CN114058716A - Method for analyzing diversity of tick-borne pathogens - Google Patents
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Abstract
The invention provides an analysis method of tick-borne pathogen diversity, belonging to the technical field of molecular biology and comprising the steps of collecting a specimen; extracting DNA of a specimen; primary screening of tick-borne pathogens, wherein detection and screening are completed by carrying out specific primer design and PCR amplification on the DNA; sequencing; and biological information analysis, wherein the biological information comprises homology analysis, construction of a phylogenetic tree and statistics and analysis on the composition of the complex infection conditions of different parasitic ticks. The use of the assay in microbial diversity analysis, preferably pathogen diversity analysis. The analysis method is used for solving the problems that the diversity of parasitic animals and pathogens of the ticks and the complex infection condition of the pathogens lack the systematic analysis and the research of the gene level; the reliability and the accuracy of the analysis result are high, the method is favorable for developing the targeted prevention and control work of the tick-borne infectious disease, and the disease caused by the cicada vector microorganisms can be further controlled and reduced.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to an analysis method for tick-borne pathogen diversity.
Background
Ticks are blood-sucking arthropods which are obligately parasitic on the body surface of mammals and are also important transmission vectors of various zoonotic infectious diseases. Tick, as a blood-sucking parasite, can survive freely in nature and also parasitize on the body surfaces of humans and animals such as cattle, sheep, horses, camels, rats, etc. Although tick borne infectious diseases are mostly natural epidemic diseases, the tick borne infectious diseases are easily popularized in the nature due to wide distribution of animal hosts and transmission vectors.
The pathogens of tick-borne infections are of a wide variety, including viruses, bacteria, and parasites. Because the host of ticks is diverse and can carry multiple pathogens simultaneously, the chances of causing a complex infection also increase. In recent years, new and recurrent tick-borne diseases, such as fever with thrombocytopenia syndrome, human granulocytopenia, monocytic ehrlichiosis, novel fever, and the like, are continuously appearing in China and surrounding countries and regions, and attract worldwide attention. Tick-borne infectious diseases are relatively mild in clinical characteristics in the early stage of infection and are often easy to ignore; in the later period, the joint deformation and the lower limb muscular atrophy can be caused, and serious patients can cause damage to multiple systems and multiple organs of the human body, and even cause the life-long disability or death.
The inner Mongolia is in the north of China in the middle and the west, the east-west span is large, the ecological environment mainly comprises grassland, semi-desert grassland and Gobi, various livestock are herded and become the main breeding areas and the best habitat of ticks, and the epidemic risk of tick-borne infectious diseases is increased. In recent years, in inner Mongolia areas, new infectious diseases such as Lyme disease spirochaete and rickettsia maculatus are separated from tick bodies successively, so that the existence of tick-borne infectious diseases is proved.
The incidence of zoonosis in areas with ticks is high, and the current detection method and technology for ticks pathogens are imperfect, the pathogens are various in types and distribution, and the detection accuracy is still to be improved, so the scientific detection and analysis for ticks pathogens and tick-borne infectious diseases are still to be deeply researched and explored. In order to carry out systematic analysis and gene level research on the parasite and pathogen diversity of the ticks and possible pathogen complex infection conditions, a reliable and accurate analysis method related to the gene level tick-borne pathogen diversity is provided.
Disclosure of Invention
The invention provides an analysis method of tick-borne pathogen diversity, which is used for solving the problems of lack of systematic analysis and gene level research on the diversity of parasitic animals and pathogens of ticks and possible pathogen complex infection conditions. The analysis method improves the reliability and accuracy of the result, deeply excavates the diversity of pathogens of the tick-borne infectious disease, facilitates related personnel to further know the epidemiological characteristics and the propagation rule of the tick-borne infectious disease, is more favorable for developing the targeted prevention and control work of the tick-borne infectious disease, and can further control and reduce the occurrence of diseases caused by cicada vector microorganisms.
In a first aspect, the present invention provides a method for analyzing tick-borne pathogen diversity, comprising:
s1, collecting a specimen;
s2, extracting DNA of the specimen;
s3, primary screening of tick-borne pathogens, wherein the primary screening is to complete detection and screening by carrying out specific primer design and PCR amplification on the DNA;
s4, sequencing;
and S5, analyzing biological information, wherein the biological information comprises homology analysis, construction of a phylogenetic tree, and statistics and analysis of the composition of the complex infection conditions of different ticks.
Through above technical scheme, through collection, extraction, preliminary screening, sequencing and analysis to the tick, parasitic animal and the pathogen diversity of tick have been studied from the genetic aspect to and the pathogen combined infection condition, detect high-efficiently, the precision is high, can realize carrying out the simultaneous detection to the multiple different target molecules in the sample, furthest's the variety that remains the sample, reduce bias and error nature, greatly reduced detection and analysis cost has improved analysis appraisal rate of accuracy and relevance ratio.
In particular embodiments, the markers in step S1 include free ticks and parasitic ticks that are parasitic on the body surfaces of livestock and other animals; the free tick specimen is collected by a flag distribution method, and the parasitic tick specimen is collected by a livestock examination method. Preferably, the period of tick specimen collection is the annual peak tick activity period, i.e. the 4-6 months of the year.
In a specific embodiment, the step of DNA extraction in step S2 comprises: soaking the collected tick sample in sodium hypophosphite and 75% ethanol and iodophor for 5-10min, sterilizing, washing with sterile water, naturally drying, dissecting with a stereomicroscope one by one, picking salivary gland, storing in PBS solution, extracting DNA with genome extraction kit, and storing the extracted DNA at-20 deg.C.
In a specific embodiment, in step S3, the target genes of the preliminary screening step include: citrate synthase gltA, flagellin flaB, phosphodiesterase glpQ, outer membrane protein omp1 and major surface protein msp 2.
In specific embodiments, the specific primers designed in step S3 include a forward primer set and a reverse primer set; the forward primer set consists of one or more of the following forward primers:
primer F1: the primer sequence is 5'-CGAACTTACCGCTATTAGAATG-3';
primer F2: the primer sequence is 5'-TGGTGGAGCTCATAAGTTACA-3';
primer F3: the primer sequence is 5'-GCTAAGGAGTTAGCTTATGA-3';
primer F4: the primer sequence is 5 '-ATYAGTGSAAARTAYRTRCCAA-3';
primer F5: the primer sequence is 5'-GCTGAAGAGCTTGGAATGCACC-3';
primer F6: the primer sequence is 5 '-CATACGCTTCTGCYTTRGGMGCTGA-3';
the reverse primer group consists of one or more of the following reverse primers:
primer R1: the primer sequence is 5'-CTTTAAGAGCGATAGCTTCAAG-3';
primer R2: the primer sequence is 5'-AGTTACATTTCCTGCACCTAC-3';
primer R3: the primer sequence is 5'-AGAAGATCATAACAAGCATTG-3';
primer R4: the primer sequence is 5 '-TTARAARGYAAAYCTKCCTCC-3';
primer R5: the primer sequence is 5'-TGATCAGTATCATTCTAATAGCA-3';
primer R6: the primer sequence is 5 '-GCAACCTGTGYCATACCTTCTTSTG-3'.
In a specific embodiment, the 25 μ L reaction of PCR amplification in step S3 contains: 5 XBuffer 4 uL, 5.0mM dNTP1 uL, 0.1-0.2mM mercaptoethylamine, 0.15-0.25mM theophylline, 10 uM primer pair mixture formed by the forward primer and the reverse primer, 0.5 uL of each primer pair mixture, 100ng of genome DNA and the balance dH2O。
The base preference of template molecules amplified by PCR can cause low amplification efficiency, more amplified fragments and repeated sequences are obtained, further, information of some sequence fragments is lost, the amplification yield is reduced, nonspecific bands appear in electrophoresis, and the result accuracy is reduced. Mercaptoethylamine and theophylline added into an amplification system possibly exert a synergistic effect, improve the specificity reaction of PCR, inhibit GC base pairing, promote effective combination of a primer and a template, improve the amplification efficiency and the amplification yield, reduce the generation of repeated sequences, provide more sequence information, further reduce the bias and the error, and improve the accuracy of analysis and identification and diversity evaluation results.
In a specific embodiment, the conditions for PCR amplification in step S3 are as follows: denaturation at 94 ℃ for 15-30s, annealing at 53-55 ℃ for 15-30s, and extension at 70-72 ℃ for 30s, with 35 cycles.
In a specific embodiment, the step S5 of constructing the phylogenetic tree uses an adjacency method, the repetition parameter is set to 100 times, and boottrap is set to 1000.
In a second aspect, the invention also provides the use of the above method of analysis of tick-borne pathogen diversity in microbial diversity analysis, including pathogen diversity analysis. The analysis method is suitable for various microorganism samples such as conventional samples, trace samples and the like, and different microorganism samples can be used for ensuring the richness of the samples and the authenticity of the abundance of different species of the samples through collection, extraction, primary screening, sequencing and analysis, so that the application range of microorganism diversity research can be expanded.
Preferably, examples of microorganisms for microbial diversity analysis include, but are not limited to: bacteria, fungi, archaea, pathogens, spirochetes, mycoplasma, rickettsia, chlamydia, actinomycetes, etc.
In a specific embodiment, the mass of the genomic DNA of the microbial sample obtained by lysis or extraction of the microbial sample is not less than 1 ng. Preferably, the mass of genomic DNA of the microbial sample is 10-500 ng.
The method for analyzing the diversity of tick-borne pathogens provided by the invention realizes the following beneficial effects by means of gene level detection and systematic evolution analysis:
1) the method deeply excavates the potential transmission rule of the tick-borne infectious disease, is convenient for relevant personnel to further know the epidemiological characteristics and the transmission rule of the tick-borne infectious disease, is more favorable for developing the targeted prevention and control work of the tick-borne infectious disease, and can further control and reduce the occurrence of diseases caused by cicada vector microorganisms.
2) The method has the advantages of simple sampling, high detection efficiency, high precision and high detection result accuracy; meanwhile, a plurality of samples are subjected to preliminary screening, sequencing and analysis, so that the diversity of the samples can be retained to the greater extent, the sample composition can be analyzed more comprehensively, the bias and the error of the traditional investigation method are reduced, the analysis and identification accuracy and the detection rate are improved, the distribution difference and the diversity of parasitic animals and pathogens of ticks and the pathogen complex infection condition can be well reflected, and the method is a reliable diversity analysis investigation technology.
3) The method has good flexibility and repeatability, can modify and/or add or subtract the types of detected pathogens according to needs, can simultaneously detect various different target molecules in a sample, greatly reduces the detection and analysis cost, and can be applied to quality monitoring, epidemiological investigation and early warning of microorganism samples.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and those skilled in the art can also obtain other drawings according to the drawings without creative efforts.
FIG. 1 is a phylogenetic analysis diagram of gltA gene fragment of tick merdian;
FIG. 2 is a phylogenetic analysis diagram of a tick merkexin rOmpA gene fragment;
FIG. 3 is a diagram of phylogenetic analysis of the tick-mediated rickettsia omp1 gene fragment;
FIG. 4 is a diagram of genetic evolution analysis of a fragment of the tick-borne Borrelia flaB gene;
FIG. 5 is a schematic diagram showing the constitution of the complex infection status of different parasitic ticks;
FIG. 6 is an electrophoretogram of PCR products of different experimental groups, A-example 1, B-comparative example 1, C-comparative example 2, D-comparative example 3.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art based on the embodiments of the present invention without any inventive step, also belong to the scope of protection of the present invention.
The various specific primers used in the PCR amplification of the present invention were synthesized by Nanjing Kingsrei Biotech. The Kit (QIAamp DNA Mini Kit) was purchased from Qiagen, Germany, the PCR reagent and DNA recovery and purification Kit was purchased from TaKaRa, Japan, the TA cloning Kit was purchased from Thermo Fisher, the DNA marker was purchased from Tiangen Biochemical technology (Beijing) Ltd, Escherichia coli (Escherichia coli) competent cell DH 5. alpha. was purchased from TOYOBO, Japan, and the Rsa I and Alu I enzymes were purchased from NEB, USA.
In a more specific embodiment, in step S3, a target gene with higher sensitivity and specificity is used to detect a pathogen carried by ticks and a PCR preliminary screening is performed. Specifically, the pyrexia maculans uses genes such as citrate synthase a (gltA), borrelia flagellin B (flaB), phosphodiesterase (glpQ), erigeron epicarp protein 1 (omp 1), and anaplasma as a target gene, such as major surface protein 2 (msp 2).
In a more specific embodiment, in the step S4 sequencing, rickettsial gltA gene primary screening positive samples were Restriction Fragment Length Polymorphism (RFLP) classified with Rsa i and Alu i, followed by gltA gene sequencing, and rickettsial outer membrane protein a (rlompa) gene amplification for sequencing confirmation.
In a more specific embodiment, in the step S4 sequencing, an amplification band of the gene of the amorphous msp2 and the Elektrom omp1 is recovered according to the instruction of a DNA recovery and purification kit, connected to a pCR2.1 vector, transformed into Escherichia coli, and screened and separated into positive clones on a plate containing IPTG and X-gal; after a large amount of culture, extracting plasmids, carrying out enzyme digestion by EcoRI to confirm the inserted fragment, and then sending the fragments to Nanjing Kingsrei Biotech company for bidirectional sequencing.
In a more specific embodiment, in step S5 bioinformatic analysis, BLAST homology alignment was performed on NCBI, homology analysis was performed using MEGA7.0, Clustal W, phylogenetic trees were constructed using the adjacency method with the repetition parameter set to 100 times and boottrap set to 1000; meanwhile, statistics and analysis are carried out on the diversity distribution conditions of parasitic animals and pathogens of the ticks and the composition of the complex infection conditions of different parasitic ticks.
In more specific embodiments, the genomic DNA of the microorganism sample is not limited to that obtained by lysis or extraction from the microorganism sample, but can be sample genomic DNA that has been obtained by lysis of the microorganism sample or extracted from the microorganism sample by others using techniques well known in the art. Further, the cells of the microbial sample providing the genomic DNA are prokaryotic cells or eukaryotic cells.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein, which are not described in detail, all employ conventional techniques in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related fields, and the equipment, reagent materials, etc., employed are conventional equipment and conventional reagent materials used by those skilled in the art. If the kit is used, the operation is carried out according to the instruction of the corresponding kit. And will not be described in detail herein.
The present invention will be described in further detail with reference to examples.
Example 1:
a method for analyzing tick-borne pathogen diversity, comprising the steps of:
1) collecting a specimen: collecting tick samples in the environment at the peak period of tick activity (4-6 months), wherein the tick samples comprise free ticks and parasitic ticks parasitic on the body surfaces of livestock and other animals. The free tick specimen is collected by a flag distribution method, and the parasitic tick specimen is collected by a livestock examination method. And recording information such as acquisition time, place, method, host type and the like.
Wherein, the tick specimen collecting region comprises CeLinGuo Lei union, Wulanscobu City, Bayan Yan Er City, Ordos City, Uhai City and Alason union; the collection host comprises cattle, sheep, wandering dog, horse and camel; tick species include procymidone (Dermacentor nuttalli), Hyalomma subarachnoidea (Hyalomma asiaticum), Hyalomma marginalis (h.
2) DNA extraction of the specimen: soaking collected tick insect specimen in sodium hypophosphite and 75% ethanol and iodophor for 5min, sterilizing, washing with sterile water, naturally drying, dissecting with a stereomicroscope one by one, taking out salivary gland, storing in PBS solution, extracting DNA with genome extraction Kit (QIAamp DNA Mini Kit), and storing at-20 deg.C.
3) Primary screening of tick-borne pathogens: and detecting the pathogen carried by the tick by adopting a target gene with higher sensitivity and specificity and carrying out PCR primary screening, wherein the primary screening comprises the steps of carrying out specific primer design, PCR amplification, detection and screening on the DNA.
Specifically, the physalis maculata uses citrate synthase a (gltA), borrelia uses phosphodiesterase (glpQ), flagellin B (flaB), ericsson coat protein 1 (omp 1), anaplasma uses major surface protein 2 (msp 2) and other genes as target genes.
The designed specific primers comprise a forward primer group and a reverse primer group; the forward primer set consists of one or more of the following forward primers:
primer F1: the primer sequence is 5'-CGAACTTACCGCTATTAGAATG-3';
primer F2: the primer sequence is 5'-TGGTGGAGCTCATAAGTTACA-3';
primer F3: the primer sequence is 5'-GCTAAGGAGTTAGCTTATGA-3';
primer F4: the primer sequence is 5 '-ATYAGTGSAAARTAYRTRCCAA-3';
primer F5: the primer sequence is 5'-GCTGAAGAGCTTGGAATGCACC-3';
primer F6: the primer sequence is 5 '-CATACGCTTCTGCYTTRGGMGCTGA-3';
the reverse primer group consists of one or more of the following reverse primers:
primer R1: the primer sequence is 5'-CTTTAAGAGCGATAGCTTCAAG-3';
primer R2: the primer sequence is 5'-AGTTACATTTCCTGCACCTAC-3';
primer R3: the primer sequence is 5'-AGAAGATCATAACAAGCATTG-3';
primer R4: the primer sequence is 5 '-TTARAARGYAAAYCTKCCTCC-3';
primer R5: the primer sequence is 5'-TGATCAGTATCATTCTAATAGCA-3';
primer R6: the primer sequence is 5 '-GCAACCTGTGYCATACCTTCTTSTG-3'.
The 25. mu.L reaction system for PCR amplification described above contained: 5 XBuffer 4 uL, 5.0mM dNTP1 uL, 0.15mM mercaptoethylamine, 0.2mM theophylline, 10 uM primer pair mixtures formed corresponding to the forward primer and the reverse primer, 0.5 uL of each primer pair mixture, 100ng of genomic DNA, and the balance dH2O。
The conditions for the PCR amplification are as follows: denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, for 35 cycles.
After the PCR amplification is finished, detecting the PCR product by using 2% agarose gel electrophoresis, and recovering and purifying the qualified PCR product by using the DNA recovery and purification kit.
4) Sequencing: the initial screening positive samples of the rickettsial gltA gene are classified by Restriction Fragment Length Polymorphism (RFLP) with Rsa I and Alu I, then subjected to gltA gene sequencing, and amplified to carry out sequencing confirmation on the rickettsial outer membrane protein A (rOmpA) gene.
Recovering an anaplasmic msp2 and an Elektrococcus omp1 gene amplification strip according to the instruction of a DNA recovery and purification kit, connecting the amplification strip to a pCR2.1 vector, transforming Escherichia coli, and screening and separating positive clones on a plate containing IPTG and X-gal; after a large amount of culture, extracting plasmids, carrying out enzyme digestion by EcoRI to confirm the inserted fragment, and then sending the fragments to Nanjing Kingsrei Biotech company for bidirectional sequencing.
5) And (3) biological information analysis: after sequencing, performing BLAST homology alignment on NCBI, performing homology analysis by using MEGA7.0 and Clustal W, and constructing a phylogenetic tree (shown in attached figures 1-4) by using an adjacent method, wherein a repetition parameter is set to 100 times, and Bootstrap is set to 1000 times; meanwhile, statistics and analysis are carried out on the diversity distribution conditions of parasitic animals and pathogens of the ticks and the composition of the complex infection conditions of different parasitic ticks.
Wherein, the sequence of the rickettsial gltA gene is 581bp, and the homology with R.raoultii (DQ365804) or R.aesclimani (KT873466) is 100 percent; the rOmpA genes are all 367bp long, and have 100% homology with R.raoultii (AH015610) or R.aesclimani (U83466). Borrelia flaB gene sequences with homology of 90.6-100% and 95.6-100% with the current lyme disease major pathogens b.garrinii (AB035602) and b.afzeliipko (NC008277), respectively. The sequence of the Elektrococcus omp1 gene has the highest homology with E.muris, only 65-69%.
FIG. 1 is a phylogenetic analysis diagram of gltA gene fragment of tick merdian. FIG. 2 is a phylogenetic analysis diagram of a tick merkezi rOmpA gene fragment. FIG. 3 is a diagram of phylogenetic analysis of the tick-mediated rickettsia omp1 gene fragment. FIG. 4 is a diagram of genetic evolution analysis of a fragment of the tick-borne Borrelia flaB gene.
Note:ameaning that there is no Chinese nomenclature so far.
As can be seen from fig. 1 to 4, phylogenetic trees constructed in the adjacency based on the rickettsial gltA and rmompa sequences showed that rickettsial infected with haemaphysalis pratensis, haemaphysalis asiatica and haemaphysalis marginalis all clustered with r. The phylogenetic tree constructed by the sequence of the ehrlichia flora omp1 shows that the ehrlichia flora infected by the prairie ticks is far away from the currently known ehrlichia flora, and forms an independent cluster. The phylogenetic tree of borrelia flora flaB sequences shows that 1 from the original ticks and hyalomma subadonovani in 10 obtained sequences is clustered with b.garrini; another 1 of the raw ticks clustered with b.afzelii; the remaining 8 sequences are in different branches from the flaB gene sequences of b.garinii and b.afzelii.
The omp1 polygene family is the major outer membrane protein gene group characteristic of the ehrlichia flora, and the copy number of the gene group is 22. The OMP1 protein structure has complete amino acid segment and incomplete segment, the N end and C end of the amino acid sequence are high conservation region, the central region is variable region, the designed primer can amplify the segment scattered in the genome simultaneously to raise PCR sensitivity. In the embodiment, the omp1 gene group is detected from only 1 tick sample, and the omp1 gene group has low homology with the known ehrlichia flora and is probably an undiscovered new species to be further researched and verified.
The statistical and analysis results of the diversity distribution conditions of parasitic animals and pathogens of the ticks are shown in table 1, wherein 3590 free ticks, 1695 parasitic ticks of cattle as hosts, 1541 parasitic ticks of sheep as hosts, 621 parasitic ticks of wandering dogs as hosts, 1820 parasitic ticks of horses as hosts and 942 parasitic ticks of camels as hosts are collected in tick specimens.
TABLE 1 analysis of the diversity of different parasitic ticks carrying pathogens
The composition of the complex infection status of different parasitic ticks was counted and analyzed as shown in fig. 5. FIG. 5 is a schematic diagram showing the complex infection status of different parasitic ticks. The results show that the infection ratio of 1 pathogen in free ticks is 27.5%, the infection ratio of 2 composite pathogens is 21.9%, the infection ratio of 3 composite pathogens is 15.0%, and the infection ratio of 4 composite pathogens is 7.4%; the infection ratio of 1 pathogen in the parasitic ticks of the cattle is 64.7 percent, and the infection ratio of the compound 2 pathogens is 3.4 percent; the infection ratio of 1 pathogen in the sheep parasitic ticks is 68.9 percent, the infection ratio of the compound 2 pathogens is 7.3 percent, and the infection ratio of the compound 3 pathogens is 3.0 percent; the infection ratio of 1 pathogen in the camel parasitic ticks is 79.1 percent, and the infection ratio of the compound 2 pathogens is 3.8 percent; the proportion of 1 pathogen infection in parasitic ticks of horses and wandering dogs is 59.6% and 63.0%, respectively. The complex infection of tick-borne diseases is more common, and the complex infection rate of free ticks is far higher than that of parasitic ticks.
Comparative example 1:
an analysis method of tick-borne pathogen diversity, which differs from example 1 only in that: in step 3), the 25. mu.L reaction system for PCR amplification contains: mu.L of 5 XBuffer 4. mu.L, 5.0mM dNTP 1. mu.L, 0.5. mu.L of each primer pair mixture formed by 10. mu.M forward primer and reverse primer, 100ng of genomic DNA, and the balance dH2O。
Comparative example 2:
an analysis method of tick-borne pathogen diversity, which differs from example 1 only in that: in step 3), the 25. mu.L reaction system for PCR amplification contains: 5 XBuffer 4 uL, 5.0mM dNTP1 uL, 0.15mM mercaptoethylamine, 10 uM primer pair mixtures formed corresponding to the forward primer and the reverse primer, 0.5 uL of each, 100ng of genomic DNA, and the balance dH2O。
Comparative example 3:
an analysis method of tick-borne pathogen diversity, which differs from example 1 only in that: in step 3), the 25. mu.L reaction system for PCR amplification contains: 5 XBuffer 4. mu.L, 5.0mM dNTP 1. mu.L, 0.2mM theophylline, 10. mu.M primer pair mixtures corresponding to the forward primer and reverse primer, 0.5. mu.L each, 100ng genomic DNA, and the balance dH2O。
Experimental example 1:
the experimental method comprises the following steps: according to the amplification systems of example 1 and comparative examples 1-3, the amplification products were collected and detected by 2% agarose gel electrophoresis under the same conditions, and the electrophoresis pattern is shown in FIG. 6. Each experimental group was provided with 3 replicates.
FIG. 6 is an electrophoretogram of PCR products of different experimental groups, A-example 1, B-comparative example 1, C-comparative example 2, D-comparative example 3. The results show that the electrophoretogram of the PCR product of example 1 is clear, bright, single and no non-specific band is generated; the method shows that the synergistic effect can be exerted by adding mercaptoethylamine and theophylline into a PCR reaction system, the specific reaction of PCR is improved, the generation of non-specific products is inhibited, the amplification efficiency and the amplification yield are improved, the generation of repeated sequences is reduced, the sequencing quality is higher, the bias and the error are reduced, and the accuracy of analysis and identification and diversity evaluation results is improved.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the same; while the invention has been described in detail and with reference to the foregoing embodiments, those skilled in the art will appreciate that; the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for analyzing tick-borne pathogen diversity, comprising:
s1, collecting a specimen;
s2, extracting DNA of the specimen;
s3, primary screening of tick-borne pathogens, wherein the primary screening is to complete detection and screening by carrying out specific primer design and PCR amplification on the DNA;
s4, sequencing;
and S5, analyzing biological information, wherein the biological information comprises homology analysis, construction of a phylogenetic tree, and statistics and analysis of the composition of the complex infection conditions of different ticks.
2. The method of claim 1, wherein: the S1 standard includes free ticks and parasitic ticks parasitized on the body surfaces of livestock and other animals; the free tick specimen is collected by adopting a flag distribution method, and the parasitic tick specimen is collected by adopting a livestock examination method.
3. The method of claim 1, wherein: the step of extracting DNA in S2 comprises the following steps: soaking the collected tick sample in sodium hypophosphite and 75% ethanol and iodophor for 5-10min, sterilizing, washing with sterile water, naturally drying, dissecting with a stereomicroscope one by one, picking salivary gland, storing in PBS solution, extracting DNA with genome extraction kit, and storing the extracted DNA at-20 deg.C.
4. The method of claim 1, wherein: in S3, the target genes in the preliminary screening step include: citrate synthase gltA, flagellin flaB, phosphodiesterase glpQ, outer membrane protein omp1 and major surface protein msp 2.
5. The method of claim 1, wherein: the specific primers designed in S3 comprise a forward primer group and a reverse primer group;
the forward primer set consists of one or more of the following forward primers:
primer F1: the primer sequence is 5'-CGAACTTACCGCTATTAGAATG-3';
primer F2: the primer sequence is 5'-TGGTGGAGCTCATAAGTTACA-3';
primer F3: the primer sequence is 5'-GCTAAGGAGTTAGCTTATGA-3';
primer F4: the primer sequence is 5 '-ATYAGTGSAAARTAYRTRCCAA-3';
primer F5: the primer sequence is 5'-GCTGAAGAGCTTGGAATGCACC-3';
primer F6: the primer sequence is 5 '-CATACGCTTCTGCYTTRGGMGCTGA-3';
the reverse primer group consists of one or more of the following reverse primers:
primer R1: the primer sequence is 5'-CTTTAAGAGCGATAGCTTCAAG-3';
primer R2: the primer sequence is 5'-AGTTACATTTCCTGCACCTAC-3';
primer R3: the primer sequence is 5'-AGAAGATCATAACAAGCATTG-3';
primer R4: the primer sequence is 5 '-TTARAARGYAAAYCTKCCTCC-3';
primer R5: the primer sequence is 5'-TGATCAGTATCATTCTAATAGCA-3';
primer R6: the primer sequence is 5 '-GCAACCTGTGYCATACCTTCTTSTG-3'.
6. The method of claim 1, wherein: the 25. mu.L reaction system for PCR amplification in S3 contained: 5 XBuffer 4 uL, 5.0mM dNTP1 uL, 0.1-0.2mM mercaptoethylamine, 0.15-0.25mM theophylline, 10 uM primer pair mixture formed by the forward primer and the reverse primer, 0.5 uL of each primer pair mixture, 100ng of genome DNA and the balance dH2O。
7. The method of claim 1, wherein: the conditions for PCR amplification in S3 were as follows: denaturation at 94 ℃ for 15-30s, annealing at 53-55 ℃ for 15-30s, and extension at 70-72 ℃ for 30s, with 35 cycles.
8. The method of claim 1, wherein: and constructing a phylogenetic tree in the S5 by adopting an adjacency method, setting repetition parameters as 100 times, and setting Bootstrap as 1000.
9. Use of the method of analysis of tick-borne pathogen diversity according to any of claims 1-8 in microbial diversity analysis, including pathogen diversity analysis.
10. Use according to claim 9, characterized in that: the mass of the genome DNA of the microorganism sample is not less than 1ng, and the genome DNA is obtained by cracking or extracting the microorganism sample.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115553287A (en) * | 2022-10-27 | 2023-01-03 | 北京标驰泽惠生物科技有限公司 | Tick preserving fluid and preparation method and application thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101613757A (en) * | 2009-07-09 | 2009-12-30 | 中国农业科学院兰州兽医研究所 | Detect the test kit and the detection method of Lyme disease cause of disease in the tick body |
| CN103173567A (en) * | 2013-03-27 | 2013-06-26 | 中国检验检疫科学研究院 | Suspension-array multiple non-diagnostic detection method for tick-borne pathogens |
| US20130323795A1 (en) * | 2007-01-10 | 2013-12-05 | General Electric Company | Endonuclase-assisted isothermal amplification using contamination-free reagents |
| WO2014197607A1 (en) * | 2013-06-05 | 2014-12-11 | The Regents Of The University Of California | Detection of tick-borne diseases |
| CN109280699A (en) * | 2018-09-06 | 2019-01-29 | 海盐昆植生物技术有限公司 | A kind of new varieties identification method based on ddRAD method |
| US20190093149A1 (en) * | 2017-09-27 | 2019-03-28 | The United States Of America, As Represented By The Secretary Of Agriculture | Assays for detecting multiple tick-borne pathogens |
| WO2020186223A1 (en) * | 2019-03-14 | 2020-09-17 | The Broad Institute, Inc. | Sherlock assays for tick-borne diseases |
| CN113444773A (en) * | 2021-08-17 | 2021-09-28 | 黑龙江省牡丹江林业中心医院 | Method and kit for detecting tick pathogen nucleic acid based on liquid chip |
-
2021
- 2021-12-08 CN CN202111491616.XA patent/CN114058716A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130323795A1 (en) * | 2007-01-10 | 2013-12-05 | General Electric Company | Endonuclase-assisted isothermal amplification using contamination-free reagents |
| CN101613757A (en) * | 2009-07-09 | 2009-12-30 | 中国农业科学院兰州兽医研究所 | Detect the test kit and the detection method of Lyme disease cause of disease in the tick body |
| CN103173567A (en) * | 2013-03-27 | 2013-06-26 | 中国检验检疫科学研究院 | Suspension-array multiple non-diagnostic detection method for tick-borne pathogens |
| WO2014197607A1 (en) * | 2013-06-05 | 2014-12-11 | The Regents Of The University Of California | Detection of tick-borne diseases |
| US20190093149A1 (en) * | 2017-09-27 | 2019-03-28 | The United States Of America, As Represented By The Secretary Of Agriculture | Assays for detecting multiple tick-borne pathogens |
| CN109280699A (en) * | 2018-09-06 | 2019-01-29 | 海盐昆植生物技术有限公司 | A kind of new varieties identification method based on ddRAD method |
| WO2020186223A1 (en) * | 2019-03-14 | 2020-09-17 | The Broad Institute, Inc. | Sherlock assays for tick-borne diseases |
| CN113444773A (en) * | 2021-08-17 | 2021-09-28 | 黑龙江省牡丹江林业中心医院 | Method and kit for detecting tick pathogen nucleic acid based on liquid chip |
Non-Patent Citations (7)
| Title |
|---|
| DJURSUN KARASARTOVA ET AL.: "Bacterial and protozoal pathogens found in ticks collected from humans in Corum province of Turkey", 《PLOS NEGL TROP DIS》, vol. 12, no. 4, pages 1 - 19 * |
| ZHENYU SHEN ET AL.: "Development of a tick-borne pathogen QPCR panel for detection of Anaplasma, Ehrlichia, Rickettsia, and Lyme disease Borrelia in animals", 《J MICROBIOL METHODS》, vol. 151, pages 83 - 89 * |
| 乌兰图雅 等: "内蒙古中西部草原蜱媒病原体多样性及基因型分析", 《中国寄生虫学与寄生虫病杂志》, vol. 39, no. 01, pages 29 - 31 * |
| 刘丹 等: "内蒙古地区2015—2019年不同寄生蜱的种群分布及病原体多样性分析", 《现代预防医学》, vol. 48, no. 08, pages 1345 * |
| 刘丹 等: "内蒙古大兴安岭林区牙克石段的蜱种及蜱携病原体感染调查", 《中国人兽共患病学报》, vol. 37, no. 09, pages 845 - 850 * |
| 徐蕾 等: "PCR增强剂在核酸体外扩增检测技术的研究进展", 《生物技术进展》, vol. 10, no. 02, pages 139 * |
| 黄邵军 等: "基于高通量测序技术分析浙江省余姚市蜱中携带细菌菌群多样性", 《疾病监测》, vol. 35, no. 07, pages 642 - 645 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115553287A (en) * | 2022-10-27 | 2023-01-03 | 北京标驰泽惠生物科技有限公司 | Tick preserving fluid and preparation method and application thereof |
| CN115553287B (en) * | 2022-10-27 | 2023-08-08 | 北京标驰泽惠生物科技有限公司 | Tick preservation solution and preparation method and application thereof |
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