CN108950020B - Application of Echinococcus granulosus mitochondrion ND6 gene - Google Patents
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Abstract
The invention relates to the technical field of biology, and discloses a series of new applications of echinococcus granulosus mitochondria ND6 gene. The invention takes the Echinococcus granulosus mitochondrion ND6 gene as a molecular diagnosis marker, detects whether the dog is infected with the Echinococcus granulosus by PCR, has high reliability, specificity and sensitivity, can detect the Echinococcus granulosus in the dog excrement in time at the early stage of infection, and can be used for epidemiological investigation of the infection of the dog with the Echinococcus granulosus.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of echinococcus granulosus mitochondria ND6 gene.
Background
Echinococcus granulosus (Eg) has a complex life history and is accomplished by intermediate host owners or by some domestic animals (such as herbivores like cattle, sheep) and the end-host canines. Echinococcus granulosus, a medium-taenidium larvae, is predominantly parasitic in the liver and lungs of the intermediate host, causing Echinococcosis granulosa (Echinococcosis cysticercosis). The disease is a parasitic disease of zoonosis, is distributed worldwide, wherein China belongs to one of high-incidence areas, mainly prevails in Qinghai, Xinjiang, Tibet, northwest pasturing areas of Sichuan and Ningxia, and currently, at least 368 popular counties are in China. The disease is listed as an animal epidemic disease which is preferentially prevented and mainly prevented in China 'national middle and long term animal epidemic disease prevention and treatment plan' (2012 and 2020).
Canines are the most important end-use host and source of infection for echinococcus granulosus. After the dogs swallow the echinococcus, the protocephalus can develop into adults in the small intestine after 40-50 days, and the life of the adults in the dogs is 5-6 months. The adult pregnancies and the worm eggs are discharged along with the dog dung and pollute food, water sources and the environment, and the intermediate host is infected by eating the worm eggs through the mouth, thereby causing cystic echinococcosis. If the condition that the dogs are infected with the echinococcus granulosus can be timely and accurately detected, the method has important significance for preventing and controlling human and animal echinococcosis.
The traditional method for detecting the canine echinococcus granulosus infection mainly comprises the following steps: a dissection method, an arecoline purgation method and a worm egg floating inspection method. The detection result of the autopsy method is visual, but the operation is complicated, and the condition of missed detection can occur; arecoline purgation has good application value, but the catharsis rate is only about 70%, and environmental pollution may be caused. Although the egg floating inspection method is simple, convenient and easy to implement, the condition of missed inspection still occurs. In addition, the specificity of this method is not high because the eggs of Echinococcus tapeworm are morphologically indistinguishable from those of other tapeworms.
The principle of the fecal antigen ELISA detection method is that the polypide parasitizes in the intestinal tract of a host, and the polypide tissues (called fecal antigens) such as metabolites, secretions, tablets, eggs and the like of the polypide can be discharged out of the body along with the feces of the host, and the fecal antigens can be detected by preparing specific antibodies. The method is simple and easy to implement, has no strict requirements on detection samples, and is suitable for popularization and use in the basic level; and Zhangxu et al reported that dogs infected with 5 worms were positive 16 days after infection, and had early diagnostic value. The specificity of the method can reach 97%, but the sensitivity is influenced by the infection degree, wherein the sensitivity can reach 92.0% when the number of infected insects is more than 100, but the sensitivity is only 29.0% when the number of infected insects is less than 100. In addition, the method may cause serious cross reaction among similar polypide in tapeworm species of the family Zodiaceae, so that a high false positive may be generated in the detection result of the coproantigen ELISA method.
A loop-mediated isothermal amplification (LAMP) technology is used for designing 4 pairs of primers aiming at 6 regions of a target gene, and a target fragment can be amplified by Bst DNA polymerase with strand displacement activity for about 1 hour under a constant temperature condition (65 ℃). The method has the advantages of simple required equipment, quick reaction, convenient and fast product detection and strong specificity. Chenoplume et al have reported that echinococcus granulosus infection in dogs with greater than 10000 bands is detectable 18d after infection. In addition, when the canine feces were examined, the activity of Bst DNA polymerase was hardly affected by the fecal inhibitor, which also made the LAMP method more sensitive than the conventional PCR. However, due to the excessively high sensitivity, the experimental result of the method is false positive due to a small amount of gene pollution, and the method needs to be verified by an additional detection method; furthermore, the LAMP method has a plurality of electrophorograms, and is not easily recognized even when nonspecific amplification occurs. Meanwhile, the LAMP method cannot perform genotyping by gene sequencing because of a non-single band. In addition, the method requires that the length of the target sequence is controlled below 300bp, the requirement on the primers is high, and a large amount of work is required for screening proper primers.
Animal wastes are easy to collect and store, the number of infection sources in the wastes is large, pathogen genetic materials of the animal wastes can be derived from parasite eggs, cells, tissue fragments and the like of parasite bodies, and a target gene can be extracted by using a small amount of wastes, so that the waste PCR detection method is gradually an important way for detecting animal diseases. Compared with pathogenic detection and immunological detection methods, the PCR detection method has the advantages of rapidness, accuracy, sensitivity, specificity and the like, and can also realize the identification of the taenia taenidis with difficultly distinguished morphology. After the PCR technique was reported by Bretag et al (1993) to detect Echinococcus multilocularis infection in fox feces DNA, the technique was also widely used in the detection of Echinococcus canicola eggs. Dinkel et al (1998) amplified the Echinococcus multilocularis 12S rRNA fragment (373bp) using nested PCR detection with a minimum of 1 egg detected, whereas 11 other species of tapeworms, Echinococcus granulosus, were not amplified non-specifically and were 100% specific. Abbasi et al (2003) performed PCR amplification on the target repeat (EgG1 Hae III) (133bp), which has high sensitivity and can detect one worm EgG at the lowest. However, the specificity is poor, and meanwhile, the amplified tapeworm vesiculosus, tapeworm multiceps and tapeworm ovipositae also have the same 133bpDNA repetitive sequence. Stefania et al (2004) carry out PCR amplification on a 12SrDNA fragment (255bp) of the Echinococcus granulosus G1 strain, the sensitivity reaches the detection of one parasite egg, and simultaneously, other 5 genotypes of the Echinococcus granulosus and other 14 tapeworm genes are amplified, only the Echinococcus granulosus G1 type is specifically amplified, and the detection result is not false positive. In addition, Dinkel et al (2004) also designed primers according to the mitochondrial 12S rRNA gene sequence (254bp) of Echinococcus granulosus G1 strain, and identified different genotypes (16 types in total) of Taenia and Echinococcus granulosus, wherein the specificity of the primers is 100%, and the sensitivity of the primers is 0.25pg of DNA content.
Although the PCR technology is widely applied to clinical detection due to its characteristics of high sensitivity, specificity, etc., it is very important to select a proper and reliable molecular diagnostic marker for detecting animal diseases.
Disclosure of Invention
In view of the above, the present invention aims to provide a novel application of the echinococcus granulosus mitochondrial ND6 gene, such that the ND6 gene has high accuracy, sensitivity and specificity when used as a molecular diagnostic marker for detecting echinococcus granulosus, and can detect echinococcus granulosus earlier.
In order to achieve the above purpose, the invention provides the following technical scheme:
the application of Echinococcus granulosus mitochondrion ND6 gene as a molecular diagnostic marker for detecting Echinococcus granulosus;
the echinococcus granulosus mitochondrion ND6 gene (ND6) has short full sequence length (456bp), simple structure, no intron, moderate evolution rate, rich sequence variation and mutation rate obviously higher than that of nuclear DNA, is widely applied to population genetic structure analysis of yaks, fishes and insects at present, but is not reported to be used for detecting the echinococcus granulosus infected by dogs. The invention utilizes PCR technology, takes the ND6 whole gene sequence screened from mitochondrial genome as a molecular diagnostic marker and amplifies the gene, and establishes a PCR method for detecting Echinococcus granulosus DNA in dog feces.
In China, parasitic tapeworms in the small intestine of dogs include echinococcus granulosus, and other tapeworms such as echinococcus multilocularis, taenia multiceps, taenia buqueta, taenia sojae, taenia canicola, and tapeworm Menispermum. Additionally, toxocara canis is also a very common parasite. The invention takes ND6 gene as molecular diagnostic marker to carry out PCR detection on the above eight parasites respectively, the result shows that no target band is amplified except for Echinococcus granulosus, which indicates that ND6 gene is taken as molecular diagnostic marker, has high species specificity and does not generate cross reaction. In order to further increase the specificity, the invention uses ND6 gene as main molecular diagnostic marker, and adds upstream partial tRNA-GLU sequence (38bp) and downstream partial tRNA-TYR sequence (64bp) to form better molecular diagnostic marker.
When the sensitivity of the molecular diagnostic marker ND6 gene is measured, the target gene is proportionally diluted (273ng-4pg), and the result shows that the brightness of the target band is gradually reduced along with the gradual reduction of the DNA concentration until the positive band completely disappears when the target band is diluted to 4 pg. In the existing report, Abbasi (2003) detected that the DNA content of the gene (EgG1 Hae III) of a single Echinococcus granulosus EgG is 8pg, which indicates that the ND6 gene is used as a molecular diagnostic marker with higher sensitivity, and even only 1 Echinococcus granulosus EgG can be detected.
In addition, in order to verify the reliability of taking the ND6 gene as a molecular diagnostic marker, the invention carries out PCR detection on the dog feces of the artificially infected protozoan coenuruses, and the result shows that when the dogs are artificially infected with about 50000 protozoan coenuruses, the PCR can amplify the target gene from the dog feces on the 13 th day of infection, which shows that the ND6 gene as the molecular diagnostic marker can diagnose the infection condition of the echinococcus granulosus canis at early stage.
Based on various excellent effects of the ND6 gene as a molecular diagnosis marker, the invention also provides the following novel applications:
the application of Echinococcus granulosus mitochondrion ND6 gene in the preparation of molecular diagnostic markers for detecting Echinococcus granulosus;
the echinococcus granulosus mitochondrion ND6 gene is used as a molecular diagnostic marker in the preparation of a kit for detecting echinococcus granulosus;
the application of the upstream and downstream primers for amplifying the Echinococcus granulosus mitochondrial ND6 gene in the preparation of a kit for detecting Echinococcus granulosus;
wherein the Echinococcus granulosus mitochondrial ND6 gene is an ND6 wild-type gene; or a sequence consisting of 1-100bp upstream of the ND6 wild-type gene, 1-100bp downstream of the ND6 wild-type gene and the ND6 wild-type gene; or a sequence in which one or more bases are deleted, added or substituted in the sequence of the ND6 wild-type gene; or a modified sequence of the wild type gene ND 6; the deletion, addition or substitution of one or more bases and the modification do not affect the PCR amplification of the amplification primer designed with the ND6 gene as a target gene. The upstream and downstream 1-100bp sequences of the ND6 wild-type gene are a partial tRNA-GLU sequence and a partial tRNA-TYR sequence respectively.
According to the application, the invention also provides a kit for detecting the Echinococcus granulosus, which comprises an upstream primer and a downstream primer for amplifying the Echinococcus granulosus mitochondrial ND6 gene; the upstream Primer and the downstream Primer for amplifying the Echinococcus granulosus mitochondrion ND6 gene can be designed according to the existing software such as Primer Premier 5.0, in the specific embodiment of the invention, the upstream Primer and the downstream Primer which are designed by taking the sequence consisting of 1-100bp upstream of the ND6 wild type gene, 1-100bp downstream of the ND6 wild type gene and 1-100bp downstream of the ND6 wild type gene as target genes are provided, and the upstream Primer and the downstream Primer are shown as SEQ ID NO: 1-2; meanwhile, an upstream primer and a downstream primer which are designed only by taking the ND6 wild-type gene as a target gene are also provided, and the upstream primer and the downstream primer are shown as SEQ ID NO. 3-4;
in addition, the kit may further comprise an enzyme for PCR amplification, dNTP, ddH2O and one or more than two components in the stabilizer. In the specific embodiment of the invention, the kit comprises upstream and downstream primers shown in SEQ ID NO 1-2 or SEQ ID NO 3-4, Taq PCR MasterMix and ddH2O and the stabilizer BSA.
According to the technical scheme, the Echinococcus granulosus mitochondrion ND6 gene is used as a molecular diagnosis marker, whether the dog is infected with the Echinococcus granulosus is detected through PCR, the kit has high reliability, specificity and sensitivity, can detect the Echinococcus granulosus in the dog excrement in time at the early infection stage, and can be used for epidemiological investigation of the infection of the dog with the Echinococcus granulosus.
Drawings
FIG. 1 shows a PCR amplification gel of 18 day old infusorian DNA; wherein M is DL2000DNA molecular weight standard; 1 is DNA of 18 days old infusorian; 2 is a positive control; 3 is a negative control;
FIG. 2 is a diagram showing PCR amplification gels of different samples of Taenia DNA; wherein M is DL2000DNA molecular weight standard; 1 is Echinococcus granulosus DNA; 2 is Echinococcus multilocularis DNA; 3 is Taenia multiceps DNA; 4 is the tapeworm DNA of Meng's loop; 5 is a taenia pisiformis DNA; 6 is bubbly tapeworm DNA; 7 is a Diphyllophora canicola DNA; 8 is dog toxocara canis; 9 is a negative control;
FIG. 3 is a PCR amplification gel diagram of 5 parts of naturally infected bean-shaped taenia canine feces, 5 parts of naturally infected multi-taenia canine feces and 2 parts of naturally infected vesicular-shaped taenia canine feces; wherein M is DL2000DNA molecular weight standard; 1 is a positive control; 2-6 is the DNA of the taenia pisiformis dog feces; 7-11 is Taenia multiceps dog excrement DNA; 12-13 is the DNA of the bubbly tapeworm dog feces;
FIG. 4 shows a PCR amplification gel map of 24 clinical dog faeces; wherein M is DL2000DNA molecular weight standard; 1-24 is clinical dog excrement DNA;
FIG. 5 shows a PCR amplification gel of different dilutions of Echinococcus granulosus DNA; wherein M is DL2000DNA molecular weight standard; 1 is the initial DNA; 2-11 are DNA diluted by 1:10, 1:100, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000 and 1:128000 times in sequence;
FIG. 6 shows a PCR amplification gel of 3d-18d feces from 2 artificially infected dogs; wherein, (a) and (b) represent the results of 3d-18d feces of 2 artificially infected dogs, respectively, and M is a DL2000DNA molecular weight standard; 1-16 were canine fecal DNA from day 3 to day 18 of artificial infection in sequence.
Detailed Description
The invention discloses application of echinococcus granulosus mitochondrion ND6 gene, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the techniques of the invention can be implemented and applied by modifying or appropriately combining the applications described herein without departing from the spirit, scope and spirit of the invention.
The application of the Echinococcus granulosus mitochondrial ND6 gene provided by the present invention is further described below.
Example 1: primer design
Based on the ND6 gene sequence of Echinococcus granulosus mitochondrial whole genome (SEQ ID NO: KJ559023.1) in GenBank, the Primer Premier 5.0 software was used to design1 pair of primers. Upstream primer A1: 5'-TTTCGTGCTGTAGATGGT-3' (SEQ ID NO: 1), downstream primer A25'-CACAGATTTCAAAGGGTT-3' (shown in SEQ ID NO: 2), and the amplified fragment is 558bp (consisting of 456bp of the complete ND6 wild-type gene, 38bp of the upstream partial tRNA-GLU sequence and 64bp of the downstream partial tRNA-TYR sequence, and the sequence is shown in SEQ ID NO: 5). The primer sequence was synthesized by Biochemical engineering (Shanghai) Co., Ltd.
Example 2: PCR amplification
The amplification system was 25 μ L: 2 XTaq PCR MasterMix 12.5. mu.L, primer A1、A21.0. mu.L each of template DNA 1.0. mu.L, ddH2O8.7. mu.L, 0.4% BSA 0.8. mu.L (BSA as stabilizer), gently mixed, and then subjected to instantaneous centrifugation for 15s with ddH2O was used as a blank control instead of the template DNA. Amplification conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 45s, annealing at 58 ℃ for 45s, and extension at 72 ℃ for 30s for 30 cycles; finally, extension was carried out at 72 ℃ for 6 min. After the reaction, 10. mu.L of the amplified product was identified by 1% agarose gel electrophoresis. The parallel experiments were repeated 3 times.
Example 3: echinococcus granulosus DNA PCR amplification
With synthetic primer A1、A2Amplifying by the method of example 2 by using DNA extracted from 18-day-old infusorium as a template, wherein the result is shown in figure 1, the size of a band is consistent with the expected result, the band is clear, and the negative control has no band; in addition, the result is expected after gene sequencing by reagent companies.
Example 4: specific assay
1. Specificity test of different tapeworm DNA samples
Echinococcus granulosus, Echinococcus montelus (Spirometera mansonii), Taenia pisiformis (Taenia pisiformis), Taenia canis (Dipyllidium caninum), Taenia multiceps (Taenia multiceps) and Taenia vesicularis (Taenia hydatigene) sample DNA and Toxocara canis sample DNA are provided by the animal parasitic disease research center of Sichuan university of agriculture; echinococcus multilocularis (Echinococcus multilocularis) sample DNA was gifted by the Chinese CDC Shanghai parasitic disease institute, Yoshiwan doctor.
With synthetic primer A1、A2The DNA of the above Echinococcus granulosus, Echinococcus multilocularis, Echinococcus montelukas, Taenia sojae, Taenia canina, Taenia multiceps, Taenia buqueta and ascaris canis was amplified according to the method of example 2, and the results are shown in FIG. 2.
The PCR amplification reaction of the echinococcus granulosus DNA sample generates a single target band, and the DNA samples and negative controls of echinococcus multocida, tapeworm Mene, zona pisiformis, dog polypore tapeworm, taenia multiceps, zona bubbica and dog toxocara canis do not have the amplification band.
2. Detection of different stool samples
Dog dung of tapeworm bean (5 parts), tapeworm multiceps (5 parts) and tapeworm vesicularis (2 parts) are provided by animal parasitic disease research center of Sichuan university of agriculture;
24 parts of clinical dog dung is collected from an epidemic area of the onchachiosis in Ganzui, Sichuan province and provided by a Ganzui animal epidemic disease prevention and control center, and the results are positive after the detection of the antigen detection kit of the onchafer of the dog dung.
Fecal DNA was extracted according to Qiagen DNeasy PowerSoil kit (Qiagen, Carlsbad, Calif.). The extracted DNA was immediately used for PCR or stored at-20 ℃ until use.
New upstream and downstream primers (shown in SEQ ID NO: 3-4) for the ND6 wild-type gene were designed using Primer Premier 5.0 software, and 3 dog feces samples were PCR-positive after PCR detection of 24 clinical dog feces according to the method of example 2 (FIG. 3). The 24 clinical dog feces are detected again by utilizing the loop-mediated isothermal amplification technology, the result is consistent with that of the invention, and the defect of high false positive in the existing feces antigen ELISA detection method is also proved.
On the other hand, when PCR detection was performed on 5 parts of naturally infected canine feces with tapeworm bean, 5 parts of naturally infected canine feces with tapeworm multiheaded and 2 parts of naturally infected canine feces with tapeworm vesicular, no sample exhibited an amplified band (FIG. 4).
Example 5: sensitivity measurement
The initial concentration of Echinococcus granulosus template DNA was determined using a Thermo Scientific NanoDrop spectrophotometer (Thermo, New York, USA), after which the extracted template DNA was diluted at 1:10, 1:100, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000 fold, New upstream and downstream primers (shown in SEQ ID NO: 3-4) for the ND6 wild-type gene were designed using Primer Premier 5.0 software and tested as in example 2, with the results shown in FIG. 5.
It was determined that the initial DNA content of the Echinococcus granulosus template was 273ng, the content of eggs in the template decreased with increasing dilution, the intensity of the DNA band decreased gradually, a very faint band was amplified at a dilution of 1/64000 (4pg), but no band was amplified at a dilution of 1/128000 (2 pg).
Example 6: PCR assay of dog feces of artificial infected metacercaria
After 2 dogs had been medicated to repel parasites, about 50000 cases of metacercaria were fed to each dog. After 3 days of infection, feces of 2 dogs were collected each day and placed at-80 ℃ for 2 weeks or more for harmless treatment. Dogs were euthanized at 18d and their small intestine dissected to collect the baby worms. The DNA extraction and detection according to example 2 were carried out sequentially on dog feces on different infection days (primer A)1、A2) The results are shown in FIG. 6.
According to the determination, the PCR amplification results of the feces of 2 dogs are negative when the metacercaria is infected at 3d to 11d, the PCR amplification result of only one dog is positive at 12d, and the PCR amplification results of two dogs show positive target bands at 13d to 18d (figure 6).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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Claims (6)
1. The echinococcus granulosus mitochondrion ND6 gene is used as a molecular diagnostic marker in the preparation of a kit for detecting echinococcus granulosus; the ND6 gene is ND6 gene with a sequence number of KJ559023.1 in GenBank.
2. The use of claim 1, wherein the Echinococcus granulosus mitochondrial ND6 gene is the ND6 wild-type gene; or a sequence consisting of 1-100bp upstream of the ND6 wild-type gene, 1-100bp downstream of the ND6 wild-type gene and the ND6 wild-type gene; the ND6 wild type gene is ND6 gene with a sequence number of KJ559023.1 in GenBank.
3. A kit for detecting Echinococcus granulosus is characterized by comprising upstream and downstream primers for amplifying Echinococcus granulosus mitochondrial ND6 gene, wherein the ND6 gene is a sequence consisting of 1-100bp upstream of the ND6 wild-type gene, 1-100bp downstream of the ND6 wild-type gene and 1-100bp downstream of the ND6 wild-type gene, and the ND6 wild-type gene is the ND6 gene with the sequence number of KJ559023.1 in GenBank.
4. The kit of claim 3, further comprising an enzyme for PCR amplification, dNTP, ddH2O and one or more than two components in the stabilizer.
5. The application of the upstream and downstream primers for amplifying the Echinococcus granulosus mitochondrial ND6 gene in the preparation of a kit for detecting Echinococcus granulosus; the ND6 gene is ND6 gene with a sequence number of KJ559023.1 in GenBank.
6. The use of claim 5, wherein the upstream and downstream primers are shown in SEQ ID NO 1-2 or SEQ ID NO 3-4.
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| Copro-PCR prevalence of Echinococcus granulosus infection in dogs in Kerman, south-eastern Iran;S R Mirbadie等;《J Helminthol》;20170130;第92卷(第1期);第17-21页 * |
| Echinococcus granulosus mitochondrion, complete genome;Wang,N.等;《GenBank》;20141218;Accession No: KJ559023.1 * |
| 基于cox2基因的细粒棘球绦虫环介导等温扩增检测方法的初步建立;张艳艳 等;《中国寄生虫学与寄生虫病杂志》;20170430;第35卷(第2期);第169-172页 * |
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