CN112176077A - Dual PCR detection method for clonorchis sinensis and accessory didymis orientalis cysticercus infection - Google Patents
Dual PCR detection method for clonorchis sinensis and accessory didymis orientalis cysticercus infection Download PDFInfo
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Abstract
The invention provides a double PCR detection method for clonorchis sinensis and oriental secondary testicular cyst infection, a double PCR detection method for simultaneously detecting freshwater fish and shrimp clonorchis sinensis and oriental secondary testicular cyst infection, and a preparation method and a use method of the kit.
Description
Technical Field
The invention provides a double PCR detection method for clonorchis sinensis and accessory didymis orientalis cysticercosis infection, a double PCR detection method for simultaneously detecting clonorchis sinensis and accessory didymis orientalis cysticercosis infection of freshwater fish and shrimp, and provides a preparation method and a use method of the kit, belonging to the technical field of parasite detection.
Background
Clonorchis sinensis is a food-borne parasitic disease of both human and animal caused by clonorchis sinensis parasitizing in the hepatobiliary tract of human and mammal, mainly caused by hepatobiliary disease. Clonorchis sinensis mainly causes hepatobiliary diseases and surrounding inflammations, and can cause cirrhosis, ascites and even canceration in severe cases. The disease is brought into one of 'overlooked tropical diseases' by the World Health Organization (WHO), is distributed worldwide, is infected by about 3500 or more than ten thousand people worldwide, and is mainly popular in regions with rich fresh water resources. The life history of clonorchis sinensis has two intermediate hosts, namely freshwater snail and freshwater fish and shrimp, and there are over 139 kinds of freshwater fish infected with clonorchis sinensis metacercaria, among which there are 102 kinds in China.
The east schistosomiasis japonica adults are mainly parasitized in gallbladders and bile ducts of poultry such as chickens and ducks and the like, so that the gallbladders and the bile ducts are thickened, the bile ducts are blocked, bile cannot flow smoothly and other pathological changes are caused; death can occur when severe. The life history, pathogenic effect and clinical manifestation of the medicament are very similar to those of clonorchis sinensis. It has been reported that humans are infected with fresh water fish that are either eaten live or semi-live and contain metacercaria of clonorchis sinensis and metacercaria of testicles of eastern origin.
The freshwater fish and shrimp are the common intermediate host of clonorchis sinensis and epididymis orientalis. The prevalence areas of both flukes overlap and often cause mixed infections in endemic areas. In addition, the morphological structure of the soft-shelled turtle is similar, and false detection is easy to cause, so that the infection conditions of the freshwater fish of the clonorchis sinensis and the oriental secondary testicular larva cysticercosis are simultaneously detected and identified, and the soft-shelled turtle worm has important significance for preventing and controlling clonorchiasis sinensis and oriental secondary testicular larva.
At present, the detection method of clonorchis sinensis and oriental secondary testicular metacercaria mainly comprises microscopic examination. The microscopic examination is widely applied, mainly comprises a direct pressing microscopic examination method and a pepsin digestive microscopic examination method, but the forms of the two methods are similar, the microscopic examination has higher requirements on the experience of detection personnel, and the method has the defects of missed detection, false detection and the like; the double PCR detection method has the advantages of high efficiency, specificity, simplicity and the like, can simultaneously detect the infection condition of the clonorchis sinensis and the cysticercus of the secondary clonorchis orientalis in the same sample, greatly improves the efficiency of detecting the sample, and plays an increasingly important role in epidemiological investigation. Therefore, it is urgently needed to develop a dual PCR detection kit for Clonorchis sinensis and Leydig sinensis metacercaria with good specificity and high sensitivity.
Disclosure of Invention
The invention provides a dual PCR detection method for clonorchis sinensis and accessory didymis orientalis cysticercosis infection, which is used for detecting clonorchis sinensis and accessory didymis orientalis cysticercosis.
The invention relates to a primer sequence designed according to an ITS1 gene of clonorchis sinensis and an NADH dehydrogenase subunit 2 gene of the oriental epididymis.
The invention also provides a preparation method of the clonorchis sinensis and the oriental metacercaria double PCR detection kit, which comprises the following steps:
(1) PCR reaction solution: 2.5mM dNTPs, 10 pmol/. mu.L final concentration of primer Clonorchis sinensis upstream primer F1Clonorchis sinensis downstream primer R1Oriental subthrest clonorchis upstream primer F2Oriental secondary testis worm downstream primer R2Sterilization of ddH2O, PCR buffer and DNA template, adding Taq enzyme according to 2.0U of each PCR reaction (20 mu l), and not containing Taq enzyme in the reaction solution;
(2) dual PCR primers
The specific primer of clonorchis sinensis: f1: ACA GTA CAC AAA GCC CAA ACA CCT C, respectively;
R1：TAT CAG TCG TAC CCG GGA TAA GGC；
specific primer of east secondary testis worm F2: TGG TTG TAT GGG GTG TTT CGA C;
R2:ACC AAA GAA CAG GGG AAA GAA AC；
(3) positive control: the positive control DNA template is clonorchis sinensis and oriental secondary clonorchis sinensis metacercaria genome DNA, and the PCR product is compared and whether the PCR operation process is correct or not is monitored;
(4) negative control: the template for the negative control was sterilized ddH2O, aiming at eliminating the nucleic acid pollution in the reaction solution and the false positive problem in the operation process;
the kit of the present invention may further contain Agarose (Agarose), bromophenol blue spotting buffer, Taq enzyme and ethidium bromide (E.B) 10. mu.g/. mu.l, and may further contain a PCR reaction tube.
The invention relates to a detection method of a clonorchis sinensis and accessory didymis orientalis cysticercus infection double PCR detection method, which comprises the following steps:
1) pretreatment of the sample: grinding the specimen into paste;
2) extraction of tissue genomic DNA:
extracting the genomic DNA of clonorchis sinensis and epididymis orientalis from freshwater fish by using a tissue genomic DNA extraction kit sold by a biological company;
3) and (3) PCR amplification:
(1) preparing PCR reaction tubes with equal number of samples to be detected, wherein the tubes comprise primers, Taq enzyme and sterilized ddH2O, and the like, tightly covering the cover, and uniformly mixing on a vortex device for later use;
(2) the negative and positive controls were added to the labeled tubes, respectively, and then the samples were added sequentially and labeled, and placed in the PCR instrument. The PCR conditions were: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, annealing at 56 deg.C for 30s, and extension at 72 deg.C for 45s, for 33 cycles, and extension at 72 deg.C for 7 min;
4) observation of PCR products:
agarose was weighed, mixed with the electrophoresis solution (1.0%) and heated in a microwave oven for two minutes, and E.B was added at a concentration of 0.5. mu.g/mL. And respectively adding samples in the sample adding holes after the gel is cooled, performing electrophoresis for about 20 minutes under the voltage of 120V, and observing the experimental result under a gel imaging system or an ultraviolet lamp. If the PCR product of the sample clonorchis sinensis is 618bp in an electrophoretogram, and if a band exists at 376bp of the PCR product of the east clonorchis sinensis, the sample is positive.
The invention has the positive effects that: the invention designs double PCR primers according to the partial conserved sequences of the ITS1 gene of clonorchis sinensis and the NADH dehydrogenase subunit 2 gene of the oriental epididymis. The sensitivity is improved by optimizing amplification conditions, and the result can be directly observed after the amplification product is subjected to 1.0% agarose gel electrophoresis. The detection method can be used for simultaneously and rapidly detecting the infection of the freshwater fish clonorchis sinensis and the oriental secondary testicular cyst, has the characteristics of simple operation, high sensitivity, strong specificity and the like, and is accurate and objective in result judgment.
Drawings
FIG. 1 is an optimized graph of double PCR annealing temperatures (M: DL2000 marker; 1-5: annealing temperatures in sequence 52, 54, 56, 58, 60);
FIG. 2 is a diagram showing a dual PCR specificity test (M: DL2000 marker; 1, Clonorchis sinensis and Oriental Clonorchis sinensis; 2, Clonorchis sinensis; 3, Oriental Clonorchis sinensis; 4, Fasciola hepatica; 5, neospora; 6: Cryptosporidium; 7, Eimeria tenella);
FIG. 3 is a test chart of the dual PCR sensitivity of clonorchis sinensis of the present invention (M: DL2000 marker, 1: 36ng/ul, 2: 3.6ng/ul, 3: 0.36ng/ul, 4: 0.036ng/ul, 5: 0.0036ng/ul, 6: 0.00036 ng/ul);
FIG. 4 is a diagram of the double PCR sensitivity test of the east subcorchidius sinensis of the present invention (M: DL2000 marker; 1: 61ng/ul, 2: 6.1ng/ul, 3: 0.61ng/ul, 4: 0.061ng/ul, 5: 0.0061 ng/ul).
The specific implementation mode is as follows:
the present invention is further illustrated by the following examples, which do not limit the present invention in any way, and any modifications or changes that can be easily made by a person skilled in the art to the present invention will fall within the scope of the claims of the present invention without departing from the technical solution of the present invention.
Example 1
The specific detection sequence of clonorchis sinensis and oriental secondary testicular larva: the ITS1 gene of clonorchis sinensis and the NADH dehydrogenase subunit 2 gene partial sequence of the oriental epididymis.
Example 2
Specific PCR detection primers were designed according to the sequences of clonorchis sinensis ITS1 and the NADH dehydrogenase subunit 2 gene of the accessory Clonorchis sinensis in example 1 as follows:
the specific primer of clonorchis sinensis:
F1：ACA GTA CAC AAA GCC CAA ACA CCT C；
R1：TAT CAG TCG TAC CCG GGA TAA GGC；
specific primers of east inferior testudinis:
F2:TGG TTG TAT GGG GTG TTT CGA C；
R2:ACC AAA GAA CAG GGG AAA GAA AC；
the primers can amplify specific detection gene segments in different systems, and target bands are bright and have no non-specific miscellaneous bands. The purpose of simultaneously detecting whether the same sample contains clonorchis sinensis and the oriental secondary testicular metacercaria is realized.
Example 3
The double PCR detection kit comprises the following components:
(1) PCR reaction solution: 2.5mM dNTPs, 10 pmol/. mu.L final concentration of primer Clonorchis sinensis upstream primer F1Clonorchis sinensis downstream primer R1Oriental subthrest clonorchis upstream primer F2Oriental secondary testis worm downstream primer R2Sterilization of ddH2O, PCR buffer and DNA template, 2.0U of Taq enzyme was added for each PCR reaction (20. mu.l), and the reaction solution contained no Taq enzyme.
(2) Positive control: the positive control used by the kit of the invention is clonorchis sinensis and oriental subthreshold sinensis genome DNA, and the functions of the kit are to compare PCR products and detect whether the PCR operation process is correct.
(3) Negative control: the template for the negative control was sterilized ddH2O, aiming to eliminate the nucleic acid contamination in the reaction solution and the false positive problem during the operation.
The kit of the present invention may further contain Agarose (Agarose), bromophenol blue spotting buffer, Taq enzyme and ethidium bromide (E.B) 10. mu.g/. mu.l, and may further contain a PCR reaction tube.
Example 4
Optimization of double PCR reaction conditions:
optimization of primer annealing temperature: setting the annealing temperatures of the primers as follows: 520C、540C、560C、580C、600C, results display 560C is the optimum annealing temperature (see figure 1).
Test example 1
Dual PCR specificity assay
Taking clonorchis sinensis, oriental epididymis, fasciola hepatica, neospora, cryptosporidium and eimeria tenella genomes as templates, and respectively amplifying the clonorchis sinensis, the epididymis orientalis, the fasciola hepatica, the neospora, the cryptosporidium and the eimeria tenella genomes by using the established amplification system.
The samples were added sequentially and labeled and placed in a PCR instrument. The PCR reaction conditions are as follows: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, annealing at 56 deg.C for 30s, and extension at 72 deg.C for 45s, for 33 cycles, and extension at 72 deg.C for 7 min; the amplification products were analyzed by electrophoresis in 1% agarose and the results were visualized on a gel imaging system.
The results show that: the clonorchis sinensis and the epididymis orientalis can respectively present specific bands with sizes corresponding to 618bp and 376bp, other samples have no amplification and accord with specific detection standards, and the dual PCR method has good specificity (see figure 2).
Test example 2
Sensitivity test
The extracted total nucleic acid is quantified on a nucleic acid analyzer, and the genome concentration of the clonorchis sinensis is respectively diluted to 36ng/ul, 3.6ng/ul, 0.36ng/ul, 0.036ng/ul, 0.0036ng/ul and 0.000362 ng/ul. Reaction conditions were the same as those in the specific detection example. The products were analyzed by electrophoresis in 1% agarose and the results were visualized on a gel imaging system. The result shows that the detection threshold value is 3.6pg/ul, and completely accords with the sensitivity detection standard. The detection threshold of the same east Clonorchis sinensis is 6.1pg/ul (see the attached figures 3 and 4)
Test example 3
Sample detection
161 parts of pseudorasbora parva fish flesh samples in the vinblastic region are collected, and the samples are detected by using a tabletting microscopic examination method and a double PCR detection method respectively. And performing double PCR amplification according to the system and the conditions, repeating the operation for 3 times, and detecting 16 positive clonorchis sinensis by PCR and 2 positive clonorchis sinensis by eastern times.
Test example 4
Stability and repeatability test of dual PCR detection kit
Storing the positive template, the double PCR reaction solution, the primers and the like at the temperature of-20 ℃, and storing other articles such as bromophenol blue and the like at normal temperature. The kits were removed when the storage time was 1 month, 2 months, 3 months, 5 months and 6 months, and the stability of the kits was identified with known positive samples. 8 positive samples are taken and repeatedly detected by the kit for 3 times, and meanwhile, negative control is carried out, the result shows that a specific target band exists at the position with the size of 618bp or 376bp, and the negative control has no amplification band, so that the stability and the repeatability of the kit are good.
Test example 5
Shelf life test of dual PCR detection kit
The kits stored at 4 ℃ and-20 ℃ for 1 month, 3 months, 5 months, 7 months and 9 months, respectively, were removed and tested for known positive samples. The results show that the kit can still amplify clear target bands without impurity bands when being stored for 9 months at 4 ℃ and-20 ℃.
Sequence listing
<110> Jilin university
<120> double PCR detection method for clonorchis sinensis and accessory didymis orientalis cysticercus infection
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 618
<212> DNA
<213> Clonorchis sinensis (Clonorchis sinensis)
<400> 1
acagtacaca aagcccaaac acctcagtta atctgagcat ttggcacggg tcgtcatgcc 60
cgttgttctt gcagccttgc ctgcctaggg cggagcgatt ctagttccgt catctgtctt 120
gcagcattgt ctgcctaggg cggagcgatc ctagttccgt catgttctac atgtatgttc 180
cgcatgcatg ctgcagcgtt gtctgcctac ggtggagcgt ctctagttct accaattcct 240
ggctatacct ggcacgtgta cccaatatat atgatgtgcc tacgtacagt cgcgtttcgg 300
cagggtgcct acccgtctga tgctctcggt atgctcgctt ccgttggtgg ccagtccata 360
ttgggggtga cgggatgtgc tgtcagaatg gacagtgcta ggcttaatga gtgggcatga 420
tgtgtctcga gctacggctc acccaccgcc ctgatgttgt tgttcatttc aaaccgtttt 480
acactgttaa agtgtttcag gttggcgtgg cctgactggc tggccggctt gtctcactgc 540
cccgacatgc acccggtgtt ctacactgga ctgcatgtgc agtcgcccgg cggtgcctta 600
tcccgggtac gactgata 618
<210> 2
<211> 376
<212> DNA
<213> Oriental subthrest Clonorchis (Metarchis orientalis)
<400> 2
tggttgtatg gggtgtttcg acggttttga agaggtcttt tctttttttt ggttttattt 60
tgaggggggg ttgaagtttg tttttggttg aggtttgttg tttcttgaca tttttgttta 120
ttggtttctt tttctggtta tatacttatg gttgggttta ttattggtgt catacgatga 180
ttagttcaag agcttctttt gttataatgt ctatcgagct ttctccggat ttgcttttgt 240
atgttttttt gttttatttt ttgtgggcct cattggtgat tttgttgttg agtcgtcttg 300
agggagtgag ggtgttggag tctggatgtt gtttttttct tattgctctt ttggtttctt 360
tcccctgttc tttggt 376
<210> 4
<211> 25
<212> DNA
<213> Clonorchis sinensis upstream primer (Clonorchis sinensis)
<400> 4
acagtacaca aagcccaaac acctc 25
<210> 4
<211> 23
<212> DNA
<213> Clonorchis sinensis downstream primer (Clonorchis sinensis)
<400> 4
accaaagaac aggggaaaga aac 23
<210> 5
<211> 22
<212> DNA
<213> upstream primer of Oriental subthrest fluke (Methorchis orientalis)
<400> 5
tggttgtatg gggtgtttcg ac 22
<210> 6
<211> 23
<212> DNA
<213> Oriental epididymis downstream primer (Methorchis orientalis)
<400> 6
accaaagaac aggggaaaga aac 23
Claims (2)
1. A preparation method of a clonorchis sinensis and oriental epididymis metacercaria dual PCR detection kit is characterized in that dual PCR primers are as follows:
the specific primer of clonorchis sinensis:
F1：ACA GTA CAC AAA GCC CAA ACA CCT C；
R1：TAT CAG TCG TAC CCG GGA TAA GGC；
specific primers of east inferior testudinis:
F2:TGG TTG TAT GGG GTG TTT CGA C；
R2:ACC AAA GAA CAG GGG AAA GAA AC。
2. the preparation method of the double PCR detection kit for clonorchis sinensis and accessory didymis orientalis metacercaria according to claim 1, which comprises the following steps:
(1) PCR reaction solution: 2.5mM dNTPs, 10 pmol/. mu.L final concentration of primer Clonorchis sinensis upstream primer F1Clonorchis sinensis downstream primer R1Oriental subthrest clonorchis upstream primer F2Oriental secondary testis worm downstream primer R2Sterilization of ddH2O, PCR buffer and DNA template, adding Taq enzyme according to 2.0U of each PCR reaction (20 mu l), and not containing Taq enzyme in the reaction solution;
(2) double PCR primers: is as shown in claim 1;
(3) positive control: the positive control DNA template is clonorchis sinensis and oriental secondary clonorchis sinensis metacercaria genome DNA, and the PCR product is compared and whether the PCR operation process is correct or not is monitored;
(4) negative control: the template for the negative control was sterilized ddH2O；
(5) Agarose (Agarose), bromophenol blue spotting buffer, Taq enzyme and ethidium bromide (E.B) 10. mu.g/. mu.l; and (3) a PCR reaction tube.
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| CN117568494A (en) * | 2024-01-17 | 2024-02-20 | 黑龙江八一农垦大学 | Multiple PCR (polymerase chain reaction) detection primer group, kit and detection method for zoonotic metacercaria in freshwater fish |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117568494A (en) * | 2024-01-17 | 2024-02-20 | 黑龙江八一农垦大学 | Multiple PCR (polymerase chain reaction) detection primer group, kit and detection method for zoonotic metacercaria in freshwater fish |
| CN117568494B (en) * | 2024-01-17 | 2024-03-29 | 黑龙江八一农垦大学 | Multiple PCR (polymerase chain reaction) detection primer group, kit and detection method for zoonotic metacercaria in freshwater fish |
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