CN106119355A - The method of Cryptosporidium parvum oocysts suspended in a kind of FTA/PCR detection feces - Google Patents

The method of Cryptosporidium parvum oocysts suspended in a kind of FTA/PCR detection feces Download PDF

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CN106119355A
CN106119355A CN201610492725.6A CN201610492725A CN106119355A CN 106119355 A CN106119355 A CN 106119355A CN 201610492725 A CN201610492725 A CN 201610492725A CN 106119355 A CN106119355 A CN 106119355A
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feces
cryptosporidium
cryptosporidium parvum
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王荣军
张振杰
张龙现
张素梅
王海燕
菅复春
宁长申
胡苏辉
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Henan Agricultural University
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Abstract

The invention belongs to biological technical field, relate to a kind of based on the method for Cryptosporidium parvum oocysts suspended in FTA card detection feces.Step is as follows: purified pool Cryptosporidium parvum oocysts suspended, after counting dilution, is applied on FTA card standby after quantitative contamination feces；Positive fecal specimens is applied on FTA card standby；Fresh excreta is collected standby with FTA card；FTA card punches, and eluting FTA disk；With cleaned FTA card as DNA profiling, carry out PCR amplification with the primer designed according to Cryptosporidium 18SrRNA, then carry out agarose gel electrophoresis detection.The present invention instead of traditional commerce faeces DNA and extracts step loaded down with trivial details during test kit extraction DNA and fancy price, has with low cost, conveniently advantage.

Description

The method of Cryptosporidium parvum oocysts suspended in a kind of FTA/PCR detection feces
Technical field
The invention belongs to biological technical field, relate to a kind of based on the method for Cryptosporidium parvum oocysts suspended in FTA card detection feces.
Background technology
Cryptosporidiosis is new the Arbo infectious disease caused by Cryptosporidium, is classified as the whole world by World Health Organization (WHO) One of six big diarrhoeal diseasess.Cryptosporidium can infect 280 many animals including people, its egg capsule be widely present in animal and In environment, cause extensive outbreak of epidemic usually through water or food transmission.Cryptosporidiosis can in milk cows wide-scale distribution stream OK, causing the weight loss of milch cow, milk yield reduces, and causes serious economic loss to the development of dairy, can propagate simultaneously , there is higher public health risk, humans and animals health caused serious threat in multiple zoonosis.The quickest, simple Just, find that Cryptosporidium is the key of this disease preventing and treating accurately.
At present, the detection method of Cryptosporidium has the methods such as scatology, immunology, molecular biology.
The Food habit method of Cryptosporidium, mainly has staining and floating method, owing to Cryptosporidium volume is small, directly Footpath, in 4.5 μm-5.5 μm, is difficult to distinguish under the microscope, and recall rate is low and is easily caused erroneous judgement.At present, the most established right The panimmunity detection method of cryptosporidiosis, existing detection method mainly has: enzyme-linked immunosorbent assay (ELISA), Immunofluorescence (IFA), immunoblot assay (Western blotting) etc..Along with the development of molecular biology, both at home and abroad Some scholars utilize polymerase chain reaction (PCR), gene chip equimolecular biology techniques in succession, divide Cryptosporidium nucleic acid Son has carried out substantial amounts of research, and makes important progress, and in Cryptosporidium parvum oocysts suspended detection, the diagnosis of cryptosporidiosis, popular Sick learning in investigation and Species identification and worm strain typing is used widely.
The extracted amount of Cryptosporidium parvum oocysts suspended DNA and purity, directly affect the expanding effect of downstream PCR, the extraction of its DNA Mainly affected by of both egg capsule cracking degree and Impurity elution ability.And cow manure exists a large amount of intestinal and comes off epithelium The PCR inhibitive factor such as cell, food debris, humic acid, protein.Therefore Cryptosporidium in a kind of optimal cow manure is weighed The extracting method of egg capsule DNA, has highly important effect to the detection of feces Cryptosporidium.Mainly pass through business both at home and abroad at present Change faeces DNA and extract test kit, obtain Cryptosporidium parvum oocysts suspended DNA in cow manure, but, this method extracts DNA step Loaded down with trivial details, the longest, during DNA extraction, it is easily formed cross-contamination, thus has influence on detection accuracy.Additionally, use big Amount chemical reagent, to environment.
FTA (Flinders Technology Associates) is a kind of cotton fiber card through chemistry special handling, It is widely used at present in the DNA profiling preparation of molecular biology research the most both at home and abroad.FTA filter membrane is by denaturant and chela Mixture permeates on fibre substrate, can automatically be cracked after capturing microorganism or cell, and sample DNA adsorbs at FTA On card, by buffer solution, remove other compositions in sample, and the FTA card having adsorbed DNA can be used for directly as template PCR reacts.And the infectious agent in sample within FTA card upper 5 second just can complete deactivation, there is higher biological peace Quan Xing.
In recent years, both at home and abroad about FTA/PCR method detection Cryptosporidium parvum oocysts suspended applied research be limited only to beverage, earth's surface The water Cryptosporidium parvum oocysts suspendeds such as water, and the substantial amounts of humic acid contained in fecal specimens, food debris, the gut epithelium that comes off The impurity such as cell, protein seriously inhibits the PCR with FTA card as DNA profiling to expand.Normal according to Whatman business recommendations Rule elution process cannot wash above-mentioned PCR inhibitive factor, washes the side that the PCR inhibitive factor in feces is numerous research To, but result all cannot preferably reach cleaning performance, and the present invention, by adding different organic solvents, improves FTA card and uses Cleaning way in description, washes the PCR inhibitive factor in feces, allows and is loaded with the FTA card of cryptosporidium dna after cleaning DNA profiling can be directly used as, carry out follow-up PCR and expand equimolecular biological study.
Summary of the invention
It is an object of the invention to provide and a kind of rapidly and efficiently detect the side of Cryptosporidium parvum oocysts suspended in feces based on FTA/PCR Method, has high sensitive, high biological safety.
For achieving the above object, the present invention is by the following technical solutions:
The method of Cryptosporidium parvum oocysts suspended in a kind of FTA/PCR detection feces, step is as follows:
(1) purified pool Cryptosporidium parvum oocysts suspended, after counting dilution, is applied on FTA card standby after quantitative contamination feces；
(2) positive fecal specimens is applied on FTA card standby；
(3) fresh excreta is collected with FTA card standby；
(4) punch on the FTA card in step (1), (2) and (3), and eluting FTA disk；
(5) with cleaned FTA card as DNA profiling, PCR is carried out with the primer designed according to Cryptosporidium 18SrRNA Amplification, then carries out agarose gel electrophoresis detection.
In described step (1), purified pool method uses saturated saline floatation and cesium chloride gradient centrifugation jointly to make With, quantitative contamination refers to the egg capsule of collection is diluted to 1 × 10-1/mL-1×105/ mL egg capsule suspension, takes 1mL and is mixed into 1g feces In.
Described step (2) positives feces refers to cryptosporidium andersoni positive feces, giardia lamblia positive feces, microsporidian sun Two or more in property feces or coccidiosis positive feces.
In described step (4), elution step is:
A. the FTA disk of acquisition is respectively put in centrifuge tube, adds detergent solution and boil 5min, abandon detergent solution, Adding washes of absolute alcohol 5min, period the most slightly shakes, and then discards dehydrated alcohol；
B. add the FTA card cleanout fluid of 300 μ L Whatman companies, incubated at room 5min, add 500 μ L1 × TE solution, Incubated at room 5min；
C. repeating step b once, the FTA disk room temperature after cleaning is placed 1 hour or 10min in 56 DEG C of incubators, extremely FTA disk is completely dried, for follow-up test.
In described step a, detergent is the SDS solution of mass fraction 10%, and the volume adding dehydrated alcohol is 500 μ L.
In described step (5), PCR amplification uses Chao Shi PCR, and reaction system is 50 μ L.
A kind of FTA/PCR detection feces in Cryptosporidium parvum oocysts suspended method for genotype be C.parvum, The detection of the Cryptosporidium parvum oocysts suspended of C.andersoni, C.bovis or C.rynane.
The beneficial effects of the present invention is:
(1) FTA card improves detection sensitivity with Chao Shi PCR after being combined, and detection limit is up to 1 × 101Individual egg capsule/gram Feces；
(2) easy and simple to handle, only need to carry FTA and snap into after plant gathers fecal sample, through FTA eluent with other are organic After solution cleans, FTA card can be used as DNA profiling and carry out PCR amplification；
(3) having higher biological safety, the distinctive chelating agen of FTA card and decomposition agent can make rapidly micro-life the short time Thing and parasite egg capsule lose activity, and reduce the propagation chance of harmful cause of disease, protect the safety of experiment operator, have Higher public health meaning；
(4) the conventional elution process of FTA card, it is impossible to clean the PCR inhibitive factor in feces, the present invention is by adding difference Organic solvent, improve the cleaning way in FTA card operation instructions, washed the PCR inhibitive factor in feces, made cleaning After be loaded with the FTA card of cryptosporidium dna and can be directly used as DNA profiling, carry out follow-up PCR amplification equimolecular and grind biology Studying carefully, the present invention instead of traditional commerce faeces DNA and extracts step loaded down with trivial details during test kit extraction DNA and fancy price, tool Have with low cost, conveniently advantage.
Accompanying drawing explanation
Fig. 1 is Cryptosporidium nest-type PRC reaction condition schematic diagram.
Fig. 2 is based on 18S rRNA gene loci FTA/PCR method detection Cryptosporidium In Dairy Cattle: the sensitivity of positive fecal specimens Property test electrophoretogram；Wherein, M represents DNA Marker (DL2000)；Swimming lane 1-7:1 × 105-1×10-1The individual hidden spore of egg capsule/g Worm positive fecal specimens；P: positive control；N: negative control.
Fig. 3 is to detect Cryptosporidium In Dairy Cattle based on 18S rRNA gene loci FTA/PCR method: positive fecal specimens special Property test electrophoretogram；Wherein, M represents DNA Marker (DL2000)；Swimming lane 1: Cryptosporidum parvum positive fecal specimens；Swimming lane 2: cryptosporidium andersoni positive fecal specimens；Swimming lane 3: giardia lamblia positive fecal specimens；Swimming lane 4: microsporidian positive feces sample Product；Swimming lane 5: coccidiosis positive fecal specimens；Swimming lane 6: containing distilled water fecal specimens；P: positive control；N: negative control.
Fig. 4 is based on 18S rRNA gene loci FTA/PCR method detection Cryptosporidium In Dairy Cattle: the repetition of positive fecal specimens Property test electrophoretogram；Wherein, M represents DNA Marker (DL2000)；Swimming lane 1-5: nest-type PRC first set product electrophoretogram；Swimming Road 6-N: nest-type PRC second overlaps product electrophoretogram；Swimming lane 1-3,6-8: Cryptosporidium positive fecal specimens；4, P: positive control； 9, N: negative control.
Fig. 5 is cow manure sample detection figure；Wherein swimming lane 1-20 is detected sample, and M represents DL2000Marker, P: Positive control；N: negative control.
Detailed description of the invention
Following example are used for illustrating the present invention, but need not limit the scope of the present invention.If not specializing, embodiment is equal According to routine test.Such as Sambrook equimolecular Cloning: A Laboratory Manual (Sambrook J&Russell DW, Molecular Cloning:a laboratory manual, 2001), or the condition according to manufacturer's description suggestion.
Embodiment agents useful for same is commercially available, and material therefor the applicant's laboratory all has preservation.
Main agents: FTA card (FTATM Classic Card WhatmanTM, lot No.3983740, UK)；FTA washes De-liquid (FTA purification Reagent, WhatmanTM, lot No.9611474, UK)；BSA(Bovine Serum Albumin, SIGMATM, lot No.SLBN1377V, USA)；Dehydrated alcohol (Ethanol absolute, Tianjin bigization forever Learn reagent company limited)；SDS (dodecyl sodium sulfate, C12H25O4SNa, GENVIEWTM, lot No.4803010120)；Beat Hole device (punching diameter 2mm)；DL2000DNA Marker, 6 × Loading buffer, TaKaRa Synbiotics AB； DNAGreen, TIANDZ；KOD-plus, dNTPS, TOYOBO.
Key instrument: PCR instrument, Gene Company Limited, model 9700；Voltage stabilization and current stabilization electrophresis apparatus, Beijing six One instrument plant, model DYY-5；Ultraviolet imager, SYNGENE, model InGenius LHR；Digital camera, Canon Inc., type Number IXUS115HS；Quickly vortex mixer, GENIE, model VORTEX-2；Superclean bench, the Suzhou limited public affairs of safe and sound air technique Department；Dual pure water distillation apparatus, Shanghai Yarong Biochemical Instrument Plant, model SZ-93；Pipettor, BRAND.
One, extract DNA based on FTA card and carry out the method for Cryptosporidium parvum oocysts suspended in PCR detection cow manure, including sensitivity Test, specific test, replica test, specifically comprise the following steps that
(1) sensitivity tests:
1. the purification of Cryptosporidium parvum oocysts suspended, collects the Calves Feces after Cryptosporidium parvum oocysts suspended infects successfully, and feces is through second Obtain the purest after ether grease removal, saturated sucrose concentration sucrose gradient centrifugation floating, discontinuous precipitation, cesium chloride centrifugation precipitation Through Cecil McMaster flat board egg capsule counting method, Cryptosporidium parvum oocysts suspended, remembers that egg capsule concentration is 1 × 105/ mL egg capsule.
2. quantitative contamination feces, egg capsule doubling dilution step 1. obtained becomes 1 × 105/mL-1×10-1/ mL egg capsule, Take the egg capsule liquid 1mL after doubling dilution to be uniformly blended in the 1g cow manure the most afterwards for Cryptosporidium feminine gender.
3. being applied on FTA card by the cow manure sample after Cryptosporidium parvum oocysts suspended pollutes, room temperature dries FTA card.With Mono-hole punch punching on the FTA card being loaded with Cryptosporidium positive fecal specimens obtains the FTA disk of diameter 2mm.
4. the step 3. middle FTA disk obtained is respectively put in 2mL centrifuge tube, boils 5min with 10%SDS solution, abandon Removing SDS solution, add 500 μ L washes of absolute alcohol 5min, period the most slightly shakes, then discards dehydrated alcohol.
5. the FTA card cleanout fluid of 300 μ L Whatman companies, incubated at room 5min are added.
6. 500 μ L1 × TE solution, incubated at room 5min are added.
7. repeat step 5.-the most once.
8. the FTA disk room temperature after cleaning is placed 1 hour or 10min in 56 DEG C of incubators, completely dry to FTA disk Dry.
9. FTA card step 8. obtained, as DNA profiling, directly as in 0.2mmEP pipe, carries out nest-type PRC expansion Increase.Primer sequence such as SEQ ID NO:1, the SEQ ID that PCR primer sequence designs based on Cryptosporidium 18SrRNA with reference to Yan Ruo peak Shown in NO:2, SEQ ID NO:3 and SEQ ID NO:4；For all samples, two-wheeled PCR reaction all uses 50 μ L systems, PCR Reaction system is shown in Table 1.In first round reaction system, add bovine serum albumin (BSA) in order to neutralize PCR inhibitive factor, Second takes turns in reaction without adding.PCR reaction condition, is shown in Fig. 1.
Take 5 μ LPCR products the most respectively in 1% agarose gel electrophoresis liquid, carry out electrophoresis with 110V voltage, then at purple Imaging observed result in outer Labworks image acquisition and analysis software.
Pcr amplification product delivers to the order-checking of order-checking company, and analyzes sequencing result.
Table 1: Cryptosporidium nest-type PRC reaction system
Two, specific test:
The cryptosporidium andersoni ovum that Cryptosporidium parvum Oocysts and the present inventor's laboratory of above-mentioned (1) purification are deposited Capsule, giardia lamblia egg capsule, microsporidian egg capsule, coccidian oocyst and sterilizing distilled water are separately added into mix homogeneously in fecal specimens, so After spread upon on FTA card, ensuing process step with in above-mentioned (1) 3.-10. step identical, PCR primer sequence such as SEQ ID Shown in NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, PCR reaction system is shown in Table 1, and PCR reaction condition is shown in figure 1。
Three, replica test:
With the good FTA card of the step process in above-mentioned (1) as DNA profiling, carry out based on Cryptosporidium 18S gene loci Three nested PCR amplification, expand each sample parallel in triplicate, to detect repeatability or the stability of FTA/PCR method every time. Test procedure with in above-mentioned (1) 3.-10. step identical, PCR primer sequence is shown in Table 1, and PCR reaction system is shown in Table 1, and PCR reacts bar Part is shown in Fig. 1.
Sensitivity tests, specific test, replica test interpretation of result:
Cryptosporidium parvum oocysts suspended sensitivity tests in FTA/PCR method detection cow manure sample, is based on Cryptosporidium 18S RRNA gene loci, the nest-type PRC test carried out for DNA profiling with FTA card.Test Cryptosporidium parvum oocysts suspended is dilute with 10 times of multiple proportions It is interpreted into 105-10-17By the result of agarose gel electrophoresis and sequencing analysis, individual gradient scope, shows that swimming lane 1-5 all occurs The purpose band of about 250bp, it was demonstrated that FTA/PCR method detection cow manure sample in Cryptosporidium parvum oocysts suspended detection limit up to To 1 × 101Individual egg capsule/g feces, result Pass Test is expected, is met testing requirement.Result sees appendix Fig. 2.
Shown by the result of agarose gel electrophoresis and sequencing analysis, hidden in FTA/PCR method detection cow manure sample In sporozoon egg capsule specific test, all there is the purpose bar of about 250bp in Cryptosporidum parvum and cryptosporidium andersoni swimming lane Band, and all there is not band in other worm strains, it was demonstrated that FTA/PCR method specificity is preferable, and result sees appendix Fig. 3.Result of the test meets Test expection, meets testing requirement.
Shown by the result of agarose gel electrophoresis and sequencing analysis, hidden in FTA/PCR method detection cow manure sample In sporozoon egg capsule replica test, all there is the purpose band of about 250bp in swimming lane, it was demonstrated that FTA/PCR method repeatability is preferable, Result sees appendix Fig. 4.Experimental result Pass Test is expected, meets testing requirement.
Four, the method 20 parts of cow manures to Sichuan Beyer Co., Ltd censorship to the present inventor's laboratory set up with this test Sample detects, and the Cryptosporidium positive rate of detection is 10% (2/20), extracts with commercialization feces nucleic acid extraction kit The PCR that DNA is template amplification Cryptosporidium positive rate be 10% (2/20).
The two testing result identical (as shown in Figure 5), but simplify commercialization feces nucleic acid extraction kit and extract DNA's Tedious steps, and decrease and extracting the cross-contamination produced during DNA, have save time, advantage easily.

Claims (7)

1. the method for Cryptosporidium parvum oocysts suspended in a FTA/PCR detection feces, it is characterised in that: step is as follows:
(1) purified pool Cryptosporidium parvum oocysts suspended, after counting dilution, is applied on FTA card standby after quantitative contamination feces；
(2) positive fecal specimens is applied on FTA card standby；
(3) fresh excreta is collected with FTA card standby；
(4) punch on the FTA card in step (1), (2) and (3), and eluting FTA disk；
(5) with cleaned FTA card as DNA profiling, PCR amplification is carried out with the primer designed according to Cryptosporidium 18SrRNA, Then agarose gel electrophoresis detection is carried out.
2. the method for Cryptosporidium parvum oocysts suspended in FTA/PCR detection feces as claimed in claim 1, it is characterised in that: described step Suddenly in (1), purified pool method uses saturated saline floatation and cesium chloride gradient centrifugation jointly to act on, quantitative contamination refer to by The egg capsule collected is diluted to 1 × 10-1/mL-1×105/ mL egg capsule suspension, takes 1mL and is mixed in 1g feces.
3. the method for Cryptosporidium parvum oocysts suspended in FTA/PCR detection feces as claimed in claim 1, it is characterised in that: described step Suddenly (2) positives feces refers to cryptosporidium andersoni positive feces, giardia lamblia positive feces, microsporidian positive feces or coccidiosis sun Two or more in property feces.
4. the method for Cryptosporidium parvum oocysts suspended in FTA/PCR detection feces as claimed in claim 1, it is characterised in that: described step Suddenly in (4), elution step is:
A. the FTA disk of acquisition is respectively put in centrifuge tube, adds detergent solution and boil 5min, abandon detergent solution, add nothing Water-ethanol cleans 5min, and period the most slightly shakes, then discards dehydrated alcohol；
B. add 300 μ L FTA card cleanout fluid, incubated at room 5min, add 500 μ L1 × TE solution, incubated at room 5min；
C. repeating step b once, the FTA disk room temperature after cleaning is placed 1 hour or 56o10min in C incubator, to FTA circle Sheet is completely dried, for follow-up test.
5. the method for Cryptosporidium parvum oocysts suspended in FTA/PCR detection feces as claimed in claim 4, it is characterised in that: described step In rapid a, detergent is the SDS solution of mass fraction 10%, and the volume adding dehydrated alcohol is 500 μ L.
6. the method for Cryptosporidium parvum oocysts suspended in FTA/PCR detection feces as claimed in claim 1, it is characterised in that: described step Suddenly in (5), PCR amplification uses Chao Shi PCR, and reaction system is 50 μ L.
7. in a FTA/PCR detection feces, the method for Cryptosporidium parvum oocysts suspended for genotype isC. parvum、C. andersoni、C. bovisOrC. rynaneThe detection of Cryptosporidium parvum oocysts suspended.
CN201610492725.6A 2016-06-27 2016-06-27 The method of Cryptosporidium parvum oocysts suspended in a kind of FTA/PCR detection feces Pending CN106119355A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113016713A (en) * 2021-03-03 2021-06-25 新疆医科大学第一附属医院 Method for separating echinococcus granulosus eggs from dog feces and hatching oncosphere in vitro to carry out primary infection animal molding

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
付素兰等: "一种快速、简便的模板提取方法用于 PCR 检测粪便中的金黄色葡萄球菌的研究", 《河北医药》 *
刘振伟等: "隐孢子虫卵囊纯化技术研究进展", 《热带医学杂志》 *
张于光等: "《濒危动物保护遗传学研究》", 30 September 2015 *
李凌等: "《生物化学与分子生物学实验指导（第 2 版）》", 31 August 2015 *
田利光等: "应用 FTA/PCR 检测粪便中隐孢子虫的实验研究", 《国际医学寄生虫病杂志》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113016713A (en) * 2021-03-03 2021-06-25 新疆医科大学第一附属医院 Method for separating echinococcus granulosus eggs from dog feces and hatching oncosphere in vitro to carry out primary infection animal molding

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Application publication date: 20161116