based on primers, characterized in that: PD-F / PD-R sequences are as shown in SEQ ID NO. 1 and 2, and the gel-recovered product was ligated to pMD TM 18-T vector and transformed into DH5α competent cells, and the recombinant plasmid was extracted and the concentration was determined by UV nucleic acid and protein analyzer; S3: Set up the reaction system: Each reaction contains 10 μL of 2× Super Real Pre Mix Plus, 0.6 μL of forward primer, 0.6 μL of reverse primer, 2 μL of template DNA, and 6.8 μL of ddH2O; S4: Perform the following reaction program: incubate at 95°C for 15 minutes, followed by 40 cycles of denaturation at 95°C for 10 seconds and annealing / extension at 60–66°C for 30 seconds.

2. The real-time fluorescence quantitative PCR detection method for Pseudomonas aeruginosa according to claim 1, wherein: The sequences of MH5F / MH5R are shown in SEQ ID NOs. 3 and 4.

3. The real-time fluorescence quantitative PCR detection method for Pseudomonas aeruginosa according to claim 1, wherein: The positive plasmids that were sequenced correctly were used as standards. The concentration and purity were determined using an ultraviolet nucleic acid and protein analyzer. The DNA copy number was calculated according to the formula: copy number = Attia constant × plasmid concentration / DNA length × 660, where Attia constant is 6.02 × 10 23 ; Dilute the standard sample with RNase-free water in a 10-fold gradient, pipette several times to mix the solution thoroughly, dilute 10 gradient samples, and store at -20℃ for later use.

4. The real-time fluorescence quantitative PCR detection method for Pseudomonas devorei according to claim 1, wherein: In the standard copy number calculation formula, the DNA length is fixed at 595 bp. During the gradient dilution, 10 μL of plasmid solution is added to 90 μL of ddH2O and mixed.

5. A detection kit, characterized in that: include: The primer pair as defined in any one of claims 1 to 4: MH5F / MH5R; 2×Super Real Pre Mix Plus premix; A recombinant plasmid standard containing the ITS gene of Pseudomonas devoiryi.

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