CN106434861A - Primer capable of distinguishing and detecting low pathogenic babesia motasi and kit - Google Patents

Primer capable of distinguishing and detecting low pathogenic babesia motasi and kit Download PDF

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CN106434861A
CN106434861A CN201610338651.0A CN201610338651A CN106434861A CN 106434861 A CN106434861 A CN 106434861A CN 201610338651 A CN201610338651 A CN 201610338651A CN 106434861 A CN106434861 A CN 106434861A
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babesia
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primer
mohs
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刘爱红
刘军龙
关贵全
罗建勋
殷宏
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Lanzhou Veterinary Research Institute of CAAS
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Abstract

The invention discloses a primer capable of distinguishing and detecting low pathogenic babesia motasi and a kit. The primer sequences are SEQ ID No.1 and SEQ ID No.2, and the primer in the kit is a probe primer SEQ ID No.3. The primer has the advantages of being high in sensitivity, speed and specificity when used for detection and is widely applied in qualitative and quantitative measurement of pathogene.

Description

Primer and the kit of detection low pathogenicity babesia motasi can be distinguished
Technical field
The present invention relates to a kind of primer detecting parazoon to and kit.Exactly, the present invention relates to one can Distinguish primer and the detection kit of detection low pathogenicity babesia motasi.
Background technology
It has been reported that Babesia have more than 100 and to plant, wherein name and be recognized and infect the Babesia of sheep and have sheep Babesia（B. ovis）, Mohs Babesia（B. motasi）, coarse Babesia（B. crassi）With some BABEIs this Worm species indeterminate.Sheep Babesia is by Rh（Rhipicephalus）Hard tick propagate, be small-sized BABEI pathogenic by force This worm, its biological characteristics is basically identical with ox Babesia；Coarse Babesia be large-scale BABEI that a kind of pathogenicity is low this Worm, breeds in the way of quadripartion or binary fission, and its communication media tick and circulation way are unclear, at present only in Iran With Turkey's discovery；And Mohs Babesia be the Babesia infecting sheep is distributed the most extensively, characteristic of division the most complicated Polypide, they are pathogenic different because of its difference being distributed, and as stronger in having of separating with Mediterranean in southern Europe causes a disease Property, and separate in Northern Europe pathogenic more weak, and both Mohs Babesias are all by Haemaphysalis punctata（Haemaphysalis punctata）The large-scale Babesia propagated, at the Mohs Babesia that Sahara separates, between known in form Mohs Babesia and sheep Babesia between, but its communication media is not the tick of Haemaphysalis.Cause according to Babesia Characteristic of disease, the difference to sheep and goat appeal, morphology, serology and molecular classification Evolution analysis result, Uilenberg (2006) thinks and at least contains two or more kinds or subspecies in Mohs Babesia.
China before 1996, the report of only 2-3 example sheep Babesia Gibsoni, then respectively in Gansu, Hebei, the Liao Dynasty Rather, there is the report of morbidity in the ground such as Yunnan, Shanxi, Henan and Xinjiang.The Epidemic Scope of this disease is in expanding trend, Ke Nengyu year by year Tick victor distribution extensively have certain relation.This research team is in last century end, to China's part provinces and regions sheep BABEI This parasitosis has carried out reconnaissance investigation.By blood inoculation and tick propagation experimentation, successively in China Liaoning, Hebei, Gansu, The ground such as Hubei, Xinjiang separates the Babesia strains obtaining 9 strain sheep.Epidemiology survey finds that this disease is popular extensively in China General, some local harm are serious, and positive rate is more than 30%, and breeding sheep and lamb that other places is introduced by cause of disease are pathogenic relatively strong, sternly Remake the breed improvement of about sheep and the development of sheep husbandry.With pyriform worm（Including Babesia and Taylor worm）Ribosomes 18S RRNA gene carries out molecular classification research discovery, Lintan, Gansu, fiber crops when, Zhuhe, sky Ning County and Hebei, Hubei and Liaoning point Affiliation close to strain all has with Mohs Babesia, thus name for Mohs Babesia (Liu et al., 2007; Guan et al., 2009), and carry out with ribosomes 28S rRNA and transcribed spacer ITS gene, mitochondria CoI gene etc. The research of molecular classification finds:The Mohs Babesia of China can be divided into again two Asias to prop up；One is the Mohs bar of low pathogenicity The Lintan strain of bass worm and Mohs Babesia God blessings strain, two be highly pathogenic Mohs Babesia Ning County's strain and Mohs BABEI this Worm Hebei strain (Niu et al., 2009a).2010, Guan etc. (Guan et al., 2010e) confirm Mohs BABEI this The positive serum of sheep is infected in worm Lintan strain whole worm antigen and Mohs Babesia God blessings strain a strong cross reaction, and with not The positive serum of family name's Babesia Ning County's strain and Hebei strain does not has cross reaction.The conclusion phase of these results and Uilenberg Symbol：The Mohs Babesia of China is likely at least there are two different subspecies.Confirm through propagation experimentation, Mohs Babesia Communication media between different subspecies also differs, and the communication media of some subspecies is not yet studied clear at present.
The Babesia Gibsoni of people is reported in nineteen sixty, mainly the Babesia disease pathogen vole by two kinds of infection people the earliest Babesia（B. microti）With difference Babesia (B. divergens) cause, patient is often accompanied by severe anemia, intermittently The concurrent various syndromes such as high heat, DOMS, tic, can cause death during severe infections.In recent years research showed in the past not Infect the Babesia of people, due to entomophila distribution constantly extensively, host's model of the pathogenic cause of disease of carrier's Babesia Enclosing and also expanding, the new pathogenic cause of disease of people's Babesia occurs continuous（Schnittger L,et al., 2012）, this disease Progressively become a kind of global mankind and newly send out parasitic disease by public attention.Recording according to current document, existing mainly have four Big class Babesia has the ability of Zoonosis, and the first kind is vole Babesia, and it is a kind of close with rodent Related Zoonosis parasite, U.S. most people infect Babesia case and are caused by it；Equations of The Second Kind is new life NameB. duncani, form is similar to vole Babesia, but the two affiliation is farther out；3rd class includes difference BABEI This worm（B. divergence）With class difference Babesia,B. venatorum（Also referred to as EU1）；4th class is Korea S Newfound Babesia KO1 type, much like with Chinese Mohs Babesia Hebei pathogen strain form, molecular classification analysis Show that both homologys are up to 98%, and nucleotide sequence only differs from 13 bases.
Present stage, China is primarily present sheep Babesia Mohs Babesia and the sheep Babesia U sp of two kinds Xinjiang Strain.But between the different Local Isolates of Mohs Babesia, owing to it is pathogenic, communication media and serology have one Determining difference, binding molecule taxology shows that Mohs Babesia exists two different subspecies, i.e. the Mohs BABEI of low pathogenicity This worm subspecies, such as Lintan strain and God blessings strain；Highly pathogenic Mohs Babesia subspecies, such as Ning County's strain and Hebei strain.
The goldstandard of Babesia detection is the mode using blood film dyeing microscopic examination, but blood film dyeing microscopic examination result is by very Multifactorial impact, recall rate is low, and accuracy is poor.Standard PCR, sleeve type PCR and real-time based on SYBR Green dyestuff Though the nuclei aoid methods such as fluorescent PCR have an application, but Standard PCR to there is sensitivity low, poor specificity, the shortcoming such as detection cycle length； Sleeve type PCR is highly sensitive, but the PCR of two steps reacts loaded down with trivial details, easily pollutes, and is not suitable for large-scale epidemiology survey.
Content of the invention
The present invention provides one that prior art can be overcome not enough, can distinguish detect low pathogenicity babesia motasi primer and Kit.
Gene order is by the primer of the detection the distinguished low pathogenicity babesia motasi of the present invention：SEQ ID No.1 and SEQ ID No.2.
Include that sequence is in the kit of the detection the distinguished low pathogenicity babesia motasi of the present invention：SEQ ID No.1、 The primer of SEQ ID No.2 and probe primer SEQ ID No.3.
For convenience of using, also have in the kit of the detection the distinguished low pathogenicity babesia motasi of the present invention：Standard male Property plasmid template pGEM-Teasy-18S rRNABLT and pGEM-Teasy-18S rRNABNX.
The condition that the kit of the detection the distinguished low pathogenicity babesia motasi of the present invention enters performing PCR amplification is：
The condition expanding with primer and the kit of the present invention is：
The condition of the Mohs Babesia PCR amplification of low pathogenicity is：95 DEG C of denaturations 30s, then 95 DEG C of 5s, 58 DEG C of 10s, 72 DEG C of 30s, 40 circulations；
The condition of highly pathogenic Mohs Babesia PCR amplification is：95 DEG C of denaturations 30s, then 95 DEG C of 5s, 60 DEG C of 10s, 72 DEG C of 30s, 40 circulations.
Present invention suggestion when concrete application should have following three part compositions：1）The Mohs bar of specific detection low pathogenicity The standard positive plasmid template pGEM-Teasy-18S rRNABLT of bass worm subspecies, forward primer SEQ ID No.1 and reversely Primer SEQ ID No.2（10μΜ）Each 0.5 μ L, fluorescence probe SEQ ID No.3（5μM）1.0μL；2）Specific detection is high to be caused The standard positive plasmid template pGEM-Teasy-18S rRNABNX of the Mohs Babesia subspecies of characteristic of disease, forward primer SEQ ID No.4 and reverse primer SEQ ID No.5（10μΜ）Each 0.5 μ L, fluorescence probe SEQ ID No.6（5μM）1.0μL；3）It is suitable for Public reagent fluorescence quantitative PCR reaction solution, negative quality control standard product in two kinds of detection methods.Quantitative fluorescent PCR therein is anti- Answer liquid by Premix Ex TaqTM（2×）12.5 μ L, ROX Reference Dye II（50×）0.5 μ L, sterile purified water 8.0μL.
The present invention is a kind of TaqMan Real-Time Fluorescent Quantitative PCR Technique.Fluorescence PCR assay sensitiveness height, high specificity, inspection Degree of testing the speed is fast.TaqMan probe fluorescent quantitative PCR technique is that addition one is special while PCR amplification adds pair of primers The fluorescence probe of property, this probe is an oligonucleotides, and two ends mark a reporter fluorescence group and a quenching fluorescence base respectively Group.When probe is complete, the fluorescence signal that reporter group is launched is quenched group absorptions；When just starting, probe combines at DNA On any one strand；During PCR amplification, probe is digested degraded, makes report by 5 ' to 3 ' 5 prime excision enzyme activities of Taq enzyme Fluorophor separates with quenching fluorescence group, thus fluorescence monitoring system can receive fluorescence signal, i.e. often expands a DNA Chain, just has a fluorescence molecule to be formed, it is achieved that the accumulation of fluorescence signal forms Complete Synchronization with PCR primer.The method is sensitive Property height, high specificity, detection cycle are short, compared with Standard PCR, there is not the harm to human body of cross pollution and nucleic acid dye It etc. shortcoming, is gradually applied to the detection of various pathogen in recent years.
The present invention is with pyriform worm（Including Taylor worm and Babesia）Ribosomes 18S rRNA is target, and excavation is suitable as For the target gene 18S rRNA of pathogeny antidiastole, set up can distinguish China in animal reservoir and natural medium tick body now simultaneously The Mohs Babesia Lintan of the different subspecies of the Mohs Babesia deposited, i.e. low pathogenicity, God blessings strain and highly pathogenic Mohs Babesia Ning County, the TaqMan real-time fluorescence quantitative PCR diagnostic techniques of Hebei strain.This technology is highly sensitive with it, speed fast, The advantages such as high specificity, can be widely applied in the qualitative and quantitative context of detection of pathogen.And the method is to close System carries out amplification and real-time detection, avoids environmental pollution, shorten the detection time, be particularly suited for acute and subclinical The detection of the batch samples infecting and diagnosis.
Brief description
The Mohs Babesia subspecies real-time fluorescence quantitative PCR specific amplification curve of Fig. 1 low pathogenicity.
The highly pathogenic Mohs Babesia subspecies real-time fluorescence quantitative PCR specific amplification curve of Fig. 2.
Detailed description of the invention
1. construction recombination plasmid standard items.
（1）According to the Mohs Babesia Lintan strain ribosomes 18S rRNA gene order infecting sheep, use Primer Premier 5.0 and Oligo 7 designs primer, and the purpose clip size of pre-acquired is Mohs Babesia subspecies Lintan strain respectively 661bp and Mohs Babesia subspecies Ning County strain 666bp, by precious biotinylated biomolecule engineering（Dalian）Co., Ltd synthesizes.Primer pair Sequence is：BLTC-S（Forward primer）：5 '-AGAAACGGCTACCACATC-3 ' SEQ ID No.7, BLTC-AS（Reversely draw Thing）： 5-CTTGCGACCATACTCCC-3’ SEQ ID No.8.
（2）The Mohs Babesia subspecies Lintan strain that preserves with laboratory and the DNA of Mohs Babesia subspecies Ning County strain Expanding for template, using the PCR reaction system of 50 μ L, reaction condition is 94 DEG C of denaturations 4min, then 94 DEG C of 1min, 56 DEG C 1min, 72 DEG C of 1min, 30 circulations, 72 DEG C extend 7min.Take 5 μ L amplified production agarose gel electrophoresis to identify.Identify Reclaim purification kit for positive PCR primer Ago-Gel（TaKaRa, Dalian）It is purified recovery, product will be reclaimed It is connected to pGEM-Teasy carrier（Promega, America）After be transformed into competent escherichia coli cell JM-109（TaKaRa, Dalian）In, carry out blue hickie screening, picking positive colony（Day shift）Enter performing PCR and order-checking is identified.
（3）Plasmid is extracted to the clone being accredited as the positive, uses NanoDrop 2000/2000C（Thermo Scientific, America）Ultramicrospectrophotometer measures plasmid concentration, and is converted into copy/μ L, in this, as Plasmid standard.The plasmid standard that will build, serial dilution is final concentration of 1.0 × 109-1.0×100Copy/μ L, To carry out real-time fluorescence quantitative PCR amplification.
2. real time fluorescence quantifying PCR method
（1）Responsiveness
1）The Mohs Babesia subspecies Lintan of low pathogenicity, God blessings strain:Quantitative PCR reaction system is 25 μ L：Premix Ex TaqTM（2×）12.5 μ L, forward primer SEQ ID No.1 and reverse primer SEQ ID No.2（10μΜ）Each 0.5 μ L, fluorescence Probe SEQ ID No.3（5μM）1.0 μ L, ROX Reference Dye II (50 ×) 0.5 μ L, genomic DNA template 2 μ L, go out Bacterium distilled water 8.0 μ L.Reaction condition is：95 DEG C of denaturations 30s, then 95 DEG C of 5s, 58 DEG C of 10s, 72 DEG C of 30s, 45 are followed Ring.
2）Highly pathogenic Mohs Babesia subspecies Ning County, Hebei strain:Quantitative PCR reaction system is 25 μ L：Premix Ex TaqTM（2×）12.5 μ L, forward primer SEQ ID No.4 and reverse primer SEQ ID No.5（10μΜ）Each 0.5 μ L, glimmering Light probe SEQ ID No.6（5μM）1.0 μ L, ROX Reference Dye II (50 ×) 0.5 μ L, genomic DNA template 2 μ L, Sterile purified water 8.0 μ L.Reaction condition is：95 DEG C of denaturations 30s, then 95 DEG C of 5s, 60 DEG C of 10s, 72 DEG C of 30s, 45 are followed Ring.1）With 2) reaction system in the middle of, at the end of each elongating temperature circulating set fluorescence signal acquisition point carry out Detection in real time, each template concentrations is done 3 repetitions, the calculating coefficient of variation of averaging, is tested and be all provided with negative control.Its result shows Show：Template amount is linearly related to corresponding Ct value, expands why curve is S-type.
（2）Specific analysis
1) the Mohs Babesia subspecies Lintan of low pathogenicity, God blessings strain:Utilize above-mentioned set up real-time fluorescence quantitative PCR side Method, each sample detects Mohs Babesia subspecies Lintan strain and God blessings strain in triplicate respectively；Set Mohs Babesia simultaneously The strain of subspecies Ning County and Hebei strain, sheep Babesia U sp Xinjiang Strain, the Theileria luwenshuni of sheep, T.uilenbergi, sheep Taylor Worm, sheep are negative control without slurry, clean sheep genome and aqua sterilisa.Result shows:Only 1-Mohs Babesia subspecies Lintan strain, 2-Mohs Babesia subspecies God blessings strain are positive, Mohs Babesia subspecies Ning County's strain and Hebei strain, sheep bar Bass worm species indeterminate Xinjiang Strain, other related cause of diseases of sheep and water are all negative（Fig. 1）.
2) highly pathogenic Mohs Babesia subspecies Ning County, Hebei strain:Utilize above-mentioned set up real time fluorescent quantitative PCR method, each sample detects Mohs Babesia subspecies Ning County's strain and Hebei strain in triplicate, sets Mohs Babesia simultaneously The strain of subspecies Lintan and God blessings strain, sheep Babesia U sp Xinjiang Strain；The Theileria luwenshuni of sheep, T.uilenbergi, sheep Taylor Worm, sheep are negative control without slurry, clean sheep genome and aqua sterilisa.Result shows:Only 1-Mohs Babesia subspecies Ning County's strain and 2-Mohs Babesia subspecies Hebei strain are positive, and Mohs Babesia subspecies Lintan strain, God blessings strain, sheep Other related cause of diseases of Babesia U sp Xinjiang Strain and sheep and water are all negative（Fig. 2）.
（3）Stability is analyzed with repeatability
For different detection methods, when being analyzed, selected concentration is also different；Sub-to the Mohs Babesia of low pathogenicity Planting Lintan, God blessings strain, taking concentration is 1.0 × 109-1.0×107The standard items of 3 concentration of copy/μ L carry out analysis of stability Analysis；To highly pathogenic Mohs Babesia subspecies Ning County, Hebei strain, taking concentration is 1.0 × 109-1.0×108Copy/μ L The standard items of 2 concentration carry out stability analysis.Two kinds of methods all first carry out batch interior replica test, dilute same batch Standard items, each concentration duplicate detection four times, analyze Ct value the coefficient of variation.Then replica test between carrying out again criticizing, to 3 The standard items of the dilution of individual different batches detect, and judge its stability through analyzing the Ct value coefficient of variation.It is iteratively repeated this Two test three times, it is thus achieved that data difference nonsignificance, illustrate that the testing result between its different batches has it comparable Property, there is good repeatability.
<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
<120>Primer and the kit of detection low pathogenicity babesia motasi can be distinguished
<160> 8
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence（Forward primer OBLMT-S）
<400>
tgccggttca tgaatttg 18
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence（Reverse primer OBLMT-AS）
<400>
gcaagtcata ctgcgtatc 19
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence（Probe primer OBLMT-Prob）
<400>
ctgcgtcctt catcgttgtg t 21
<210> 4
<211> 19
<212> DNA
<213>Artificial sequence（Forward primer OBHN-S）
<400>
aggcagtaat tgctaagtg 19
<210> 5
<211> 19
<212> DNA
<213>Artificial sequence（Reverse primer OBLMT-AS）
<400>
gcgaaaccaa atggtaatc 19
<210> 6
<211> 20
<212> DNA
<213>Artificial sequence（Probe primer OBHN-Prob）
<400>
cgctcactaa tggagaccgc 20
<210> 7
<211> 18
<212> DNA
<213>Artificial sequence（Forward primer BLTC-S）
<400>
agaaacggct accacatc 18
<210> 8
<211> 17
<212> DNA
<213>Artificial sequence（Reverse primer BLTC-AS）
<400>
cttgcgacca tactccc 17

Claims (3)

1. can distinguish the primer pair of detection low pathogenicity babesia motasi, it is characterised in that gene order is by primer：SEQ ID No.1 and SEQ ID No.2.
2. can distinguish the kit of detection low pathogenicity babesia motasi, it is characterised in that in kit, include that sequence is：SEQ ID No.1, the primer of SEQ ID No.2 and probe primer SEQ ID No.3.
3. the using method of the kit of the detection the distinguished low pathogenicity babesia motasi described in claim 2, its feature exists It in the condition entering performing PCR amplification is：95 DEG C of denaturations 30s, then 95 DEG C of 5s, 58 DEG C of 10s, 72 DEG C of 30s, 45 circulations.
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CN109593826A (en) * 2019-01-14 2019-04-09 中国农业科学院兰州兽医研究所 A kind of Mohs Babesia detection kit

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