CN111381021A - Early diagnosis method based on detection of hydatid specific antigen and coding gene thereof - Google Patents
Early diagnosis method based on detection of hydatid specific antigen and coding gene thereof Download PDFInfo
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Abstract
The invention discloses an early diagnosis method based on detecting echinococcus specific antigen and its coding gene, which relates to the echinococcosis diagnosis technical field, and utilizes the specific reaction after combining the antibody and the antigen, combines the diluted feces with the help of the echinococcus antibody enzyme labeling conjugate, after reacting for a period of time, utilizes an enzyme labeling instrument to detect the light absorption value of the reaction liquid, and compares with the light absorption value of the reaction liquid of the normal operation, so as to obtain the result, namely, whether the examinee has the echinococcus specific antigen, starts with the blood serum of the examinee, the free DNA molecule extracted from the blood serum is more stable, which is beneficial to improving the accuracy of the diagnosis data, utilizes the second generation sequencing technology of DNA to sequence the free DNA molecule, then utilizes the PCR technology to expand the DNA sequence, and compares with the reference sample sequence of the echinococcosis patient through the bioinformatics analysis technology, so that the conclusion whether the subject suffers from echinococcosis can be drawn.
Description
Technical Field
The invention relates to the technical field of echinococcosis diagnosis, in particular to an early diagnosis method based on detection of echinococcosis specific antigen and encoding genes thereof.
Background
Echinococcosis (echinococcosis) is a chronic parasitic disease caused by infection of human with echinococcus larvae (echinococcosis). The clinical manifestations of the disease vary depending on the cyst position, size and complications of echinococcosis, which is considered as a parasitic disease of zoonosis and called as zoonosis, but recently epidemiological investigation shows that the disease is called as endemic parasitic disease; the endemic area is characterized by occupational injury and is classified as an occupational disease of certain people; echinococcosis is a common and frequently occurring disease that is unique to ethnic minority or religious tribes on a global scale.
At present, early diagnosis methods for echinococcosis usually adopt etiologic diagnosis (imaging method including X-ray, ultrasonic examination and CT), Indirect Fluorescent Antibody Test (IFAT) and enzyme-linked immunosorbent assay (ELISA) and the like, and the diagnosis methods have advantages, but have common defects that the echinococcosis is difficult to detect in the early stage due to small focus, and meanwhile, some symptoms of cancer patients are similar to echinococcosis, and the requirements on professional technicians are high, so that the problems of missed detection and false detection are easy to occur, time and labor are wasted, and certain injury risks are caused to the patients.
In view of the above, the skilled person in the art proposes an early diagnosis method based on the detection of a hydatid-specific antigen and its encoding gene.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an early diagnosis method based on the detection of a specific antigen of echinococcosis and a coding gene thereof, which solves the problems that the early diagnosis method aiming at the echinococcosis usually has advantages through etiological diagnosis (an imaging method, including X-ray, ultrasonic examination and CT), Indirect Fluorescent Antibody Test (IFAT), enzyme-linked immunosorbent assay (ELISA) and the like, but has the common defects that the echinococcosis is difficult to detect due to small focus in the early stage, and meanwhile, some symptoms of cancer patients are similar to the echinococcosis, the requirements on professional technicians are high, the problems of missed detection and false detection are easy to occur, time and labor are wasted, and a certain injury risk is caused to the patients.
In order to achieve the purpose, the invention is realized by the following technical scheme: an early diagnosis method based on the detection of a hydatid specific antigen comprises the following steps:
s1, sampling: selecting a group with age of 20-40 as a diagnosis object, taking fresh feces discharged by the patient at 7-8 am, and placing in a special feces box;
s2, sample treatment: placing the excrement box containing the fresh excrement in the step S1 in a medical freezing container, wherein the freezing temperature is-60 ℃, the freezing time lasts for at least one week, and the insect eggs are subjected to inactivation treatment;
s3, sample dilution: taking 10-20g of the frozen excrement sample in the step S2, pouring into a dilution container, additionally adding 10-20ml of excrement diluent, and fully stirring by using a stirring rod to obtain a sample solution;
s4, centrifuging the sample liquid and taking supernatant: adding the sample liquid obtained in the step S3 into a medical centrifuge, controlling the speed of the centrifuge to be 5000-;
s5, adding medicine into the supernatant for reaction: dripping 2-4 drops of the supernatant of the sample liquid obtained in the step S4 into a reaction plate by using a rubber head dropper, additionally adding a diagnostic reagent into the reaction plate, sealing the reaction plate by using a biological preservative film, transferring the reaction plate into a thermostat, and reacting for 1-1.5 hours in a dark place to obtain a reaction liquid;
s6, color development reaction: dripping 3-5 drops of a display agent into the reaction liquid obtained in the step S5, and transferring the reaction liquid into a thermostat to react for 15-20min in a dark place;
s7, enzyme labeling test, and judging the result: and (4) reading the light absorption value of the solution after the reaction in the step S6 by using an enzyme labeling instrument under the light wave with the wavelength of 450nm, and judging whether the hydatid specific antigen exists or not according to the multiple between the light absorption value and the light absorption value of the negative solution.
Preferably, the stool box in step S1 is made of medical stainless steel, and needs to be sterilized at high temperature.
Preferably, in step S5, the diagnostic reagent is selected from a hydatid enzyme-labeled conjugate.
Preferably, the temperature inside the oven is 34-37 ℃ in step S5, and 35-37.5 ℃ in step S6.
Preferably, in step S7, after the detection by the microplate reader, the determination of the existence of the hydatid specific antigen is made when the multiple between the light absorption value of the obtained reacted solution and the light absorption value of the negative solution is 2.9 to 3.1 times.
An early diagnosis method based on detecting a chafer coding gene comprises the following steps:
A. sample acquisition: collecting a blood sample of a subject, and placing the obtained blood sample in a blood collection container;
B. obtaining serum: placing the blood sampling container containing the sample blood in the step A in a medical centrifuge, centrifuging for 3-4min to obtain blood serum, and placing the blood serum in a special serum storage bottle;
C. extracting genome DNA: extracting free DNA molecules from the serum obtained in step B by a concentrated salt method;
D. and (3) sequence determination: based on the second generation sequencing technology of DNA, the DNA molecules obtained in the step C are subjected to sequencing treatment to obtain a sample sequence of the DNA;
E. PCR amplification of DNA: d, carrying out multiple polymerase chain reaction by using relatively longer fragments in the DNA sample sequence obtained in the step D, and adding a primer pair in the reaction to obtain a DNA amplification sequence;
F. comparing and screening: comparing the DNA amplification sequence obtained in the step E with a reference sample sequence of a patient suffering from echinococcosis based on bioinformatics analysis technology;
G. the results were obtained: and F, comparing and processing to obtain whether the free DNA contains the molecules of the echinococcosis source or the host specific reaction source in the early process of the echinococcosis.
Preferably, the primer pair added in step E is an oligonucleotide primer labeled with a fluorescent dye.
Advantageous effects
The invention provides an early diagnosis method based on detection of a hydatid specific antigen and a coding gene thereof. Compared with the prior art, the method has the following beneficial effects:
1. the early diagnosis method based on detecting the hydatid specific antigen utilizes the specific reaction after combining the antibody and the antigen, in the diagnosis process, the excrement of a detected person who does not know whether the hydatid specific antigen exists is firstly frozen, the eggs of the hydatid which possibly exist in the inside are inactivated, then the excrement after being frozen for a period of time is diluted, the excrement is combined with the diluted excrement by virtue of an hydatid enzyme-labeled conjugate, after the reaction is carried out for a period of time, an enzyme-labeled instrument is utilized to detect the light absorption value of reaction liquid, and the light absorption value is compared with the light absorption value of the reaction liquid of the normal operation, so that the result can be obtained, namely whether the detected person has the hydatid specific antigen, and the method is simple, fast, convenient and effective.
2. The early diagnosis method based on detecting the echinococcosis encoding gene starts with the serum of a detected person, free DNA molecules extracted from the serum are more stable, the accuracy of diagnosis data is favorably improved, the free DNA molecules are subjected to sequencing treatment by utilizing a second-generation DNA sequencing technology, then DNA sequences are expanded by a PCR technology, and the DNA sequences are compared with reference sample sequences of patients suffering from echinococcosis by a bioinformatics analysis technology, so that the conclusion whether the detected person suffers from echinococcosis can be obtained.
Drawings
FIG. 1 is a block diagram of the process for early diagnosis of a hydatid-specific antigen according to the present invention;
FIG. 2 is a block diagram of the early diagnosis process of the codling gene of hydatid of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1, the present invention provides a technical solution: an early diagnosis method based on the detection of a hydatid specific antigen comprises the following steps:
s1, sampling: selecting a group with age of 30 years as a diagnosis object, taking fresh excrement discharged by the patient at 8 am, and placing the fresh excrement in a special excrement box;
s2, sample treatment: placing the excrement box containing the fresh excrement in the step S1 in a medical freezing container, wherein the freezing temperature is-60 ℃, the freezing time lasts for at least one week, and the insect eggs are subjected to inactivation treatment;
s3, sample dilution: taking 15g of the frozen excrement sample in the step S2, pouring the sample into a dilution container, adding 15ml of excrement diluent, and fully stirring by using a stirring rod to obtain a sample solution;
s4, centrifuging the sample liquid and taking supernatant: adding the sample liquid obtained in the step S3 into a medical centrifugal machine, controlling the speed of the centrifugal machine to be 6000rpm, and centrifuging for 18min to obtain a supernatant of the sample liquid, and taking out the supernatant for later use;
s5, adding medicine into the supernatant for reaction: dripping 2-4 drops of the supernatant of the sample liquid obtained in the step S4 into a reaction plate by using a rubber head dropper, additionally adding a diagnostic reagent into the reaction plate, sealing the reaction plate by using a biological preservative film, transferring the reaction plate into a thermostat, and reacting for 1 hour in a dark place to obtain a reaction liquid;
s6, color development reaction: dripping 4 drops of the developer into the reaction liquid obtained in the step S5, and transferring the reaction liquid into a thermostat to react for 20min in a dark place;
s7, enzyme labeling test, and judging the result: and (4) reading the light absorption value of the solution after the reaction in the step S6 by using an enzyme labeling instrument under the light wave with the wavelength of 450nm, and judging whether the hydatid specific antigen exists or not according to the multiple between the light absorption value and the light absorption value of the negative solution.
Further, the feces box in step S1 is made of medical stainless steel and needs to be sterilized at high temperature.
Further, in step S5, the diagnostic reagent is an enzyme-labeled conjugate of a hydatid antibody.
Further, in step S5, the internal temperature of the oven was 35 ℃ and in step S6, the internal temperature of the oven was 37 ℃.
Further, in step S7, after the detection by the microplate reader, when the multiple between the light absorption value of the obtained reacted solution and the light absorption value of the negative solution is 3.1 times, it is determined that the hydatid specific antigen exists.
Referring to fig. 2, an early diagnosis method based on detecting the codogenic gene of hydatid includes the following steps:
A. sample acquisition: collecting a blood sample of a subject, and placing the obtained blood sample in a blood collection container;
B. obtaining serum: placing the blood sampling container containing the sample blood in the step A in a medical centrifuge, centrifuging for 4min to obtain blood serum, and placing the blood serum in a special serum storage bottle;
C. extracting genome DNA: extracting free DNA molecules from the serum obtained in step B by a concentrated salt method;
D. and (3) sequence determination: based on the second generation sequencing technology of DNA, the DNA molecules obtained in the step C are subjected to sequencing treatment to obtain a sample sequence of the DNA;
E. PCR amplification of DNA: d, carrying out multiple polymerase chain reaction by using relatively longer fragments in the DNA sample sequence obtained in the step D, and adding a primer pair in the reaction to obtain a DNA amplification sequence;
F. comparing and screening: comparing the DNA amplification sequence obtained in the step E with a reference sample sequence of a patient suffering from echinococcosis based on bioinformatics analysis technology;
G. the results were obtained: and F, comparing and processing to obtain whether the free DNA contains the molecules of the echinococcosis source or the host specific reaction source in the early process of the echinococcosis.
Further, the primer pair added in step E is an oligonucleotide primer labeled with a fluorescent dye.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. An early diagnosis method based on the detection of a hydatid specific antigen, which is characterized by comprising the following steps:
s1, sampling: selecting a group with age of 20-40 as a diagnosis object, taking fresh feces discharged by the patient at 7-8 am, and placing in a special feces box;
s2, sample treatment: placing the excrement box containing the fresh excrement in the step S1 in a medical freezing container, wherein the freezing temperature is-60 ℃, the freezing time lasts for at least one week, and the insect eggs are subjected to inactivation treatment;
s3, sample dilution: taking 10-20g of the frozen excrement sample in the step S2, pouring into a dilution container, additionally adding 10-20ml of excrement diluent, and fully stirring by using a stirring rod to obtain a sample solution;
s4, centrifuging the sample liquid and taking supernatant: adding the sample liquid obtained in the step S3 into a medical centrifuge, controlling the speed of the centrifuge to be 5000-;
s5, adding medicine into the supernatant for reaction: dripping 2-4 drops of the supernatant of the sample liquid obtained in the step S4 into a reaction plate by using a rubber head dropper, additionally adding a diagnostic reagent into the reaction plate, sealing the reaction plate by using a biological preservative film, transferring the reaction plate into a thermostat, and reacting for 1-1.5 hours in a dark place to obtain a reaction liquid;
s6, color development reaction: dripping 3-5 drops of a display agent into the reaction liquid obtained in the step S5, and transferring the reaction liquid into a thermostat to react for 15-20min in a dark place;
s7, enzyme labeling test, and judging the result: and (4) reading the light absorption value of the solution after the reaction in the step S6 by using an enzyme labeling instrument under the light wave with the wavelength of 450nm, and judging whether the hydatid specific antigen exists or not according to the multiple between the light absorption value and the light absorption value of the negative solution.
2. The method of claim 1, wherein the fecal cassette of step S1 is made of stainless steel and is sterilized at high temperature.
3. The method as claimed in claim 1, wherein the diagnostic reagent is selected from the group consisting of a hydatid-specific enzyme-labeled conjugate in step S5.
4. The method of claim 1, wherein the temperature inside the incubator is 34-37 ℃ in step S5, and 35-37.5 ℃ in step S6.
5. The early diagnosis method based on the detection of the hydatid specific antigen as claimed in claim 1, wherein in step S7, after the detection by the microplate reader, if the multiple between the light absorption value of the reacted solution and the light absorption value of the negative solution is 2.9-3.1 times, the existence of the hydatid specific antigen is determined.
6. An early diagnosis method based on the detection of a chafer coding gene is characterized by comprising the following steps:
A. sample acquisition: collecting a blood sample of a subject, and placing the obtained blood sample in a blood collection container;
B. obtaining serum: placing the blood sampling container containing the sample blood in the step A in a medical centrifuge, centrifuging for 3-4min to obtain blood serum, and placing the blood serum in a special serum storage bottle;
C. extracting genome DNA: extracting free DNA molecules from the serum obtained in step B by a concentrated salt method;
D. and (3) sequence determination: based on the second generation sequencing technology of DNA, the DNA molecules obtained in the step C are subjected to sequencing treatment to obtain a sample sequence of the DNA;
E. PCR amplification of DNA: d, carrying out multiple polymerase chain reaction by using relatively longer fragments in the DNA sample sequence obtained in the step D, and adding a primer pair in the reaction to obtain a DNA amplification sequence;
F. comparing and screening: comparing the DNA amplification sequence obtained in the step E with a reference sample sequence of a patient suffering from echinococcosis based on bioinformatics analysis technology;
G. the results were obtained: and F, comparing and processing to obtain whether the free DNA contains the molecules of the echinococcosis source or the host specific reaction source in the early process of the echinococcosis.
7. The method for early diagnosis based on the detection of the codling gene of the chaulmoogra as claimed in claim 6, wherein the primer pair added in step E is an oligonucleotide primer labeled with a fluorescent dye.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007309851A (en) * | 2006-05-19 | 2007-11-29 | Adtec Kk | Method for diagnosing echinococcus alveolar hydatid disease |
| CN106970210A (en) * | 2017-02-22 | 2017-07-21 | 中国农业科学院上海兽医研究所 | A kind of toxoplasmosis indirect ELISA diagnostic reagent kit |
| WO2019007251A1 (en) * | 2017-07-02 | 2019-01-10 | 中南大学湘雅医院 | Method for detecting parasitic infection and kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007309851A (en) * | 2006-05-19 | 2007-11-29 | Adtec Kk | Method for diagnosing echinococcus alveolar hydatid disease |
| CN106970210A (en) * | 2017-02-22 | 2017-07-21 | 中国农业科学院上海兽医研究所 | A kind of toxoplasmosis indirect ELISA diagnostic reagent kit |
| WO2019007251A1 (en) * | 2017-07-02 | 2019-01-10 | 中南大学湘雅医院 | Method for detecting parasitic infection and kit |
Non-Patent Citations (1)
| Title |
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| 祁小英: "犬粪包虫抗原检测调查及防治" * |
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