CN107164479B - Primer pair combination and kit for detecting and identifying human tissue hydatid pathogen - Google Patents
Primer pair combination and kit for detecting and identifying human tissue hydatid pathogen Download PDFInfo
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Abstract
The invention discloses a multiplex PCR method capable of simultaneously detecting and identifying three human histoechinococcus granulosus types G1-G3, Echinococcus multilocularis and G6-G10. The invention designs 3 pairs of specific primers aiming at 3 pathogenic mitochondrial genes, adds the specific primers into the same PCR reaction system, complementarily combines the specific primers with target genes of corresponding insect species, generates target bands with different sizes by amplification reaction, and separates and detects the target bands by gel electrophoresis. The three pairs of primers do not interfere with each other, and do not have cross reaction with 8 tapeworms with close relativity or interfere with the interpretation of results. The method has strong specificity and high sensitivity, can efficiently and accurately realize the synchronous detection of 3 pathogens, greatly saves the detection time and cost, effectively improves the working efficiency, and can be used for the typing identification of the human hydatid pathogen and the research on the molecular epidemiology of echinococcosis.
Description
Technical Field
The invention belongs to the field of disease diagnosis, and particularly relates to a primer pair combination and a kit thereof for detecting and identifying various human histobazoonosis pathogens.
Background
Echinococcosis (Hydatidosis) is a common name for echinococcosis (echinococcosis) and is a zoonosis caused by parasitism of echinococcus larvae in humans. The Echinococcus species found in the world in the past are classified into 6 species in total, namely Echinococcus granulosus (Echinococcus grandis), Echinococcus multilocularis (Echinococcus multilocularis), Echinococcus fortunei (Echinococcus vogelis), Echinococcus oligogranulosus (Echinococcus oligurnus), Echinococcus felis and Echinococcus shiquicus (Echinococcus shiquicus), wherein Echinococcus granulosus (E. grandis) are classified into 10 genotypes by genotype, namely Echinococcus granulosus G1-G10. With the application and development of molecular biology technology in echinococcus research, we divided the 10 genotypes of the original echinococcus granulosus (e.g. grandilosus) into 4 new species, which are e.grandilosus sensu stricto (G1-G3), e.equinus (G4), e.ortleppi (G5) and e.canadens (G6-G10), respectively, according to the results obtained by phylogenetic tree analysis. Echinococcosis is widely prevalent around the world and has an increasing tendency to develop over the last 20 years. China is the most serious country with global echinococcosis prevalence, the high incidence area accounts for 44% of the total area of the country, and the number of threatened people is over 500 ten thousand.
According to the reports of the existing documents, three types of echinococcosis disease which can cause human body infection and are discovered at present in China, wherein the prevalence of echinococcus granulosus G1-G3 and echinococcus multilocularis is dominant, and the echinococcus granulosus G6-G10 is in a sporadic state in various places, but the infection risk is not ignored. The adult Echinococcus and its larva of different species have differences in morphology, biological characteristics and pathogenicity. Wherein the Echinococcus granulosus G1-G3 and Echinococcus granulosus G6-G10 larvae can cause human cystic echinococcosis, and Echinococcus multocida larvae can cause human cystic echinococcosis. Cystic echinococcosis can occur in multiple organs of the whole body, most common in liver and lung, and the primary focus of echinococcosis is almost in the liver. The detection and identification of the types of the echinococcosis pathogens are of great significance to epidemiological research, prevention control and treatment of diseases.
At present, PCR detection methods are available to specifically amplify target DNA sequences in lesion tissues of minimally invasive/surgical patients to confirm whether echinococcosis is infected or not and to confirm the kind of echinococcosis pathogen infected. The existing PCR detection methods mainly comprise a single PCR method and a multiple PCR method. Dinkel A et al (A PCR system for identification of Echinococcus species and genotypes, with a reference to the epidemic positioning in an apparatus [ J ].2004:48-48.) established a PCR detection method for detecting Echinococcus granulosus; boufana B et al (Development of Three PCR Assays for the Differentiation between Echinococcus shiquisus, E.grandilosus (G1genotype), and E.multilocularis DNA in the Co-Engineer Region of Qinghai-Tibet plane, China [ J ]. American Journal of veterinary Medicine & Hygene, 2013,88(4):795-802) established 3 singleplex PCR methods for the detection of Echinococcus granulosus G1 type, Echinococcus polyangiensis and Echinococcus shiwisensis; can H et al (Detection of Echinococcus grandis and Echinococcus multilocularis in circulating multiple using a non-stationary simple tube multiplex polymerase reaction. [ J ]. Mikrobiyoiji Bulteni,2016,50(2):266.) have established a real-time fluorescence multiplex PCR method for simultaneous Detection of Echinococcus multibladus and Echinococcus granulosus; cong nuan Liu et al (diagnosis between E.grandis sensu stricto, E.grandis and E.shiquisus Using a Multiplex PCR Assay [ J ]. Plos Neglected targeted Diseases,2015,9(9): e0004084.) and Rooia et al (patent CN201310102181.4) have established a Multiplex PCR method for simultaneously detecting Echinococcus granulosus G1-G3 type, Echinococcus shikoensis and Echinococcus multibladus, in which no human infection has been found at present, whose host of infection is mainly small mammals and canids.
In the method, one reaction system of the single PCR method can only detect one pathogen, and the detection procedure is complicated, time-consuming and labor-consuming, so that the method is not beneficial to the rapid detection of large-scale samples. The multiplex PCR method can synchronously detect two or more different target genes in the same system under the same reaction condition, namely, one reaction system can simultaneously detect various pathogens, thereby greatly improving the efficiency of the method. However, on one hand, the existing multiplex PCR method is not designed aiming at three human echinococcosis pathogens (Echinococcus granulosus G1-G3 type E.grandinosus sensu stricto, Echinococcus multocida E.multilocularis and Echinococcus granulosus G6-G10 type E.canadens) discovered in China, and can not meet the requirements for detecting and identifying human echinococcosis pathogens; on the other hand, the design difficulty of the primers of the multiplex PCR is far greater than that of the single PCR, and because a plurality of pairs of primers are added into one system of the multiplex PCR, cross reaction with pathogens with close relativity is easy to occur, a false positive result is generated, complementary combination is easy to occur between the primer pairs, and an interference reaction is easy to generate, the existing echinococcosis multiplex PCR method has the problems of poor specificity, unreliable method and the like.
Therefore, the invention designs specific primers aiming at the mitochondrial gene conserved regions of three pathogens, namely Echinococcus granulosus G1-G3 (E.grandicosus sensu stricoto), Echinococcus multocida (E.Multilocularis) and Echinococcus granulosus G6-G10 (E.canadensis) infected by human bodies in China, and constructs a multiplex PCR method capable of synchronously detecting various echinococcosis pathogens. The method has the advantages of strong specificity, high sensitivity, high flux, simplicity, convenience and rapidness, can obviously improve the working efficiency, reduce the time and the economic cost, and has wide application prospect in the field of further identifying typing of minimally invasive or operative echinococcosis patients and molecular epidemiological investigation of echinococcosis patients.
Disclosure of Invention
Aiming at the defects in the prior art, the first purpose of the invention is to provide a primer pair combination which can be used for detecting and identifying a plurality of human tissue echinococcosis pathogens simultaneously, the primer pair combination does not interfere with each other when performing multiplex PCR amplification in the same system and under the same condition, has strong specificity and high sensitivity, and is designed aiming at three pathogens (Echinococcus granulosus G1-G3 type, Echinococcus multocida and Echinococcus granulosus G6-G10 type, hereinafter also referred to as three purpose Echinococcus granulosus) infected by human bodies in China.
It is a second object of the present invention to provide a kit comprising a combination of primer pairs that can be used simultaneously to detect and identify a plurality of human histo-echinococcosis pathogens.
The third purpose of the invention is to provide a using method of the kit.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a primer pair combination for detecting and identifying a human tissue hydatid pathogen, the primer pair combination comprising:
echinococcus granulosus G1-G3 type specific primer pair: gtp1F is shown as a sequence table SEQ ID NO.1, and gtp1R is shown as a sequence table SEQ ID NO. 2;
echinococcus multilocularis specificity primer pair: mtb42F is shown as sequence table SEQ ID NO.3, and mtb42R is shown as sequence table SEQ ID NO. 4;
echinococcus granulosus G6-G10 type specific primer pair: cox1F is shown in a sequence table SEQ ID NO. 5; cox1R is shown in sequence table SEQ ID NO. 6.
Furthermore, the lengths of target fragments amplified by combining the primer pairs for detecting and identifying the human hydatid pathogen are 240bp, 152bp and 441bp in sequence.
A kit, which contains the primer pair combination for detecting and identifying the human tissue hydatid pathogen.
Furthermore, the kit also contains a PCR reagent for DNA amplification.
Further, the PCR reagent in the kit comprises a DNA polymerase mixture and sterile double distilled water.
The use method of the kit comprises the following steps: extracting the DNA of the biopsy tissue of the person to be detected, adding the DNA into the reagent in the kit by taking the DNA as a template to perform PCR amplification, spotting the amplified PCR product into 2 percent agarose gel to perform electrophoresis, and observing the electrophoresis result.
Further, the using method of the kit is characterized in that the PCR amplification condition is pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30 s; annealing at 56 ℃ for 30 s; extension at 72 ℃ for 40 s; a total of 35 cycles; extension at 72 ℃ for 5 min.
The design and screening of primers is the key of the multiplex PCR method, and the specificity and sensitivity of the primers directly determine the specificity and sensitivity of the whole method. The design difficulty of the primers of the multiplex PCR is far higher than that of the single PCR, because a plurality of pairs of primers are added into one system, the primers are easy to generate cross reaction with pathogens with close relativity, a false positive result is generated, complementary combination is easy to generate between the primer pairs, and an interference reaction is easy to generate. In addition, the multiplex PCR method requires that the lengths of the amplified products have certain differences, and the bands can be distinguished through electrophoresis, which also increases the difficulty of primer design. The three pairs of primers are not interfered with each other, the specificity is strong, no cross reaction is generated with 8 tapeworms with close relativity, or the size of a band generated by the cross reaction is different from that of a target band, and the interpretation of a result is not interfered. The sensitivity is high, and the detection limit of the DNA of three pathogens is higher than 0.005 ng/mul. The specific primer is utilized to carry out multiplex PCR detection on the sample according to the method of the invention, and the synchronous detection of 3 types of pathogen of Echinococcus granulosus G1-G3, Echinococcus multocida and Echinococcus granulosus G6-G10 infected by human body in China can be realized.
Drawings
FIG. 1 is an electrophoretogram of PCR amplification products of specificity experiment 1 of example 1;
FIG. 2 is an electrophoretogram of PCR amplification products of specificity experiment 2 of example 1;
FIG. 3 is an electrophoretogram of PCR amplification products of specificity experiment 3 of example 1;
FIG. 4 shows the results of the sensitivity test in example 2;
FIG. 5 shows the results of multiplex PCR detection of 10 cases of patients to be examined from surgical excision of tissue DNA with 3 pairs of mixed primers in example 3;
FIG. 6 is a graph showing the results of the experiment in the comparative example.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. It will be apparent to those skilled in the art that the embodiments may be substituted/combined according to the present disclosure and all such embodiments are within the scope of the present invention.
In the following examples, where specific experimental conditions are not indicated, molecular cloning according to conventional conditions well known to those skilled in the art, for example, Sambrook et al: a Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), a Laboratory animal general Manual (national center for Laboratory animal Specifications, month 11 2004): the conditions and experimental procedures described in the fifth edition of the basic technical guide (John Wiley & Sons, Inc, 2005), or as suggested by the manufacturer. In the examples, materials and reagents, which are not specifically described, were commercially available by a conventional method.
Experimental methods
The primer pair of the invention comprises:
echinococcus granulosus G1-G3 type specific primers,
upstream primer gtp 1F: TGGTTTGGTTTATATGTTGGTGTTT, respectively;
downstream primer gtp 1R: ATCCATAAAGGAGTTCCTAGCG
An echinococcus multilocularis specificity primer,
upstream primer mtb 42F: ACCAACTGAGGGTGGCACTA, respectively;
downstream primer mtb 42R: CCATTACTACCCTAAACAACTGACA
Echinococcus granulosus G6-G10 type specific primers,
upstream primer cox 1F: TTTTATTTACGTTTGGGGGCG, respectively;
downstream primer cox 1R: CCACCAAACCAAAAGACCTG
Standard DNA template:
respectively extracting DNA of three target echinococcus and other tapeworms with close relationship
Echinococcus granulosus type G1-G3 (E.grandinosus sensu stricoto), Echinococcus multocida (E.Multilocularis), Echinococcus granulosus type G6-G10 (E.canadensis), Echinococcus shiquicus (E.shiquicus), Taenia vesiculosus (T.hysatigenia), Taenia megaterium (T.taeniformis), Taenia asiana (T.asitica), Taenia bovis (T.saginata), Taenia suis (T.solium), Taenia longicorna (H.diminicola) and Taenia serrata (T.serialis), whole genome DNA extraction is performed according to DNA extraction kit operation instructions, PCR detection and sequencing are performed on the extracted DNA, the sequence is compared with the corresponding sequence in the NCBI gene bank, and the DNA sample after the identification is used as a DNA template.
DNA of the patient to be tested:
selecting 15-25 mg of cyst germinal layer tissue or cyst fluid of a patient to be detected, shearing, and extracting whole genome DNA by adopting a tissue DNA extraction kit according to a specification.
PCR amplification system and conditions and results observations:
PCR reaction liquid is prepared in a PCR reaction tube, and a 50uL reaction system is adopted. The method comprises the following steps: 25uL DNA polymerase mixture, 5uL DNA template with the concentration of more than 50ng/uL (the concentration for specificity experiments, the detection of samples to be detected is not limited in the specification), 1.5uL (10uM) of each specificity primer, and double distilled water to make up for 50 uL.
Putting the PCR reaction tube into a PCR instrument for reaction, wherein the thermal cycle program is as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30 s; annealing at 56 ℃ for 30 s; extension at 72 ℃ for 40 s; a total of 35 cycles; extending for 5min at 72 ℃, and storing at 4 ℃ after the reaction is finished. The PCR reaction products were electrophoresed in 2% agarose gel (containing 0.5. mu.g/ml gold view) at a voltage of 80-110v and observed by UV gel imager.
Whether the echinococcosis is infected or not and the type of the infected worm are determined according to the existence and the size of the electrophoresis strip.
EXAMPLE 1 specificity experiments
3 groups of experiments are designed in total to investigate the specificity of the echinococcosis pathogen detected and identified by the technology of the invention, and the 3 groups of experiments respectively comprise:
1. the 3 pairs of mixed primers of the invention are respectively used for carrying out the specificity experiment of amplification on the DNA templates of three target echinococcus;
2. the 3 pairs of mixed primers of the invention carry out the specificity experiment of amplification on the DNA mixed templates of three target echinococcus;
3. the 3 pairs of mixed primers of the invention are used for carrying out the specificity experiment of amplification on three target echinococcus and other 8 tapeworm DNA templates with closer relativity.
The specific operation is as described in the experimental method, and the electrophoresis results are shown in FIGS. 1 to 3.
In fig. 1: lanes 1, 2 and 3 are PCR results of three mixed primers for amplifying Echinococcus granulosus G1-G3 type, Echinococcus multilocularis and Echinococcus granulosus G6-G10 type DNA templates, respectively; lane 4 is a negative control; m is DNA molecular weight standard markerII.
In fig. 2: lane 1 shows the PCR results of the three mixed primers for amplifying mixed DNA templates of Echinococcus granulosus G1-G3 type, Echinococcus multilocularis and Echinococcus granulosus G6-G10 type; lane 2 is a negative control; m is DNA molecular weight standard markerII.
In fig. 3: lanes 1-3 show the PCR results of the mixed primers for amplifying the target species Echinococcus granulosus G1-G3, Echinococcus multilocularis, and Echinococcus granulosus G6-G10, respectively; lanes 4-11 are PCR results for amplification of Echinococcus shikoensis, Taenia vesicularis, Taenia megaterium, Taenia Asiatica, Taenia bovis, Taenia suis, Taenia longibrachiata, and Taenia serrata; m is DNA molecular weight standard markerII.
According to experimental results, the 3 pairs of primers respectively amplify 240bp, 152bp and 441bp specific single-purpose bands which sequentially correspond to Echinococcus granulosus G1-G3 type, Echinococcus multilocularis and Echinococcus granulosus G6-G10 type, and the size of amplified fragments is consistent with expectation, which shows that the method can specifically detect three kinds of purpose Echinococcus granulosus; after the three pairs of primers are mixed, the three pairs of primers cannot interfere with each other, and can only amplify the target band of the corresponding insect species; in addition, the invention can only detect three corresponding target insect species, 8 insect species which are close to other relatives have no cross reaction, or the size of a band formed by the cross reaction is a non-target band, which does not influence the judgment of a detection result, so the invention has strong specificity.
Example 2 sensitivity test
Three target echinococcus DNA are respectively used as templates, and the final concentrations of the templates are respectively as follows: amplification was performed according to the aforementioned assay at 0.7 ng/. mu.l, 0.35 ng/. mu.l, 0.18 ng/. mu.l, 0.09 ng/. mu.l, 0.04 ng/. mu.l, 0.02 ng/. mu.l, 0.01 ng/. mu.l, 0.005 ng/. mu.l.
The results of electrophoresis are shown in FIG. 4, in which the template in lanes 1-8 is Echinococcus granulosus G1-G3 type, the template in lanes 9-16 is Echinococcus multilocularis G6-G10 type, and the template in lanes 17-24 is Echinococcus granulosus G6-G10 type. The final concentrations of the template in the reaction system were 0.7 ng/. mu.l, 0.35 ng/. mu.l, 0.18 ng/. mu.l, 0.09 ng/. mu.l, 0.04 ng/. mu.l, 0.02 ng/. mu.l, 0.01 ng/. mu.l, and 0.005 ng/. mu.l, respectively. The detection limit of the invention to DNA of three target insect species is higher than 0.005 ng/mul, which shows that the invention has high sensitivity.
EXAMPLE 3 use in the detection of human lesion tissue samples
10 cases of operation patients are selected to be sent to examine liver lesion tissues for detecting echinococcus infection. The DNA extraction and the multiplex PCR amplification of the patient to be tested are carried out according to the experimental method.
The amplification band sizes of the Echinococcus granulosus G1-G3 type, Echinococcus multilocularis and Echinococcus granulosus G6-G10 type are 240bp, 152bp and 441bp respectively, and the electrophoresis results are shown in FIG. 5, wherein: lanes 1-10 are sample PCR results; lane 11 is a positive control, i.e., the PCR amplification results of 3 target worm species mixed DNA templates and 3 pairs of mixed primers; lane 12 is a negative control; m is DNA molecular weight standard markerII.
1. The bands in lanes 3, 7 and 10 are 240bp, so that the corresponding patient is positive for Echinococcus granulosus infection types G1-G3; 2. the bands in lanes 4, 5 and 9 are 152bp, so that the Echinococcus multilocularis infection corresponding to the patient is positive; the band in lane 8 is 441bp, so that the patient is positive for Echinococcus granulosus infection type G6-G10; no band in lane 6 is negative, so this patient is not infected with echinococcosis.
Comparative examples
The multiplex PCR detection method of the present invention was compared with comparative 1: Cong-Nuan Liu et al (Liu C N, Lou Z, Li L, et al, characterization between E.grandioses sensu stricoto, E.mulocularis and E.shiquisuses a Multiplex PCR Assay [ J ]. Plos New amplified polymorphic Diseases,2015,9(9): e0004084.) and comparative 2: the multiplex PCR detection method for the Echinococcus granulosus G1-G3 type, Echinococcus multilocularis and Echinococcus shikoensis disclosed in patent CN201310102181.4 is compared.
The DNA template of the invention respectively adopts Echinococcus granulosus G1-G3 type, Echinococcus multilocularis, Echinococcus granulosus G6-G10 type and three target species DNA mixtures; the procedure was as described in the experimental procedure.
The comparison 1 and the comparison 2 respectively adopt Echinococcus granulosus G1-G3 type, Echinococcus multilocularis, Echinococcus shikoensis and DNA mixtures of the three kinds of insects; the procedure was according to the respective literature methods.
The electrophoresis results are shown in FIG. 6, wherein lanes 1-4 are PCR results of the method of the present invention for amplifying the Echinococcus granulosus G1-G3 type, Echinococcus mulatta and Echinococcus granulosus G6-G10 type and three target species mixed DNAs, lanes 5-8 are PCR results of the method of comparative example 1 for amplifying the Echinococcus granulosus G1-G3 type, Echinococcus mulatta, Echinococcus shikoensis and three species mixed DNAs, and lanes 9-12 are PCR results of the method of comparative example 2 for amplifying the Echinococcus granulosus G1-G3 type, Echinococcus mulkoensis, Echinococcus shikoshikoensis and three species mixed DNAs.
Therefore, the invention can detect each target insect species respectively and can also detect the mixed infection of three insect species simultaneously. The method of comparison 1 can not detect Echinococcus multilocularis, the expected three bands do not appear in lane 8, only two bands appear, the sizes of the bands are 219bp and 471bp respectively, and the bands are Echinococcus granulosus G1-G3 type and Echinococcus shikoensis, which indicates that the method can not detect mixed infection of three kinds of insects simultaneously. In the method of comparison 2, the sizes of the specific amplification bands are 196bp, 584bp and 471bp, 3 bands appear in the 10 lanes, one of the bands is greater than 500bp, and the other two bands are close to the target bands of the echinococcus granulosus G1-G3 type and the echinococcus shikoensis, but a single target band of 584bp does not appear as expected, which indicates that the method has poor specificity for detecting the echinococcus granulosus G1-G3 type and the echinococcus shikoensis can be interfered. Only two target bands appear in lane 12, indicating that the method cannot realize the detection of mixed infection of three insect species.
In conclusion, the method can detect the single infection of the three target insect species and the mixed infection of the three insect species only by one-time reaction, has no cross reaction among the target insect species, good strip separation effect and better detection effect than the existing echinococcosis multiplex PCR method, and overcomes the defect that the prior art can not simultaneously detect and identify the three echinococcosis insect species infected by the human body in China.
SEQUENCE LISTING
<110> center for disease prevention and control in Sichuan province
<120> primer pair combination and kit for detecting and identifying human histoplasmosis pathogen
<130> 201705
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<213> Artificial sequence (gtp 1F)
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<221> Echinococcus granulosus G1-G3 type upstream primer
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<220>
<221> Echinococcus granulosus G1-G3 type downstream primer
<222> (1)..(22)
<400> 2
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<211> 20
<212> DNA
<213> Artificial sequence (mtb 42F)
<220>
<221> Echinococcus multilocularis upstream primer
<222> (1)..(20)
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<210> 4
<211> 25
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<213> Artificial sequence (mtb 42R)
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<221> Echinococcus multilocularis downstream primer
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<210> 5
<211> 21
<212> DNA
<213> Artificial sequence (cox 1F)
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<221> Echinococcus granulosus G6-G10 type upstream primer
<222> (1)..(21)
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ttttatttac gtttgggggc g 21
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<213> Artificial sequence (cox 1R)
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<221> Echinococcus granulosus G6-G10 type downstream primer
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Claims (5)
1. A primer pair combination for detecting and identifying human hydatid pathogen by multiplex PCR method, said primer pair combination comprising:
echinococcus granulosus G1-G3 type specific primer pair: gtp1F is shown as a sequence table SEQ ID NO.1, and gtp1R is shown as a sequence table SEQ ID NO. 2;
echinococcus multilocularis specificity primer pair: mtb42F is shown as sequence table SEQ ID NO.3, and mtb42R is shown as sequence table SEQ ID NO. 4;
echinococcus granulosus G6-G10 type specific primer pair: cox1F is shown in a sequence table SEQ ID NO. 5; cox1R is shown in sequence table SEQ ID NO. 6.
2. The primer pair combination for detecting and identifying human histoechinococcosis pathogen by multiplex PCR method according to claim 1, wherein the lengths of the target fragments amplified by the Echinococcus granulosus G1-G3 type specific primer pair, the Echinococcus multocida specific primer pair and the Echinococcus granulosus G6-G10 type specific primer pair are 240bp, 152bp and 441bp in sequence.
3. A kit comprising the primer pair combination for detecting and identifying the human hydatid pathogen by the multiplex PCR method according to claim 1 or 2.
4. The kit of claim 3, wherein the kit further comprises PCR reagents for DNA amplification.
5. The kit of claim 4, wherein the PCR reagents comprise a DNA polymerase mixture and sterile double distilled water.
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| CN106868153A (en) * | 2017-03-14 | 2017-06-20 | 西南民族大学 | The detection kit of the cattle and sheep echinococcosis granulosa based on POCKIT Micro fluorescent PCR platforms and application |
| CN110846309B (en) * | 2018-08-20 | 2023-03-24 | 中国农业科学院兰州兽医研究所 | Primer pair and primer probe set for identifying echinococcus multilocularis and application of primer pair and primer probe set |
| CN108950020B (en) * | 2018-08-31 | 2021-09-14 | 四川农业大学 | Application of Echinococcus granulosus mitochondrion ND6 gene |
| CN109097482B (en) * | 2018-08-31 | 2021-09-14 | 四川农业大学 | Application of Echinococcus granulosus mitochondrion ND6 gene |
| CN109762910B (en) * | 2019-01-10 | 2023-06-09 | 四川省疾病预防控制中心 | Primer and kit for simultaneously detecting two types of echinococcosis |
| CN111733254B (en) * | 2019-03-25 | 2023-08-18 | 中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心) | Biomarker DNA molecular fragment for detecting echinococcus granulosus and application thereof |
| CN110656187B (en) * | 2019-10-28 | 2023-06-02 | 中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心) | Kit for detecting pathological tissues or echinococcus in canine feces by multiple RAA and multiple PCR and detection method |
| CN111235285A (en) * | 2020-03-04 | 2020-06-05 | 南京亿科人群健康研究院有限公司 | Kit for detecting echinococcosis diagnosis |
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| CN102168100A (en) * | 2011-02-14 | 2011-08-31 | 中国疾病预防控制中心寄生虫病预防控制所 | Echinococcus granulosusglutathione transferase gene and application thereof |
| CN102363808A (en) * | 2011-11-07 | 2012-02-29 | 中国农业科学院兰州兽医研究所 | Kit for detecting echinococcus granulosus pathogen from dog excrement |
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| CN102168100A (en) * | 2011-02-14 | 2011-08-31 | 中国疾病预防控制中心寄生虫病预防控制所 | Echinococcus granulosusglutathione transferase gene and application thereof |
| CN102363808A (en) * | 2011-11-07 | 2012-02-29 | 中国农业科学院兰州兽医研究所 | Kit for detecting echinococcus granulosus pathogen from dog excrement |
| CN103173554A (en) * | 2013-03-27 | 2013-06-26 | 中国农业科学院兰州兽医研究所 | Detection kit for detecting and distinguishing multiple kinds of echinococcus |
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