CN106950073B - A method of collecting strongyloides intestinalis larva from watery stools - Google Patents
A method of collecting strongyloides intestinalis larva from watery stools Download PDFInfo
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- CN106950073B CN106950073B CN201710159044.2A CN201710159044A CN106950073B CN 106950073 B CN106950073 B CN 106950073B CN 201710159044 A CN201710159044 A CN 201710159044A CN 106950073 B CN106950073 B CN 106950073B
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/02—Devices for withdrawing samples
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Abstract
The method that the invention discloses a kind of to collect strongyloides intestinalis larva from watery stools, step are as follows: precipitating is collected by centrifugation in the stool of collection；Precipitating is mixed using physiological saline or distilled water, until centrifugation to mixed liquor clarification, then precipitating is collected by centrifugation；Precipitating is added dropwise in the middle part of the filter paper wide with beaker diameter, filter paper one end is fixed on above beaker mouth, then the other end is put into beaker；Physiological saline or distilled water are added into beaker along walls of beaker to apart from 0.5~1cm of filter paper bottom again, are placed at room temperature for 3~12 hours, collects the liquid repeated centrifugation in beaker, save precipitating to get.Collection method of the invention is used from naughty filter method, the strongyloides intestinalis larva of not impurity can be obtained, it is different from helminth larvae and collects common method Bell Mans method, Plating and Koga method method, and can be used for these three methods and go deimpurity follow-up work, facilitate extraction of subsequent DNA, RNA and protein etc. to study.
Description
Technical field
The invention belongs to Pathogen Biology technical fields, collect from watery stools specifically, the present invention relates to one kind
The method of strongyloides intestinalis larva, the research for subsequent molecular biology etc..
Background technique
Strongyloides intestinalis (Strongyloides stercoralis) is a kind of facultative parasite, and people beast can be caused total
Suffer from parasitic disease, and autogenous infection, dissemination can release souls from purgatory infection in host.The fearful place of strongyloides intestinalis is it with machine
Can be pathogenic, and can autogenous infection and dissemination release souls from purgatory infection.When patient's immunity is normal, clinical symptoms caused by the worm are light,
Easily being ignored leads to mistaken diagnosis, fails to pinpoint a disease in diagnosis.Once patient's immunity degradation is impaired, which is largely proliferated, and can lead to entire patient and broadcasts
Sexy dye is dissipated, causes each organ failure of whole body, or even dead.More frighteningly basic hospital examines doctor and clinician to it
Know little about it and hospital used in aetology method of inspection recall rate it is limited.With regard to national Distribution of Human Parasites report for the first time
It accuses display Hainan Province resident infection by Strongyloides stercoralis rate to rank first in the whole country, infection rate is 1.091%.
Helminth larvae collect common method have the methods of Bell's Mans method, Plating, Koga method, wherein Bell's Mans method and
Koga method is suitable for strongyloides intestinalis larvae collection.These types of method is mainly used for collecting the aetology of larva from forming excrement
Detection is not suitable for collecting strongyloides intestinalis larva from watery stools, secondly has impurity in their gleanings, be unfavorable for
Carry out the research such as extraction of subsequent strongyloides intestinalis larva DNA and RNA.Therefore, molecular biology experiment research and/or clinical inspection
Test the method for needing the high strongyloides intestinalis that free from admixture is collected from patient's watery stools of new easy recall rate.
Summary of the invention
Based on this, in order to overcome the defects of the prior art described above, the present invention provides one kind, and excrement is collected from watery stools
The method of quasi-colubriformis larva.
In order to achieve the above-mentioned object of the invention, this invention takes following technical schemes:
A method of collecting strongyloides intestinalis larva from watery stools, comprising the following steps:
(1), being centrifuged collection 5~10 minutes convenient for 500rpm-3000rpm greatly, collects precipitating；
(2), step (1) precipitating being mixed using physiological saline or distilled water, 500rpm-3000rpm is centrifuged 5~10 minutes,
Supernatant is abandoned, until repeating this step to mixed liquor clarification, is centrifuged 5~10 minutes then at 500rpm-3000rpm, collects precipitating；
(3), step (2) precipitating is mixed with a little physiological saline or distilled water, precipitating is added dropwise in the filter wide with beaker
In the middle part of paper, filter paper one end is fixed on above beaker mouth, then the other end is put into beaker；Again by physiological saline or distilled water
It is added into beaker along walls of beaker to apart from 0.5~1cm of filter paper bottom, is placed at room temperature for 3~12 hours, collect the liquid in beaker
It is centrifuged 5~10 minutes in 500rpm-3000rpm, collects precipitating；It is mixed and is precipitated using distilled water, 500rpm-3000rpm centrifugation 5
~10 minutes, repeat this step 2~3 time, save precipitating to get.
In wherein some embodiments, the speed of centrifugation described in step (1)-(3) is 2000rpm.
In wherein some embodiments, the capacity of beaker described in step (3) is 100ml, 250ml or 500ml, beaker
Capacity is the determination according to the precipitation capacity for being applied to step (2) on filter paper, if amount is more, that just selects the beaker of large capacity,
If amount is few, the beaker of low capacity is just selected.
In wherein some embodiments, the character of the filter paper is T-type or rectangle, and T-type filter paper is easy solid in rim of a cup
Fixed, rectangle filter paper can be fixed by other fixtures in rim of a cup, and the filter paper of other shapes is not easy to be fixed on rim of a cup.
In wherein some embodiments, in step (3), according to the time that is placed at room temperature for of how much determinations for collecting worm, about 3
Just there is within~5 hours larva, worm amount is many within 12 hours, observes liquid level every 1~2h, sees when larva enters beaker
In liquid, and determine the centrifugation time of next step.
The present invention is the one 67 years old male patient's water sample of tumour hospital, Hainan Province collected on 2 13rd, 2017 and 14 days
Excrement is sample, therefrom separates the method for strongyloides intestinalis larva.Compared with prior art, the present invention has below beneficial to effect
Fruit:
Collection method of the invention is used from naughty filter method, i.e., by centrifugal, elutriation method and improvement hook larva of a tapeworm or the cercaria of a schistosome cultivation
These three method organic assemblings can remove the other pathogens and biology unfavorable to molecular biology, not had in one
The single strongyloides intestinalis larva of impurity, method of the invention are different from helminth larvae and collect common method Bell Mans method, put down
Ware method and Koga method method, and can be used for Bell's Mans method, Plating and Koga method method these three methods go it is deimpurity
Follow-up work facilitates extraction of subsequent DNA, RNA and protein etc. to study, the molecular biology experiment service for after.
Specific embodiment
It is further discussed below the present invention combined with specific embodiments below, the present invention does not address place and is suitable for the prior art.Under
Face provides specific embodiments of the present invention, but embodiment is not intended to limit the present invention merely to this explanation is described in further detail
Claim.Reagent used in following embodiment or raw material derive from commercially available unless otherwise specified.
A kind of method that strongyloides intestinalis larva is collected from watery stools of embodiment 1
A kind of method for collecting strongyloides intestinalis larva from watery stools of the present embodiment, specifically includes the following steps:
1, object and collecting dung
67 years old male patient's watery stools of tumour hospital, Hainan Province.Family members cover 3 polybag layerings on bedpan, patient
It defecates in polybag.
2, larvae collection
Using from naughty filter method (abbreviation CWF method), i.e. centrifugal, elutriation method and improvement these three methods of hook larva of a tapeworm or the cercaria of a schistosome cultivation
Combination collection larva.Specifically:
1. the stool of collection is transferred in 15ml centrifuge tube by several times, 2000rpm is centrifuged 5~10 minutes, collects precipitating；
2. mixing step using physiological saline or distilled water 1. to precipitate, 2000rpm is centrifuged 5~10 minutes, abandons supernatant, is repeated
Until mixed liquor is clarified, 2000rpm is centrifuged 5~10 minutes this step, collects precipitating；
3. filter paper is cut into width and beaker (100ml or 250ml or 500ml) the wide T-type of diameter or rectangle.With
A little physiological saline or distilled water mix step and 2. precipitate, and are added dropwise in the middle part of filter paper, filter paper are put into beaker, filter paper upper end is adopted
It is fixed on above beaker mouth with fixing means.Physiological saline or distilled water are added along walls of beaker into beaker, so that in beaker
Physiological saline or distilled water apart from 0.5~1cm of filter paper bottom, contact precipitation is not advisable, and 3~5 hours is placed at room temperature for, every 1
~2h observes liquid level.See when larva enters in beaker liquid, and determine the centrifugation time of next step, collects in beaker
Liquid in 15ml centrifuge tube, 2000rpm be centrifuged 5~10 minutes, collect precipitating, using distilled water mix precipitate, 2000rpm from
The heart 5~10 minutes, this step 2~3 time are repeated, save precipitating, for use.
3, larva checks
The precipitating obtained with 1ml~5ml normal saline dilution step 2 after mixing, takes a drop to use method of direct smear,
Optical microphotograph is under the microscope.
4, result
Using the larva free from admixture collected after the centrifugation+elutriation+filter paper separation of the present embodiment.
Comparative example 1
A kind of method for collecting strongyloides intestinalis larva from watery stools of this comparative example, specifically includes the following steps:
1, object and collecting dung
67 years old male patient's watery stools of tumour hospital, Hainan Province.Family members cover 3 polybag layerings on bedpan, patient
It defecates in polybag.
2, larvae collection
The stool of collection is transferred in 15ml centrifuge tube by several times, 2000rpm is centrifuged 5~10 minutes, collects precipitating；
3, larva checks
The precipitating obtained with 1ml~5ml normal saline dilution step 2 after mixing, takes a drop to use method of direct smear,
Optical microphotograph is under the microscope.
4, result
There are many impurity in the larva being directly collected after centrifugation using the excrement sample of this comparative example.
Comparative example 2
A kind of method for collecting strongyloides intestinalis larva from watery stools of this comparative example, specifically includes the following steps:
1, object and collecting dung
67 years old male patient's watery stools of tumour hospital, Hainan Province.Family members cover 3 polybag layerings on bedpan, patient
It defecates in polybag.
2, larvae collection
1. the stool of collection is transferred in 15ml centrifuge tube by several times, 2000rpm is centrifuged 5~10 minutes, collects precipitating；
2. mixing step using physiological saline or distilled water 1. to precipitate, 2000rpm is centrifuged 5~10 minutes, abandons supernatant, is repeated
Until mixed liquor is clarified, 2000rpm is centrifuged 5~10 minutes this step, collects precipitating；
3, larva checks
The precipitating obtained with 1ml~5ml normal saline dilution step 2 after mixing, takes a drop to use method of direct smear,
Optical microphotograph is under the microscope.
4, result
Using having many impurity in the larva collected after the excrement sample directly centrifugation+elutriation of this comparative example.
From in the slave watery stools of embodiment 1 and comparative example 1~2 separate strongyloides intestinalis larva method comparison it is found that
Collection method of the invention is used from naughty filter method, i.e., by centrifugal, elutriation method and improvement hook larva of a tapeworm or the cercaria of a schistosome cultivation, (test tube is changed to
Beaker, test tube hook larva of a tapeworm or the cercaria of a schistosome cultivation are mainly used for the etiological examination of hookworm, are not suitable for collecting excrement class from a large amount of watery stools
Strongylid larva, this needs a large amount of test tube) for these three method organic assemblings in one, the excrement class that can obtain not impurity is round
Nematode larval, and centrifugal (comparative example 1) and centrifugal+elutriation method (comparative example 2) not can be removed impurity, be unfavorable for
Carry out the research such as extraction of subsequent DNA and RNA.
Hook larva of a tapeworm or the cercaria of a schistosome cultivation, centrifugal, Bell's Mans method, Plating, Koga method are inspection methods.
Hook larva of a tapeworm or the cercaria of a schistosome cultivation also becomes test tube filter paper cultivation, and application range is that hookworm is hatched in the excrement containing hookworm ovum
Larva, excrement are mostly forming or paste excrement, it is therefore an objective to etiological diagnosis or identification for hookworm；Centrifugal, Bei Erman
Purpose predominantly etiological diagnosis or the identification of worm or worm's ovum of family name's method, Plating, Koga method, the worm or worm that these methods are collected
Ovum has excrement slag, is unfavorable for the molecular biology experiments such as extraction of the later period for DNA, RNA and protein；Bell's Mans method, plate
Method, the Koga method scope of application are the excrement of forming or paste, are not suitable for watery stools.It is of the invention be better than from naughty filter method more than
Various methods, and can be used for Bell's Mans method, Plating and Koga method method these three methods and go deimpurity follow-up work,
Extraction of subsequent DNA and RNA etc. is facilitated to study.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (5)
1. a kind of method for collecting strongyloides intestinalis larva from watery stools, which comprises the following steps:
(1), being centrifuged collection 5~10 minutes convenient for 500rpm-3000rpm greatly, collects precipitating；
(2), step (1) precipitating is mixed using physiological saline or distilled water, 500rpm-3000rpm is centrifuged 5~10 minutes, in abandoning
Clearly, it until repeating this step to mixed liquor clarification, is centrifuged 5~10 minutes then at 500rpm-3000rpm, collects precipitating；
(3), step (2) precipitating is mixed with a little physiological saline or distilled water, precipitating is added dropwise in the filter paper wide with beaker
Portion, filter paper one end is fixed on above beaker mouth, and the other end is put into beaker；Physiological saline or distilled water are added along walls of beaker again
Enter beaker to apart from 0.5~1cm of filter paper bottom, be placed at room temperature for 3~12 hours, collects the liquid in beaker in 500rpm-
3000rpm is centrifuged 5~10 minutes, collects precipitating；It being mixed and is precipitated using distilled water, 500rpm-3000rpm is centrifuged 5~10 minutes,
Repeat this step 2~3 time, save precipitating to get.
2. the method according to claim 1 for collecting strongyloides intestinalis larva from watery stools, which is characterized in that step
(1) speed of centrifugation described in-(3) is 2000rpm.
3. the method according to claim 1 for collecting strongyloides intestinalis larva from watery stools, which is characterized in that step
(3) capacity of beaker described in is 100ml, 250ml or 500ml.
4. the method according to claim 1 for collecting strongyloides intestinalis larva from watery stools, which is characterized in that step
(3) shape of filter paper described in is T-type or rectangle.
5. the method according to claim 1 for collecting strongyloides intestinalis larva from watery stools, which is characterized in that step
(3) when being placed at room temperature in, liquid level is observed every 1~2h.
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| CN201710159044.2A CN106950073B (en) | 2017-03-17 | 2017-03-17 | A method of collecting strongyloides intestinalis larva from watery stools |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN2656987Y (en) * | 2003-11-26 | 2004-11-17 | 戴友良 | Faeces dreg inspection device |
| CN101424613A (en) * | 2008-04-08 | 2009-05-06 | 廖党金 | Parasite egg detecting method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2656987Y (en) * | 2003-11-26 | 2004-11-17 | 戴友良 | Faeces dreg inspection device |
| CN101424613A (en) * | 2008-04-08 | 2009-05-06 | 廖党金 | Parasite egg detecting method |
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