CN101943693A - Quarantine method for pre-encapsulated larvae of Trichinella spiralis in meat - Google Patents
Quarantine method for pre-encapsulated larvae of Trichinella spiralis in meat Download PDFInfo
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- CN101943693A CN101943693A CN2010102831834A CN201010283183A CN101943693A CN 101943693 A CN101943693 A CN 101943693A CN 2010102831834 A CN2010102831834 A CN 2010102831834A CN 201010283183 A CN201010283183 A CN 201010283183A CN 101943693 A CN101943693 A CN 101943693A
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Abstract
The invention discloses a quarantine method for pre-encapsulated larvae of Trichinella spiralis in meat, which comprises the following steps of: 1, sampling meat samples from different parts of carcass of an animal to be quarantined, and precooling at the temperature of 4 DEG C; 2, connecting the lower end of a funnel with a hose, clamping the tail end of the funnel by using a clamp, laying a medicinal gauze piece in the funnel, and soaking the gauze piece by using physiological saline; 3, inserting the lower part of the funnel into a water bath kettle at the temperature of 37 DEG C, and keeping the water surface 2cm lower than the bottom layer of the gauze piece; 4, mincing the precooled meat samples to be quarantined, putting the meat samples on the gauze piece in the funnel, and adding physiological saline; 5, taking the funnel out after 30 minutes, opening the clamp on the hose and collecting liquid therein in a centrifuge tube; and 6, centrifuging or precipitating the liquid in the centrifuge tube, removing the supernatant, and performing microscopic examination on the sediment under an optical microscope. The quarantine method has the advantages of directly separating pre-encapsulated infectious larva from the meat samples to be quarantined by using physiological characteristic of thermotropic movement of the pre-encapsulated infectious larva, and realizing reliable quarantine of the pre-encapsulated infectious larva.
Description
Technical field
The present invention relates to biotechnology and meat quarantine technical field, especially relate to the quarantine method of trichina one-tenth precystic stage larva in a kind of meat.
Background technology
Trichinosis main because of eat raw or half a lifetime food contain due to the meat and meat products of trichinzation larva, not only serious harm health of this disease also can cause tremendous economic loss to livestock breeding industry.Along with the change of people's eating habit, because of eat give birth to or half a lifetime animal meat cause that trichinous breaking out gradually increase; In addition, because the extensive of the development of the globalization of international trade, tourist industry and population flowed, all increased the chance that trichinosis is propagated.According to estimates, the nearly 1,100 ten thousand trichinosis patients in the whole world have now classified it as the disease (re-emerging disease) that occurs once more at present.Serosurvey shows that China has 2 000 ten thousand trichinzation persons approximately at present, and infection rate is far above world average level.Therefore, strengthening the quarantine of meat trichina has been instant challenge.
Many countries make laws, and the trichina that regulation should use direct method (trichina microscopy method or artificial digestion method) to carry out in the meat is quarantined.The domestic at present normal diaphram compressing tablet microscopy method that adopts is checked trichina, but the susceptibility of microscopy method is lower, and especially when containing the muscle specimen inspection of low-density larva, its false negative rate can not reach the meat safe requirement up to 52%.The artificial digestion method of international trichinosis council's recommendation use carries out the meat quarantine, in theory, as long as contain 1 or 1 above trichinella larvae (the minimum infection level that public health is constituted a threat to) in meat or the meat products sample,, can be detected if method of operating is correct.But, in actual application, the quarantine effect of digestion method is subjected to multiple factor affecting such as the filtration of the temperature of concentration, digestion of composition, pepsin and hydrochloric acid of preparation method, digestive juice of sample and duration, recovery larva and intermediate processing, actual susceptibility is that every gram muscle contains 3～5 larvas and just can be detected, contained in the tested sample and be enough to cause the trichinous larva of human body this moment, but false-negative result can occur during quarantine.Breaking out in 15 trichinosises that Former Yugoslavia takes place as 1985-1986, all is to have eaten through due to the qualified pork of quarantine, and 2001-2002 also is owing to eaten due to the pork of omission breaking out of taking place of Kumane city.Therefore, the reliability of assay is for guaranteeing that food security and people ' s health are very important.
Traditional view generally believes, has only encystation phase larva to be only the infective stage of trichina (except that pseudo-trichina etc. not the trichina of encystation), and the multiple meat trichina of using at present quarantine technology all is at the encystation phase result that observes of larva.Along with going deep into of research, confirmed now behind the trichinzation host that 18 days one-tenth precystic stage larva has had infectivity to new host, the precystic stage infective larvae.When the meat that contains trichina one-tenth precystic stage infective larvae is quarantined, because this phase trichinella larvae is cylindric or bar-shaped more, and there is not typical trichinella larvae Nang Bao, differentiate than difficult among being mingled in muscle fibre, cause the difficulty of microscopy to strengthen, the layman is difficult to detect, and recall rate reduces greatly; In addition; become the precystic stage infective larvae owing to there is not the protective effect of Nang Bao; the ability of its opposing digestion is starkly lower than encystation phase larva; can not tolerate the digestion of conventional formulation artificial digestion liquid; when using artificial digestion method that the meat that contains into the precystic stage infective larvae is quarantined; larva is often fragmented into several sections by digestion; part larva even meeting be fallen by digestion; muscle fibre mixes with not digesting completely in the meat sample; extremely difficult the resolution; have a strong impact on the quarantine effect of meat trichina, cause the appearance of false negative result, public health is constituted a threat to.So the method for quarantine trichina encystation phase larva can not be directly used in into the quarantine of precystic stage infective larvae.
This shows, the susceptibility of trichina microscopy method and artificial digestion method can not reach in actual applications fully to the meat safe requirement, during especially to trichina low-grade infection and/or the quarantine of one-tenth precystic stage infective larvae, more can not satisfy the public health needs.
Summary of the invention
The object of the present invention is to provide a kind of quarantine method that has high recall rate and can be widely used in trichina one-tenth precystic stage larva in the meat.
For achieving the above object, the present invention can take following technical proposals:
Trichina becomes the quarantine method of precystic stage larva in the meat of the present invention, comprises the steps:
The first step is gathered the meat sample to be checked of gross weight 2g～5g from the different parts of animal ketoboidies to be checked, at the mid-4 ℃ of precooling 20min of clean plate;
Second step, get one in funnel, the funnel lower end is connected top hose, with clip hose end is clamped; Get and be tiled in the funnel after hospital gauze is folded into more than 4 layers, earlier gauze is wetting with physiological saline, in funnel, slowly add physiological saline afterwards, its submergence gauze piece bottom is got final product;
The 3rd step, the water bath with thermostatic control pot temperature is adjusted to 37 ℃ after, the funnel bottom is inserted in the water-bath, keep the water surface to be lower than gauze piece bottom 2cm;
The 4th step, place stirrer to rub the meat sample to be checked after the first step precooling after, place on the hospital gauze piece of funnel, add physiological saline, physiological saline liquid level submergence meat sample to be checked is got final product;
The 5th step, behind the 30min, funnel is taken out from water-bath, open the clip of hose end, the liquid in the flexible pipe is collected in the centrifuge tube;
The 6th step, above-mentioned centrifuge tube placed the centrifugal 5min of hydro-extractor 500g or natural sedimentation 20～30min after, abandon supernatant, get sediment and place microscopy under the optical microscope.
If microscopy is found trichinella larvae, judge that then this meat sample is the positive meat sample of trichina; If microscopy is not found trichinella larvae, then need regather separating liquid 2 times, when continuous 3 collection results were all negative, then this meat sample of decidable did not contain trichina one-tenth precystic stage larva.
Owing to become the precystic stage infective larvae to have the physiological characteristic of dividing a word with a hyphen at the end of a line, utilize this characteristic the one-tenth precystic stage infective larvae in the meat sample to be checked directly can be separated, thereby realize becoming the quarantine of precystic stage infective larvae to temperature.
The invention has the advantages that standard to becoming the operative technique and the requirement of the quarantine of precystic stage larva in the meat, for guaranteeing that meat product safety, trichinous prevention provide a kind of new quarantine method, remedied the deficiency of existing meat trichina quarantine method to a great extent, can make the quarantine of meat trichina more comprehensive with existing detection method coupling, the result is more reliable in quarantine, for guaranteeing that meat product safety, guarantee people's health and international meat trade are all significant.Trichina involved in the present invention becomes precystic stage larva separation method also to can be used as trichina and becomes precystic stage larva collection method, can isolate into the precystic stage larva from the trichinzation animal model, and the larva in trichina particular growth stage is provided for scientific research.
Beneficial effect of the present invention is mainly reflected in:
1, tripping device used in the present invention is drawn materials conveniently, makes simply, and is cheap, is adapted at quarantine departmentss at different levels and popularizes;
2, trichina becomes precystic stage larva quarantine method principle simple, easy to operate in the meat involved in the present invention, is easy to grasp;
3, collection sample separation quarantine act involved in the present invention can be quarantined to 100 parts of meat samples to be checked simultaneously, has improved quarantine efficient greatly;
4, trichina becomes precystic stage larva quarantine method to remedy the deficiency of existing meat trichina quarantine method in the meat involved in the present invention, can make the quarantine of meat trichina more comprehensive with the coupling of existing meat trichina quarantine method, and the result is more reliable;
5, meat trichina involved in the present invention becomes precystic stage larva separation method still to can be used as a kind of trichina one-tenth precystic stage larva collection method provides the trichina particular growth larva in period for scientific research.
Embodiment
Term definition:
A, newborn larvae: be meant that the female adult of trichina has the larva of firm output in host's enteron aisle;
B, become the precystic stage larva: the trichina newborn larvae is grown after invading behind the intestinal mucosa and invading Skeletal Muscle Cell with blood flow or lymph, but does not also form the larva of Nang Bao, promptly infects the larva of back 5～26d at host's bone intramuscular;
C, one-tenth precystic stage infective larvae:, promptly infect the larva of back 18～26d at host's bone intramuscular to the existing infective one-tenth precystic stage larva of new host;
D, encystation phase larva: form complete Nang Bao and have infective trichinella larvae after invading skeletal muscle, promptly infect back 26d to the larva of several years at host's bone intramuscular.
Trichina becomes the quarantine method of precystic stage larva in the meat of the present invention, comprises the steps:
The first step is gathered the meat sample to be checked of gross weight 2g～5g from the different parts of animal ketoboidies to be checked, at the mid-4 ℃ of precooling 20min of clean plate; Its objective is meat sample temperature is reduced, increase temperature difference, help the separation of larva;
Second step, get one in funnel, the funnel lower end is connected the rubber hose that goes up 20cm～30cm, with clip hose end is clamped; Getting hospital gauze is folded into 4 layers and (not only larva is passed through but also can effectively stop muscle fibre to see through gauze piece, avoid muscle fibre too much to disturb larva quarantine effect) after be tiled in the funnel, use glue head dropper earlier that the gauze piece of tiling is wetting with physiological saline, slowly add an amount of physiological saline afterwards in funnel, addition just is advisable submergence gauze piece bottom physiological saline;
The 3rd step, the water temperature in the thermostat water bath is adjusted to 37 ℃ after, insert in the thermostat water bath funnel bottom that will have rubber hose, keeps the water surface to be lower than gauze piece bottom 2cm, so that form a tangible thermograde up and down at gauze piece;
The 4th step placed stirrer to rub for several times in the meat sample to be checked after the first step precooling, each 5 seconds, meat sample to be checked is rubbed fully till; Be placed on then on the hospital gauze piece in the funnel, should make meat sample to be checked be in the center of gauze piece during application of sample as far as possible, add an amount of physiological saline, the just complete submergence meat sample to be checked of physiological saline liquid level is got final product;
The 5th step, pick up counting: at this moment, temperature is lower owing to the process precooling for the meat sample to be checked of gauze piece top, the physiological saline of gauze piece below is because the pre-heat effect of water-bath and near 37 ℃, so just formed tangible thermograde up and down at gauze piece, owing to become the precystic stage larva to have the physiological characteristic of dividing a word with a hyphen at the end of a line to temperature, trichina in the meat sample to be checked becomes the precystic stage larva to be separated through the gauze piece of upper cold and lower heat, and trichinella larvae bored the hypomere that will be deposited on rubber hose behind the gauze piece; Note in the detachment process not touching gauze piece, the muscle fibre of avoiding rupturing sees through gauze piece and influences the quarantine effect; Behind the 30min, funnel is taken out from water-bath, open the clip of rubber hose end, the liquid in the rubber hose is collected in the centrifuge tube; Immediately the rubber hose of funnel lower end is clamped after collection is finished, and have the gauze piece top to add an amount of physiological saline again, proceed larva and separate;
The 6th step, have the centrifuge tube of liquid to place the centrifugal 5min of supporting hydro-extractor 500g or natural sedimentation 20～30min the above-mentioned collection after, abandon supernatant, get sediment and place microscopy under the optical microscope.
If microscopy is found trichinella larvae, judge that then this meat sample is the positive meat sample of trichina; If microscopy is not found trichinella larvae, then need regather separating liquid 2 times, when continuous 3 collection results were all negative, then this meat sample of decidable did not contain trichina one-tenth precystic stage larva.
When meat in enormous quantities was detected, the meat sample umber that once collects the sample quarantine can determine this group sample size as the case may be between 10 parts to 100 parts.To every animal trunk to be checked respectively after the meat sample to be checked of heavily about 2g～5g is gathered at different positions, should organize all meat samples placements to be checked quarantines together, if microscopy is found trichinella larvae, then point out this to organize and have 1 part of positive meat sample in the meat sample to be checked at least, must find out the animal trunk to be checked of this group sample correspondence according to prior numbering, and separate the quarantine act quarantine one by one separately, to determine positive meat sample; If microscopy is not found trichinella larvae, then need regather separating liquid 2 times, do not contain trichina in continuous 3 quarantine all negative this group of decidable still of result meat samples to be checked and become the precystic stage larva, can improve detection speed greatly like this.
Funnel used in the present invention, rubber hose, hospital gauze piece, glue head dropper, plate, stirrer and centrifuge tube all can be reused, and clean up after each the use, and be promptly reusable.
Animal experiment:
Select for use healthy male 6 age in week kunming mice, the cleaning level; Trichina (
Trichinella spiralisT1) be pig source, Henan trichina, go down to posterity with a cleaning level kunming mice by present patent application unit and to protect kind, adopt and feed the feeding infection, cut open and infect the above kunming mice of trichina 40d extremely, in the compressing tablet microscopy muscle encystation phase larva, counting back with infect trichinous muscle hello raise the blank kunming mice of fasting 1d.The kunming mice of 18d behind the infection trichina encystation phase larva is cutd open extremely, get muscle and carry out the compressing tablet microscopy, infect successfully to determine it, the interior trichinella larvae of this mouse muscle this moment is trichina one-tenth precystic stage larva.
Get 1 of the kunming mice muscle that infects 18d behind the trichina, other gets 9 of blank kunming mice muscle that do not infect trichina, cuts open respectively and kills, and gathers each 2g of muscle sample from every mouse trunk different parts respectively.With 10 parts of mouse muscles as meat sample to be checked, carry out the meat trichina according to step of the present invention and become the quarantine of precystic stage larva: find that altogether 1 part of meat sample is positive after the quarantine one by one, all the other 9 parts negative, conform to set result, prove that trichina becomes precystic stage larva quarantine method can be used for the meat quarantine in the meat involved in the present invention, and the quarantine reliable results.
Get 20 mouse and be divided into 2 groups (10 every group) at random, respectively by 20/only, 10/dosage peroral infection trichina encystation phase larvas.Infect back 18d and cut open mouse extremely, 2 groups of infecting mouse muscle are blended the back mixing respectively, respectively be divided into 15 equal portions, every part of 6g, every part of meat sample is divided into digestion method, pigs trichina disease diagnostic techniques (GB/T 18642-2002) digestion method and the meat trichina of the present invention of recommending with ICT successively after 3 parts again and becomes precystic stage larva quarantine act to quarantine.Other gets 20 mouse by 5/dosage peroral infection trichina encystation phase larvas, infecting back 18d cuts open extremely, mouse muscle is blended the back mixing, be divided into 30 parts, every part of 6g, after being divided into 3 parts again, every part of meat sample quarantines with above-mentioned 3 kinds of quarantine methods successively: found that, and 15 parts of mouse muscles of 18d after infecting for 20/dosage, the larva recall rate of 3 kinds of quarantine methods is 100%(χ
2=0.000, P＞0.05); 10/dosage infect 15 parts of mouse muscles of back 18d, and the recall rate of 3 kinds of methods is followed successively by 93.33%, 93.33% and 100%(χ
2=1.645, P＞0.05); 3 kinds of different quarantine methods are described at 10/the equal not statistically significants of difference with 20/dosage infected group larva recall rates, the quarantine effect is suitable.And for 5/only than the mouse muscles of 18d after 30 parts of infection of low dosage infected group, the recall rate of 3 kinds of methods is followed successively by 63.33%, 90% and 100%(χ
2=18.866, P＜0.05), the difference of recall rate has statistical significance, and the highest with the present invention.Can think that when low dosage infected, trichina became precystic stage larva quarantine act to become the quarantine effect of precystic stage larva to be better than digestion method to trichina in the meat of the present invention.
Claims (1)
1. trichina becomes the quarantine method of precystic stage larva in the meat, it is characterized in that: comprise the steps:
The first step is gathered the meat sample to be checked of gross weight 2g～5g from the different parts of animal ketoboidies to be checked, at the mid-4 ℃ of precooling 20min of clean plate;
Second step, get one in funnel, the funnel lower end is connected top hose, with clip hose end is clamped; Get and be tiled in the funnel after hospital gauze is folded into more than 4 layers, earlier gauze is wetting with physiological saline, in funnel, slowly add physiological saline afterwards, its submergence gauze piece bottom is got final product;
The 3rd step, the water bath with thermostatic control pot temperature is adjusted to 37 ℃ after, the funnel bottom is inserted in the water-bath, keep the water surface to be lower than gauze piece bottom 2cm;
The 4th step, place stirrer to rub the meat sample to be checked after the first step precooling after, place on the hospital gauze piece of funnel, add physiological saline, physiological saline liquid level submergence meat sample to be checked is got final product;
The 5th step, behind the 30min, funnel is taken out from water-bath, open the clip of hose end, the liquid in the flexible pipe is collected in the centrifuge tube;
The 6th step, above-mentioned centrifuge tube placed the centrifugal 5min of hydro-extractor 500g or natural sedimentation 20～30min after, abandon supernatant, get sediment and place microscopy under the optical microscope.
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| CN2010102831834A CN101943693A (en) | 2010-09-16 | 2010-09-16 | Quarantine method for pre-encapsulated larvae of Trichinella spiralis in meat |
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| CN2010102831834A CN101943693A (en) | 2010-09-16 | 2010-09-16 | Quarantine method for pre-encapsulated larvae of Trichinella spiralis in meat |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030036105A1 (en) * | 1998-07-24 | 2003-02-20 | Canadian Food Inspection Agency. | Serological diagnosis of protostrongylidae infections and identification of unique antigens |
| CN1487297A (en) * | 2003-01-05 | 2004-04-07 | 中国人民解放军军需大学 | Detection method and kit for trichinosis of both human body and animal |
-
2010
- 2010-09-16 CN CN2010102831834A patent/CN101943693A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030036105A1 (en) * | 1998-07-24 | 2003-02-20 | Canadian Food Inspection Agency. | Serological diagnosis of protostrongylidae infections and identification of unique antigens |
| CN1487297A (en) * | 2003-01-05 | 2004-04-07 | 中国人民解放军军需大学 | Detection method and kit for trichinosis of both human body and animal |
Non-Patent Citations (3)
| Title |
|---|
| 姜鹏等: "肉类中旋毛虫成囊前期幼虫检验方法及其影响因素", 《中国人兽共患病学报》 * |
| 徐冬梅等: "镜检法与贝氏法对肉类中旋毛虫成囊前幼虫的检验效果", 《中国病原生物学杂志》 * |
| 王中全等: "旋毛虫幼虫收集方法的探讨", 《河南医学研究》 * |
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Application publication date: 20110112 |