CN107271441A - A kind of method of utilization dark field microscope direct visual perception Cryptosporidium - Google Patents
A kind of method of utilization dark field microscope direct visual perception Cryptosporidium Download PDFInfo
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- CN107271441A CN107271441A CN201710550768.XA CN201710550768A CN107271441A CN 107271441 A CN107271441 A CN 107271441A CN 201710550768 A CN201710550768 A CN 201710550768A CN 107271441 A CN107271441 A CN 107271441A
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Abstract
The invention discloses a kind of method of utilization dark field microscope direct visual perception Cryptosporidium, this method is to be coupled Cryptosporidium parvum oocysts suspended antibody with the magnetic bead for being coated with albumin A, builds the magnetic probe with specificity capture Cryptosporidium；The Cryptosporidium parvum oocysts suspended in testing sample is captured with the probe, result is observed in dark field microscope.The present invention constructs the magnetic probe with reference to Cryptosporidium parvum oocysts suspended, establishes Cryptosporidium parvum oocysts suspended rapid detection system.Dark field microscope visual viewing method based on magnetic probe have it is easy, quick, the features such as without special installation, available for whether having Cryptosporidium in situ appraisal sample on the spot.
Description
Technical field
The invention belongs to pathogen detection field, and in particular to one kind utilizes the hidden spore of dark field microscope direct visual perception
The method of worm.
Background technology
Cryptosporidium (Cryptosporidium) is one of global six big diarrhoea pathogenics, belongs to infecting both domestic animals and human pathogen, sternly
The public health of the harm mankind is healthy again.In normal crowd, the patient of infection Cryptosporidium shows as diarrhoea, and with stomach
Spasm, nausea, low fever, digestive functional disturbance etc.；For the people of hypoimmunity, illness is changeable and serious, mostly continuation
Choleraic diarrhea, ultimately results in patient's dehydration dead.Cryptosporidium parvum oocysts suspended is widely present in the environment, can pass through water source and environment
In various other factors for example animal contact, PE pollution etc. carry out propagation cause outbreak of disease.Cryptosporidium is by the U.S.
The parasite cause of disease in bio-terrorism war agent is classified as, is also that must examine microorganism cause of disease specified in the multinational drinking water quality standard in the world
One of.In order to ensure safe drinking water and publilc health, Cryptosporidium prevention and control are own through the study hotspot as World Focusing.And detect
The distribution of Cryptosporidium in the environment is most important for Cryptosporidiosis prevention and control.
At present, the method for existing detection Cryptosporidium mainly includes：(1) in the drinking water of Environmental Protection Agency issue
The standard detecting method (EPA1623) of Cryptosporidium parvum oocysts suspended, the advantage of this method is removed during immuno magnetic cell separation
Impurity in environmental samples, it is ensured that the purity of the egg capsule extracted, sensitivity, accuracy are high, and can determine whether that egg capsule is lived
Property.But big, cumbersome, the required detection device of this method workload and consumptive material are also very expensive, it is not suitable for basic unit's experiment
The extensive conventional detection in room；(2) acid dyeing method is improved, its advantage is that step is few, simple and easy to do, result is obvious, need not answered
Miscellaneous instrument and equipment, suitable for test in laboratory work, but this kind of method false positive detection rate is high；(3) ELISA method, its advantage
It is easy, quick and sensitivity, but there is also fail to pinpoint a disease in diagnosis or mistaken diagnosis phenomenon this method；(4) PCR detects that its advantage is specific and quick
The substantial amounts of purpose fragment of amplification in perception, short time, but the design of this method primer is extremely important, because if primer specificity
Not strong and sensitive to DNA pollution, the reappearance of experiment is not also high, especially when infective dose very little, except this it
Outside, PCR detections need experimental technique and instrument with specialty, it is necessary to which technical professional, limits it and work in unit of primary level
In use.
The present invention is intended to provide in a kind of utilization dark field microscope direct visual perception water Cryptosporidium method.First will
Antibody and magnetic bead are coupled, and build the magnetic probe with specificity capture Cryptosporidium.This probe utilizes the spy of antigen-antibody
Opposite sex reaction, can combine closely Cryptosporidium parvum oocysts suspended, and the compound does directed movement under the influence of a magnetic field, so as to reach separation
The effect of sporozoite egg capsule, can form the clear golden yellow flower coronal structure for finding of naked eye under dark field microscope.The separation
Detection technique applies more wide in terms of the separation detection of bacterium, virus and cell, but directly utilizes dark field microscope
Observation is carried out to have not been reported.This method can carry out quick diagnosis and inspection to the Cryptosporidium in water sample, dairy products and excrement
Survey, shorten detection cycle, simplify detection process, the general applicability of detection means is improved, beneficial to Site Detection on the spot, so that soon
Speed, presence that is accurate, sensitive, easily detecting Cryptosporidium, timely prevention and control largely reduce Cryptosporidium to the mankind
Health threatens.
The content of the invention
The present invention is intended to provide a kind of method of utilization dark field microscope direct visual perception Cryptosporidium, can be to water
Sample, dairy products and excrement carry out quick diagnosis and detection, shorten detection cycle, simplify detection process, improve the general of detection means
All over applicability, beneficial to Site Detection on the spot so that quickly, accurately, presence that is sensitive, easily detecting Cryptosporidium, it is anti-in time
Control, largely reduces Cryptosporidium and human health is threatened.
It is a kind of hidden using dark field microscope direct visual perception the present invention seeks to set up to solve the deficiencies in the prior art
The method of sporozoite, below technical scheme：
A kind of method of utilization dark field microscope direct visual perception Cryptosporidium, this method is by commercialization Cryptosporidium
The magnetic bead that egg capsule antibody and commercialization are coated with albumin A carries out new and coupling and obtains magnetic probe, then using being mounted with for hidden
The magnetic probe of sporozoite egg capsule antibody is captured to the Cryptosporidium in testing sample, is then observed and is tied in dark field microscope
Really.
The specific step of the present invention is as follows：
(1) cleaning of magnetic bead：The magnetic bead for being coated with albumin A is cleaned, the more uniform magnetic bead of dispersiveness is obtained；
(2) coupling of antibody：Magnetic bead after cleaning in Cryptosporidium parvum oocysts suspended antibody and step (1) is mixed, carry out it is new and
Coupling, obtains the magnetic probe with special capture Cryptosporidium ability；
(3) magnetic probe in step (2) and sample to be tested are incubated a period of time, obtain mixed liquor；
(4) the above-mentioned mixed liquor of Magneto separate, is resuspended after mixture with PBS, is added drop-wise on slide, dark field microscope observation.
Preferably, methods described, step is as follows：
(1) cleaning of magnetic bead：The magnetic bead for being coated with albumin A is mixed with the PBS solution containing BSA, after Magneto separate
Stand, abandon supernatant, be resuspended in the PBS solution containing BSA, Magneto separate after ultrasound is resuspended in containing BSA after abandoning supernatant, washing
PBS solution in, obtain the more uniform magnetic bead of dispersiveness；
(2) coupling of antibody：By the magnetic bead after being cleaned in commercialization Cryptosporidium parvum oocysts suspended antibody and step (1) according to quality
Than for 1:1-2:5 mixing, normal temperature is incubated 1-6h；Then the uncombined antibody of Magneto separate washing, is finally washed with the PBS solution containing BSA
It is resuspended in after washing in PBS solution, obtains the magnetic probe with special capture Cryptosporidium；
(3) magnetic probe built with step (2) is captured to the Cryptosporidium in testing sample, and the magnetic probe adds
Dosage is that every 200 μ L sample solutions add 50-100 μ L magnetic probes；
(4) the 10-20 μ L mixed liquors in step (3) are taken to be added drop-wise on slide, dark field microscope observation.
In the present invention, the diameter of step (1) described magnetic bead can be micron order to nano level magnetic particle.
Step (1) magnetic bead cleaning BSA used concentration is 1%, and the time cleaned every time is 5min, wash number
For 3 times.
Step (2) antibody and magnetic bead coupling time are 30-60min.
Step (3) described sample, is water sample, dairy products and fecal specimens.
The time of step (3) the magnetic probe capture Cryptosporidium is 30-60min.
It is highly preferred that methods described, is comprised the following steps that：
(1) cleaning of magnetic bead：The magnetic bead that commercialization is coated with into albumin A is mixed with the PBS solution containing 1%BSA, magnetic
After separation, supernatant is abandoned, is resuspended in the PBS solution containing 1%BSA, Magneto separate after ultrasonic 5min is resuspended in after abandoning supernatant, washing
In PBS solution containing 1%BSA, the more uniform magnetic bead of dispersiveness is obtained；
(2) coupling of antibody：By the magnetic bead after being cleaned in commercialization Cryptosporidium parvum oocysts suspended antibody and step (1) according to quality
Than for 4:5 mixing, carry out new and coupling, mixed solution oscillation incubation 30-60min, Magneto separate at room temperature in constant temperature blending instrument
Afterwards, supernatant is abandoned, is resuspended in after then being washed with the PBS solution containing 1%BSA in PBS solution, is obtained with special capture ability
Magnetic probe；
(3) magnetic probe being mounted with step (2) for Cryptosporidium parvum oocysts suspended antibody is entered to the Cryptosporidium in sample
Row capture, the magnetic probe addition is addition 50-100 μ L magnetic probes in every 200 μ L capture systems, and mixed solution is mixed in constant temperature
After oscillation incubation 30-60min at room temperature in even instrument, Magneto separate, supernatant is abandoned, weight after then being washed with the PBS solution containing 1%BSA
It is suspended from PBS solution；
(4) the 10-20 μ L mixed liquors in step (3) are taken to be added drop-wise on slide, dark field microscope observation.
Beneficial effects of the present invention：
This research mainly for it is widely distributed and to the highly pathogenic Cryptosporidium of people be research object, utilize magnetic spy
Pin isolation technics is detected to it.Cryptosporidium is detected using magnetic probe, it is not necessary to special laboratory apparatus, it is only necessary to magnetic force
Separator and dark field microscope can complete experiment；In detection process, it is only necessary to be coated with the specific antibody of Cryptosporidium,
It just can complete to test using specific binding between antigen-antibody, can just operate at room temperature, experiment condition that should not be special；Short
Detection can be completed in time, and with higher sensitivity, it is adaptable to field operation on the spot.
Brief description of the drawings
Fig. 1：Magnetic probe detects the schematic diagram of small Cryptosporidium parvum oocysts suspended；
Fig. 2：:The SDS-PAGE of magnetic bead and the small Cryptosporidium parvum oocysts suspended antibody binding efficiency of goat-anti schemes；Wherein, Marker is pre-
Protein labeling is contaminated, 1 is 5 μ L antibody applied sample amounts, and 2 be 2 μ L antibody applied sample amounts, and 3 be 1 μ L antibody applied sample amounts, and 4 be 10 μ g magnetic beads and 5 μ
Applied sample amount after g antibody bindings；
Fig. 3：The dark field microscope figure that magnetic probe is combined with small Cryptosporidium parvum oocysts suspended；
Fig. 4：The simple microscope oil mirror figure that magnetic probe is combined with small Cryptosporidium parvum oocysts suspended；
Fig. 5：The simple microscope oil mirror negative control figure that unmodified antibody magnetic bead is combined with small Cryptosporidium parvum oocysts suspended；
Fig. 6：Magnetic probe and the PCR result nucleic acid electrophoresis figures of small Cryptosporidium parvum oocysts suspended joint efficiency；Wherein, M is 100bp's
Mark, 1 is that sample is ddH in PCR2O blank control group, 2 be the control group that sample is magnetic bead in PCR, and 3 be sample in PCR
For positive group of Cryptosporidium stoste, 4 be that sample is Cryptosporidium and magnetic bead mixed liquor but uncombined group in PCR, during 5 are PCR
Sample is the supernatant group after Cryptosporidium combines separation through magnetic probe, 6 be in PCR sample to be Cryptosporidium combine point through magnetic probe
From group.
Embodiment
The present invention is done below in conjunction with the accompanying drawings and further explained, but the present invention should not be limited by the examples.
The detection of Cryptosporidium, comprises the following steps in river sample：
First, material
The magnetic bead (10mg/mL) for being coated with albumin A is purchased from Creative Diagnostics companies of the U.S.；The hidden spore of goat-anti
Worm's ovum capsule antibody (4mg/mL) is purchased from Abcom companies of Britain；PCR kit is purchased from TIANGEN companies；Small Cryptosporidium suspension purchase
From Waterborne companies of the U.S.；Magnetic separtor is purchased from Promega companies of the U.S.；Constant temperature blending instrument is purchased from Germany
Eppendorf companies；Ultrasound Instrument is purchased from Chinese Kunshan Ultrasonic Instruments Co., Ltd..
2nd, experimental method
1. the cleaning of magnetic bead：100 μ L commercializations are coated with a diameter of 1 μm of magnetic bead of albumin A and add 500 μ L containing 1%BSA's
In PBS 1.5mL centrifuge tubes, sonic oscillation 5min is mixed, is put on magnetic separtor, treats that magnetic granular absorption completely, is discarded
Clearly；500 PBSs of the μ L containing 1%BSA are added into centrifuge tube again, are repeated three times；100 μ L are added again containing 1%BSA's
PBS, sonic oscillation is fully mixed, stand-by；
2. the coupling of antibody：By 100 μ L after being cleaned in 20 μ L commercializations goat-anti Cryptosporidium parvum oocysts suspended antibody and step (1)
Magnetic bead is mixed, and carries out new and coupling, after sonic oscillation is mixed, the 500rpm oscillation incubations 60min at room temperature in constant temperature blending instrument；
After the completion of association reaction, it is put into magnetic separtor, to be adsorbed complete, then supernatant discarding is washed with the PBS solution containing 1%BSA
Wash, be resuspended in PBS solution, obtain the magnetic probe with special capture ability；
3. the measure of antibody coupling efficiency
The antibody-solutions before and after the magnetic bead coupling with coating protein A are taken, SDS-PAGE methods detection magnetic bead is imitated with antibody coupling
Rate：5min is denatured in the boiling water for being placed in 95 DEG C or so, 10s is centrifuged, 4 DEG C of refrigerators are preserved, to be detected.4. water body example processing
The 200ml river samples of collection are stored at room temperature after 48h, upper strata water sample is slowly taken, extremely residue 100mL or so；Will
Remaining sample is centrifuged, 5000rpm, 10min；To the precipitation after centrifugation add PBS solutions of the 100 μ L containing 1%BSA and
Precipitation is resuspended in 100 μ L Cryptosporidium parvum oocysts suspendeds stostes, and 4 DEG C of refrigerators are preserved；
5. capture of the magnetic probe to Cryptosporidium in water sample system
It will be loaded with capturing the Cryptosporidium in sample for the magnetic probe of Cryptosporidium parvum oocysts suspended antibody.Take 100 μ
The L coated magnetic probes of goat-anti Cryptosporidium parvum oocysts suspended antibody and 100 μ L testing samples the room temperature 500rpm in constant temperature blending instrument vibrate
It is incubated 60min；After the completion of association reaction, it is put into magnetic separtor, to be adsorbed complete, the centrifugation of Aspirate supernatant to 1.5mL
It is used for the detection of joint efficiency in pipe, adds 500 μ L PBS washing probes, repeat three times, add 50 μ L PBS and knot is resuspended
Close the probe of Cryptosporidium parvum oocysts suspended.
6. result is observed
By the μ L of mixed liquor 15 in step (4), it is added drop-wise on slide, under dark field microscope, can be clearly apparent and surround
Cryptosporidium parvum oocysts suspended one is enclosed or the golden yellow of subregion spends coronal structure.
7. joint efficiency is detected
Magnetic bead and Cryptosporidium parvum oocysts suspended mixed liquor and hidden spore after the supernatant of collection in step (4), washing are resuspended
Sub- worm's ovum capsule stoste enters performing PCR identification, detects the joint efficiency of magnetic bead.According to specific gene COWP on Cryptosporidium parvum oocysts suspended
(GenBank accession no.:Z22537 sequence), the specificity using the Software for Design genes of Primier 5.0 is drawn
Thing, sense primer (F)：5′-CAAATTGATACCGTTTGTCCTTCTG-3′(SEQ ID NO.1)；Anti-sense primer (R)：5′-
GGCATGTCGATTCTAATTCAGCT-3 ' (SEQ ID NO.2), the length of amplified production is 150bp.PCR reaction systems are specific
It is as follows：
Prepare the PCR reaction systems that cumulative volume is 50 μ L
PCR response procedures：95 DEG C of 5min, 95 DEG C of 5s, 55 DEG C of 20s, 72 DEG C of 30s, 72 DEG C of 10min, altogether 30 circulations, 13
℃50min。
Pcr amplification product enters row agarose gel electrophoresis：The Ago-Gel of configuration 1.5%, electrophoretic parameters are voltage
80V, the time is 40min.
3rd, result
The principle of magnetic probe separation Cryptosporidium is as shown in figure 1, by Cryptosporidium parvum oocysts suspended antibody and the magnetic for being coated with albumin A
Pearl is coupled, and builds the magnetic probe with specificity capture Cryptosporidium.Cryptosporidium parvum oocysts suspended is captured with the probe, in details in a play not acted out on stage, but told through dialogues
Micro- Microscopic observation can form the clear golden yellow flower coronal structure for finding of naked eye.
The method being coupled according to above-mentioned antibody and magnetic bead, the concentration of magnetic bead coupled antibody, examination are determined by SDS-PAGE methods
Result is tested as shown in Fig. 2 10 μ g magnetic bead is mixed with 5 μ g antibody, the amount of magnetic bead coupled antibody is in 1.5 μ g or so.
After magnetic probe is mixed with Cryptosporidium parvum oocysts suspended, microscopy results as in Figure 3-5, can under dark field microscope
To be clearly observed golden yellow flower coronal structure, i.e., the magnetic probe particle of ring-type is combined with around Cryptosporidium parvum oocysts suspended；
It can also be seen that similar result under common light microscope；And control group does not see that what magnetic probe and egg capsule combined shows
As.
By PCR method, our efficiency to magnetic probe capture egg capsule detect that result of the test is as shown in fig. 6, hidden
In sporozoite egg capsule stoste and sample magnetic probe capture egg capsule PCR band brightness it is essentially identical, it was demonstrated that the probe can be high
The Cryptosporidium of the special identification water body of effect.
4th, conclusion
Because Cryptosporidium is can to cause the infecting both domestic animals and human pathogen of human diseases, easily cause local or a wide range of
Communality outburst, therefore should be drawn attention for the prevention and detection of Cryptosporidium.Existing detection method operation is loaded down with trivial details, consumption
Duration, cost are high, strongly professional, easily cause and fail to pinpoint a disease in diagnosis or mistaken diagnosis.This experiment is by using the high enriched character of magnetic probe, energy
It is enough that the time for detecting sample is shortened into 2-3h, pass through the observation of dark field microscope, you can realize simple, fast for Cryptosporidium
The detection of speed.Because magnetic probe can directly capture Cryptosporidium, high specificity from detection sample, this research is hidden in sample
The detection of sporozoite provide it is a kind of efficiently, the method for high specific, simplicity operation, it is adaptable to Site Detection on the spot.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this
The people of technology, is not departing from spirit and scope of the invention, can do various changes and modification, therefore, guarantor of the invention
What shield scope should be defined by claims is defined.
SEQUENCE LISTING
<110>Yangzhou University
<120>A kind of method of utilization dark field microscope direct visual perception Cryptosporidium
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213>Artificial sequence
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caaattgata ccgtttgtcc ttctg 25
<210> 2
<211> 23
<212> DNA
<213>Artificial sequence
<400> 2
ggcatgtcga ttctaattca gct 23
Claims (8)
1. a kind of method of utilization dark field microscope direct visual perception Cryptosporidium, it is characterised in that by Cryptosporidium parvum oocysts suspended
Antibody is coupled with the magnetic bead for being coated with albumin A, builds the magnetic probe with specificity capture Cryptosporidium；Caught with the probe
The Cryptosporidium parvum oocysts suspended in testing sample is obtained, result is observed in dark field microscope.
2. method according to claim 1, it is characterised in that step is as follows：
(1) cleaning of magnetic bead：The magnetic bead for being coated with albumin A is cleaned, the more uniform magnetic bead of dispersiveness is obtained；
(2) coupling of antibody：Magnetic bead after being cleaned in Cryptosporidium parvum oocysts suspended antibody and step (1) is mixed, new and coupling is carried out,
Obtain the magnetic probe with special capture Cryptosporidium ability；
(3) magnetic probe in step (2) and sample to be tested are incubated a period of time, obtain mixed liquor；
(4) the above-mentioned mixed liquor of Magneto separate, is resuspended after mixture with PBS, is added drop-wise on slide, dark field microscope observation.
3. method according to claim 2, it is characterised in that step is as follows：
(1) cleaning of magnetic bead：The magnetic bead for being coated with albumin A is mixed with the PBS solution containing BSA, stood after Magneto separate,
Supernatant is abandoned, is resuspended in the PBS solution containing BSA, Magneto separate after ultrasound is abandoned and the PBS containing BSA is resuspended in after supernatant, washing
In solution, the more uniform magnetic bead of dispersiveness is obtained；
(2) coupling of antibody：It is according to mass ratio by the magnetic bead after being cleaned in commercialization Cryptosporidium parvum oocysts suspended antibody and step (1)
1:1-2:5 mixing, normal temperature is incubated 1-6h；Then Magneto separate washing is not associated with antibody, after finally being washed with the PBS solution containing BSA
It is resuspended in PBS solution, obtains the magnetic probe with special capture Cryptosporidium；
(3) magnetic probe built with step (2) is captured to the Cryptosporidium in testing sample, the magnetic probe addition
50-100 μ L magnetic probes are added for every 200 μ L sample solutions；
(4) the 10-20 μ L mixed liquors in step (3) are taken to be added drop-wise on slide, dark field microscope observation.
4. method according to claim 3, it is characterised in that the diameter of step (1) described magnetic bead is micron order to nanometer
The magnetic particle of level.
5. method according to claim 3, it is characterised in that step (1) magnetic bead cleaning BSA used concentration is
1%, the time cleaned every time is 5min, and wash number is 3 times.
6. method according to claim 3, it is characterised in that step (2) antibody and magnetic bead coupling time are 30-
60min。
7. method according to claim 3, it is characterised in that step (3) described sample, is water sample, dairy products and excrement
Sample.
8. method according to claim 3, it is characterised in that step (3) magnetic probe captures the time of Cryptosporidium
For 30-60min.
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| CN110108628A (en) * | 2019-06-04 | 2019-08-09 | 扬州大学 | The method that a kind of pair of toxoplasma tachyzoite directly counts |
| CN114088943A (en) * | 2021-12-01 | 2022-02-25 | 江苏省淡水水产研究所 | Gold nanoprobe for detecting white spot syndrome virus of procambarus clarkii as well as preparation method and application of gold nanoprobe |
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| CN110108628A (en) * | 2019-06-04 | 2019-08-09 | 扬州大学 | The method that a kind of pair of toxoplasma tachyzoite directly counts |
| CN114088943A (en) * | 2021-12-01 | 2022-02-25 | 江苏省淡水水产研究所 | Gold nanoprobe for detecting white spot syndrome virus of procambarus clarkii as well as preparation method and application of gold nanoprobe |
| CN114088943B (en) * | 2021-12-01 | 2024-03-12 | 江苏省淡水水产研究所 | Gold nanoprobe for detecting procambarus clarkia white spot syndrome virus, and preparation method and application thereof |
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