CN111172244B - Method for rapidly identifying schistosoma japonicum infected oncomelania - Google Patents
Method for rapidly identifying schistosoma japonicum infected oncomelania Download PDFInfo
- Publication number
- CN111172244B CN111172244B CN201811342007.6A CN201811342007A CN111172244B CN 111172244 B CN111172244 B CN 111172244B CN 201811342007 A CN201811342007 A CN 201811342007A CN 111172244 B CN111172244 B CN 111172244B
- Authority
- CN
- China
- Prior art keywords
- oncomelania
- concentration
- dna
- volume
- positive
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for rapidly identifying schistosoma japonicum infected oncomelania, which comprises a rapid, nontoxic and harmless positive oncomelania DNA extraction method without cleaning and purification and a rapid RAA detection positive oncomelania DNA method. The invention comprises a rapid, nontoxic, harmless, cleaning and purifying-free positive oncomelania DNA extraction method, avoids using toxic and harmful chemical substances such as phenol, chloroform and the like to extract DNA, does not need complex and expensive lysate components such as proteinase K, guanidine isothiocyanate and the like, has few operation steps, is convenient and quick, and has low cost.
Description
Technical Field
The invention relates to the field of molecular biology detection, in particular to a rapid fluorescence detection method for rapidly extracting schistosoma japonicum DNA from schistosoma japonicum infected oncomelania and based on RAA technology.
Background
Schistosomiasis japonica is a parasitic disease of human and animals which are distributed worldwide, is one of six tropical diseases defined by World Health Organization (WHO), and is one of five parasitic diseases in China. Schistosomiasis japonica is caused by infection of final hosts such as epidemic water-exposed humans and animals with cercaria.
Schistosoma japonica (Schistosoma japonicum) belongs to the phylum of the flat animals, the class of the trematoda, the order of the complexation, the family of the schizoaceae, the genus of the schizogenus, the species of the Japanese, the male and female variants. The average length of male worms is 13mm, and the average length of female worms is 15mm. Worm's egg is round, light yellow, thin in egg shell, without cover, there is a thorn on its side, and mature egg contains cercaria. The miracidium is oblong, the in-ovo miracidium is hatched in water and invades oncomelania, and the oncomelania is the only intermediate host, so that the epidemic geographic distribution of the schistosomiasis japonica is consistent with that of the oncomelania, and the miracidium has a certain locality. The genome of schistosome consists of 8 pairs of chromosomes, 7 pairs of which are autosomes and 1 pair of sex chromosomes.
The schistosomiasis in China is caused by Japanese schistosome, and is mainly distributed in 433 counties (city and district) of 12 provinces such as Jiangsu, zhejiang, shanghai, anhui, jiangxi, hunan, hubei, sichuan, yunnan, guangxi, guangdong, fujian and the like, wherein the total area of oncomelania is 148 hundred million square meters, the cumulative number of infected people reaches 1160 ten thousand, and the number of threatened people is more than 1 hundred million. The schistosomiasis in China is actively controlled for 60 years, the epidemic situation of the schistosomiasis in China is effectively controlled, but in recent years, due to the fact that factors such as biology, nature, socioeconomic, population flow, policy guarantee and the like are greatly changed, situations of spreading and spreading of the schistosomiasis epidemic situation are presented in some places, the situations are represented by the fact that schistosomiasis patients in old epidemic areas are increased, oncomelania spreading is obvious, new oncomelania areas appear, the distribution range of infectious oncomelania is expanded, epidemic situation back rise possibly occurs in areas which part of the areas reach the control and the spread blocking of the schistosomiasis spread, the input schistosomiasis cases are increased in all places, and the schistosomiasis control work in China is far too.
In prevention and control of schistosomiasis, diagnosis and detection work are more important, and particularly, identification of whether oncomelania infects schistosome is important, so that necessary information and scientific basis are provided for the links of planning, implementation, evaluation of prevention and control effects and the like of prevention and control activities.
Because oncomelania is the only intermediate host of schistosome, it is the only transmission medium of schistosomiasis, and the elimination of schistosome infectious oncomelania can block the transmission and prevalence of schistosomiasis. Therefore, the accurate identification of whether the oncomelania infects schistosome is a key for preventing and controlling schistosomiasis.
The current methods for detecting schistosome infectious oncomelania have a plurality of methods:
1. visual inspection method
Under natural light or artificial light irradiation, whether the oncomelania infects schistosome is judged by observing the change of color and transmittance of the shell below the 4 th shell layer of the oncomelania and no gap exists between the liver tissue of the oncomelania and the shell. The method is simple and easy to operate, but needs a large number of experienced technicians to perform manual judgment, has large error, accuracy of only about 60%, high omission ratio, can not identify oncomelania at the early stage of schistosome infection, has large workload, is difficult to develop on site and the like.
2. Tabletting microscopic examination method
Crushing the shell of the oncomelania to expose the soft tissue of the oncomelania, and judging whether the oncomelania infects schistosome or not by observing whether schistosome female cercaria or cercaria exists in the tissue of the oncomelania under a microscope. The method has higher requirements on technicians, lower accuracy and easy identification errors: after the oncomelania is infected, the earliest time of microscopic examination is 9 days, particularly the oncomelania collected on site is also required to be fed in a laboratory for a period of time, so that the oncomelania can be judged by a microscope, the timeliness is too poor, the oncomelania at the stage of the dry period of the schistosome infection can not be detected yet, the microscope is required to be used in the detection process, the on-site use of the schistosomiasis control is inconvenient, and the current demand of the schistosomiasis prevention work can not be met.
3. Hatching method
The oncomelania is placed in a test tube or other glass container with clean water, incubated for 2-3 hours at room temperature (22-25 ℃), and whether schistosome cercaria is hatched in the oncomelania is observed to judge whether the oncomelania is schistosome infectious oncomelania. The method is also simple and convenient to operate, and has high detection rate on the oncomelania infectious at the late schistosome infection stage, but can not detect the oncomelania at the early schistosome infection stage.
4. Method for detecting circulating antigen
Firstly preparing a specific monoclonal antibody for resisting the schistosome circulating antigen, and then constructing an enzyme-linked immunosorbent assay (ELISA) for detecting the schistosome circulating antigen, so as to detect the schistosome infectious oncomelania. The method has the advantages that the schistosome infectious oncomelania is identified with high sensitivity and specificity, but the operation is complicated, the technical and equipment requirements are high, and the method is not suitable for being used on site for preventing and treating schistosomiasis.
5. Polymerase chain reaction (PCR method)
Patent CN104651489a discloses a method for specifically designing primers and identifying schistosome infectious oncomelania by using a Q-PCR method, but the method for extracting nucleic acid is performed by using a commercial kit, which takes a long time to complete a detection within 3-5 hours, and fails to realize field application, and whether schistosome specific DNA fragments exist in a oncomelania DNA sample is observed by performing polymerase chain reaction PCR) amplification on the oncomelania tissue DNA sample. The method for detecting the schistosome infectious oncomelania has high sensitivity and specificity, the current oncomelania sample DNA extraction needs a special commercial tissue DNA extraction kit, generally adopts proteinase K for digestion, then uses guanidine isothiocyanate and the like for cleavage, uses magnetic beads or adsorption columns for purification, and needs two to three times of cleaning to obtain the kit applied to QPCR detection, the steps are complex in multiple procedures, the whole extraction process is more than 2 hours, and meanwhile, a PCR instrument, a DNA electrophoresis instrument and other expensive equipment are needed during PCR detection, the detection time is 2 hours, the time consumption is long, the cost is high, and the kit is difficult to use on site for preventing and treating schistosomiasis.
6. Loop-mediated isothermal nucleic acid amplification (LAMP method)
The published patent CN101457258B utilizes a loop-mediated isothermal nucleic acid amplification technology to detect schistosome infectious oncomelania, utilizes 4 primers to amplify, uses proteinase K to digest the oncomelania sample DNA to extract, uses toxic and harmful chemical substances such as phenol, chloroform and the like to denature protein and purify DNA, has more steps, is troublesome to operate, has more than 2 hours of extraction time, has higher requirements on personnel, and has longer time consumption and high cost when being used for amplifying and detecting for more than 2 hours, so that the method has the advantage of difficult use on site for preventing and treating schistosomiasis.
The method for identifying the schistosome infectious oncomelania by the current molecular biology method is characterized in that the pretreatment and DNA extraction of a sample are both related to a polymerase chain reaction method (PCR method) and a loop-mediated isothermal nucleic acid amplification technology (LAMP method), and whether the method can be applied on site depends on the extraction efficiency and convenience of the DNA of a positive oncomelania sample.
Therefore, it is very significant to develop a method that is simpler and more convenient, can extract DNA rapidly and is non-toxic and harmless, and to accurately identify schistosome infectious positive oncomelania in combination with the rapid (the completion of the identification from the extraction to the completion of the identification within 1 hour).
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a method for infecting schistosoma japonicum oncomelania, which is simple to operate, short in time consumption and easy to rapidly identify.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a method for rapidly identifying schistosoma japonicum infected oncomelania is characterized by comprising the following steps of:
comprises a rapid, nontoxic and harmless positive oncomelania DNA extraction method without cleaning and purifying and a RAA rapid detection positive oncomelania DNA method;
the rapid, nontoxic and harmless positive oncomelania DNA extraction method without cleaning and purifying comprises the following steps:
1) Grinding Oncomelania Hupensis Gredler, removing shell, placing into centrifuge tube, adding sufficient amount of cleaning liquid A overflowed Oncomelania Hupensis Gredler, shaking, mixing, centrifuging at 12000rpm for 2min, and removing supernatant.
2) And fully grinding the oncomelania tissue by using a grinding rod.
3) Adding 500 mu L of lysate B into the centrifuge tube, shaking and mixing uniformly, and standing in a water bath kettle at 80 ℃ for 5-10min.
4) Placing the centrifuge tube after water bath in a centrifuge, centrifuging at 12000rpm for 5min, taking 400 mu L of supernatant in a new centrifuge tube, adding 160 mu L of BufferC, shaking and mixing uniformly, centrifuging at 12000rpm for 5min, transferring the supernatant into the new centrifuge tube, and preserving at-20 ℃ for later use to finish DNA extraction;
the RAA rapid detection method for positive oncomelania DNA comprises a specific primer and a fluorescent probe;
specific forward primer sequence:
5’-TCATCTCTGATCTGTATGCCTTTCCATTAG-3’,
specific reverse primer sequence:
5’-AGTTAACTAAAAGTCAGTCAGTCACTTACAACG-3’；
the fluorescent probe sequence is as follows:
5’-TACTCTGTTACTACCTCCTCTACTCTGGGATCTGGTCCGAAAATTT-3’
the two ends of the fluorescent probe are respectively marked with a reporting fluorescent group and a quenching fluorescent group, and the reporting fluorescent group is preferably a FAM fluorescent group; the quenching fluorescent group is preferably BHQ; the probe modified by the fluorescent group is as follows:
5’-TACTCTGTTACTACCTCCTCTACTCTGGGA/i6FAMdT//THF//iBHQdTGGTCCGAAAATTT-3’；
the isothermal amplification temperature is 35-42 ℃;
the isothermal amplification time is 5-20 min;
the volume of the amplification system is 25-100 mu L;
the volume of the DNA sample adding amount of the schistosome sample is 1-5 mu L;
the concentration of the forward primer is 0.02-0.1 mM, and the concentration of the reverse primer is 0.02-0.1 mM;
probe concentration the concentration of the probe is 0.02 to 0.07mM.
As an optimized technical scheme, the cleaning liquid A comprises NaCL and Na 3 C 6 H 5 O 7 ；
Wherein the mass percentage concentration of the NaCL is 0.1-1.5%; na (Na) 3 C 6 H 5 O 7 The mass percentage concentration of (2) is 1-2.5%.
As an optimized technical scheme, the NaCL in the cleaning liquid A has a mass percentage concentration of 0.9 percent, na 3 C 6 H 5 O 7 The mass percentage concentration of (2) is 1.5%.
The purpose of the cleaning liquid A is to wash away impurities such as soil on the soft surface of oncomelania and prevent the adverse effect on the subsequent extraction.
As an optimized technical scheme, the components of the lysate B comprise NaOH, tritonX-100, SDS and EDTA (PH=8.0); wherein the volume molar concentration of NaOH is 0.1-1mol/L, the mass percentage concentration of Triton X-100 is 0.2-2%, the mass percentage concentration of SDS is 0.1-0.4%, and the volume molar concentration of EDTA is 0.1-0.5mmol/L.
As an optimized technical scheme, the volume molar concentration of NaOH in the lysate B is 0.3mol/L, the mass percentage concentration of Triton X-100 is 1%, the mass percentage concentration of SDS is 0.2%, and the volume molar concentration of EDTA is 0.1mmol/L.
The purpose of the lysate B is to promote cell rupture, and DNA is released, wherein NaOH, triton-X100, SDS and EDTA are all the purpose of promoting cell rupture, and the lysate is free from proteinase K, guanidine isothiocyanate and other chemical substances, has simple components and low cost.
As an optimized technical scheme, the BufferC composition comprises CH3COOH, and the volume molar concentration of the CH3COOH is 1-2mol/L.
As an optimized technical scheme, the volume molar concentration of CH3COOH in the BufferC is 1.2mol/L.
As an optimized technical scheme, the isothermal amplification temperature is 39 ℃; the isothermal amplification time is 20min; the volume of the amplification system was 50 μl; the volume of the DNA sample of the schistosome is 2 mu L; the concentration of the forward and reverse primers is 0.05mM, and 2 mu L of each forward and reverse primer is added into the double-solution system; probe concentration was 0.03mM; the volume used was 0.5. Mu.L.
Wherein the reagent NaOH, naCL, na C6H5O7, CH 3 COOH, triton-X100, SDS, EDTA were the national drug purity.
Due to the adoption of the technical scheme, compared with the prior art, the method for extracting the positive oncomelania DNA has the advantages of being fast, nontoxic, harmless, free of cleaning and purifying, free of toxic and harmful chemical substances such as phenol, chloroform and the like for extracting the DNA, free of complex and expensive lysate components such as proteinase K, guanidine isothiocyanate and the like, few in operation steps, convenient, fast and low in cost; the primer probe with strong specificity is used for detecting the schistosoma japonicum DNA, the RAA technology is adopted for positive oncomelania identification, the whole identification process of sample treatment and DNA extraction is completed within 1 hour, the identification time is greatly shortened, the sensitivity is high, and the method is suitable for early detection of whether the schistosoma japonicum is infected by the oncomelania japonicum or not on site.
The invention will be further described with reference to the drawings and examples.
Drawings
FIG. 1 is a graph showing the change in fluorescence signal of the identification of the positive oncomelania of Schistosoma japonicum cercaria and the negative oncomelania of the non-infected Schistosoma japonicum cercaria at 1 day, 2 days and 3 days.
FIG. 2 is a graph showing the change of the identified fluorescence signal after DNA extraction from samples obtained by adding 1, 2, 3, 4, 5 and 10 positive oncomelania infection with Schistosoma japonicum cercaria to 50 negative oncomelania.
Detailed Description
Examples
The invention provides a method for rapidly identifying schistosoma japonicum infected oncomelania, which comprises two steps, wherein the method is established for rapidly and non-toxic, harmless and cleaning and purifying positive oncomelania DNA extraction. And secondly, establishing a RAA rapid detection positive oncomelania DNA method.
The rapid, nontoxic and harmless positive oncomelania DNA extraction method without cleaning and purifying is carried out according to the following steps:
1) Preparing a cleaning solution A according to the following components:
NaCL 0.9％
Na3C6H5O71.5％
the purpose of the cleaning liquid A is to wash away impurities such as soil on the soft surface of oncomelania and prevent the adverse effect on the subsequent extraction.
2) Preparing a lysate B according to the following components:
NaOH 0.3mol/L
Triton X-1001％
SDS 0.2％
EDTA(PH＝8.0)0.1mmol/L
the purpose of the lysate B is to promote cell rupture, and DNA is released, wherein NaOH, triton-X100, SDS and EDTA are all the purposes of promoting cell rupture, and the formula can fully rupture cells and has no influence on the subsequent amplification detection; the lysate does not use proteinase K, guanidine isothiocyanate and other chemical substances, and has simple components and low cost.
The formula is matched with the subsequent RAA technology to identify the schistosoma japonicum oncomelania infection without DNA purification, and the steps of protein denaturation and the like are carried out on the extracted DNA by using toxic and harmful chemical substances such as phenol, chloroform and the like, so that the inhibition effect on the RAA nucleic acid amplification is avoided, the operation steps can be reduced, and the cost is reduced.
3) Buffer C was prepared as follows
CH 3 COOH1.2mol/L
The purpose of Buffer C is to adjust the Buffer pH to achieve a reasonable interval of 7.4-8.4 for the extracted DNA.
Wherein the reagent NaOH, naCL, na C6H5O7, CH 3 COOH, triton-X100, SDS, EDTA were all analytically pure.
4) Preparing negative oncomelania and infecting Japanese blood fluke positive oncomelania;
5) The DNA extraction of the negative oncomelania and the positive oncomelania infected with schistosoma japonica was performed according to the following procedures.
Picking the oncomelania soft body from the oncomelania shell, placing the oncomelania soft body into a centrifuge tube, adding enough cleaning liquid A overflowed from the oncomelania soft body, shaking and uniformly mixing, centrifuging at 12000rpm for 2min, and pouring out the supernatant. The oncomelania tissue was thoroughly ground with a grinding rod. Adding 500 mu L of the lysate B into the centrifuge tube, shaking and mixing uniformly, standing in a water bath kettle at 80 ℃ for 5-10min, and fully performing pyrolysis.
6) Placing the centrifuge tube after water bath pyrolysis into a centrifuge, centrifuging at 12000rpm for 5min, taking 400 mu L of supernatant into a new centrifuge tube, adding 160 mu L of Buffer C, shaking and mixing uniformly, centrifuging at 12000rpm for 5min, transferring the supernatant into the new centrifuge tube, and preserving at-20 ℃ for later use, thus finishing DNA extraction.
The whole extraction method of the positive oncomelania DNA does not use toxic and harmful chemical substances in the process, does not need DNA cleaning and purifying steps, has few steps, short time and convenient operation, has low requirements on personnel quality, does not need a special extraction instrument, completes the whole operation within 30 minutes, and is easy to realize the rapid extraction of the DNA of the positive oncomelania sample on site.
The rapid detection method of the positive oncomelania DNA by using the RAA adopts a pair of screened specific Japanese blood fluke primers and probes to detect by adopting a fluorescence RAA method, wherein the RAA method is an isothermal nucleic acid amplification method, an isothermal amplification system is provided by Jiangsu Qiyan gene biotechnology Co., ltd, the RAA nucleic acid amplification reagent with the product number of F00001A is a reaction reagent in the form of freeze-dried powder, and a re-dissolving buffer solution is matched, wherein a solvent for dissolving the raw materials of the amplification system is the reaction buffer solution.
The specific method comprises the following steps:
according to the gene name schistosoma japonicum, a corresponding complete gene sequence (www.ncbi.nlm.nih.gov) is found in genebank, DNASTAR software is used for carrying out homology analysis and b1ast sequence analysis, and the highly conserved sequence of the schistosoma japonicum gene is screened as follows:
and (3) designing and screening the specific primer probe according to the conserved sequence to obtain the following primer probe:
forward primer sequence: 5'-TCATCTCTGATCTGTATGCCTTTCCATTAG-3' the number of the individual pieces of the plastic,
reverse primer sequence: 5'-AGTTAACTAAAAGTCAGTCAGTCACTTACAACG-3';
the fluorescent probe sequence is as follows:
5’-TACTCTGTTACTACCTCCTCTACTCTGGGATCTGGTCCGAAAATTT-3’
the two ends of the fluorescent probe are respectively marked with a report fluorescent group and a quenching fluorescent group, wherein the report fluorescent group is preferably a FAM fluorescent group; the quenching group is preferably BHQ; the middle uses THF group to connect the fluorescent group and quench the fluorescent group, the probe modified by the fluorescent group is:
5’-TACTCTGTTACTACCTCCTCTACTCTGGGA/i6FAMdT//THF//iBHQdTGGTCCGAAAATTT-3’。
in the implementation process, the temperature of moderate amplification is selected to be 39 ℃, the isothermal amplification reaction time is selected to be 20min, the volume of an amplification system is 50 mu L, and the volume of the DNA sample of the schistosome sample is 2 mu L.
Using a forward and reverse primer concentration of 0.05mM, 2. Mu.L each of the forward and reverse primers was added to the reconstituted system. The probe concentration was 0.03mM and the volume used was 0.5. Mu.L.
The Jiangsu Qiyan gene biotechnology Co., ltd. RAA isothermal amplification reagent was purchased with the product number of F00001A.
The RAA isothermal amplification reagent comprises a RAA basic fluorescent universal reaction reagent and a reaction buffer solution.
In the invention, the RAA rapid detection positive oncomelania DNA method provided by the scheme comprises the following steps:
a detection instrument capable of detecting FAM fluorescent groups is prepared, and the detection is carried out by using a detection instrument RAA-F1620 manufactured by the scientific and technological company of Xiyaku Technology, inc.
The RAA-F1620 is powered on for preheating, the reaction parameters are set, the temperature is set to 39 ℃, and the reaction time is as follows: 20min.
Preparing a reaction amplification system: adding 2 mu L of forward primer, 2 mu L of reverse primer and 0.5 mu L of probe into 43.5 mu L of reaction buffer, fully mixing, adding into RAA basic fluorescent universal reaction reagent to re-dissolve freeze-dried powder, adding 2 mu L of prepared sample DNA, fully mixing, and then placing into a prepared detection instrument RAA-F1620 to detect fluorescent signals.
According to the positive judgment method in the RAA-F1620 detection instrument, the detection instrument judges that the detection instrument is positive when the slope value K is more than or equal to 20, and judges that the detection instrument is negative when the slope value K is less than 20.
Example 1
Sample source: schistosoma japonicum cercaria-infected positive oncomelania and schistosoma japonicum cercaria-uninfected negative oncomelania cultured by schistosoma japonicum control research institute for 1 day, 2 days and 3 days.
The first step: preparing a cleaning solution A, a cracking solution B and a Buffer C according to the method of the invention;
1) Cleaning solution A was prepared as follows
NaCL 0.9％
Na3C6H5O7 1.5％
2) The cracking liquid B is prepared according to the following components
3) Buffer C was formulated as follows
CH 3 COOH 1.2mol/L
And a second step of: extraction of DNA
1) Preparing negative oncomelania and Japanese blood fluke cercaria positive oncomelania infected for 1 day, 2 days and 3 days, picking oncomelania soft bodies from oncomelania shells, respectively placing the oncomelania soft bodies into 4 clean centrifuge tubes, adding sufficient cleaning liquid A overflowed oncomelania soft bodies, shaking and uniformly mixing, centrifuging at 12000rpm for 2min, and pouring out the supernatant;
2) Fully grinding the oncomelania tissue by using a grinding rod;
3) Adding 500 mu L of lysate B into the centrifuge tube, shaking and mixing uniformly, standing for 10min in a water bath kettle at 80 ℃ and fully performing pyrolysis;
4) Placing each centrifuge tube subjected to water bath pyrolysis into a centrifuge, centrifuging at 12000rpm for 5min, taking 400 mu L of supernatant into a new centrifuge tube, adding 160 mu L of Buffer C, shaking and mixing uniformly, centrifuging at 12000rpm for 5min, transferring the supernatant into a new centrifuge tube, and preserving at-20 ℃ for later use to finish DNA extraction;
and a third step of: identification of extracted DNA Using RAA technology
1) The following schistosoma japonica primers and probes were designed and commissioned for synthesis by the division of bioengineering (Shanghai) Co., ltd:
forward primer sequence: 5'-TCATCTCTGATCTGTATGCCTTTCCATTAG-3' the number of the individual pieces of the plastic,
reverse primer sequence: '-AGTTAACTAAAAGTCAGTCAGTCACTTACAACG-3';
the fluorescent probe sequence is as follows:
5’-TACTCTGTTACTACCTCCTCTACTCTGGGATCTGGTCCGAAAATTT-3’
preparing the synthesized forward and reverse primers to a concentration of 0.05mM and a probe concentration of 0.03mM for standby;
detecting by using a detection instrument RAA-F1620 manufactured by the scientific and technological company of Xidetian science and technology; the RAA-F1620 is powered on for preheating, the reaction parameters are set, the temperature is set to 39 ℃, and the reaction time is as follows: 20min;
4) Taking RAA isothermal amplification reagent with the commodity number of F00001A (purchased from Sonchi gene biotechnology Co., ltd.) and preparing a reaction system: absorbing 43.5 mu L of reaction buffer solution, adding 2 mu L of forward primer, 2 mu L of reverse primer and 0.5 mu L of probe to prepare premix, adding the premix into RAA basic fluorescent universal reaction reagent to redissolve freeze-dried powder, adding 2 mu L of sample DNA extracted in the first step, fully and uniformly mixing, and then placing the mixture into a prepared detection instrument RAA-F1620 for detecting fluorescent signals.
5) Whether an exponential amplification signal appears or not is observed to judge positive, or positive can be judged according to a positive judging method in an RAA-F1620 detecting instrument, and negative can be judged when the slope value K is more than or equal to 20 and the slope value K is less than 20; the amplification condition is shown in figure 1, the negative oncomelania is not peaked, the oncomelania positive oncomelania infected for 1 day, 2 days and 3 days is peaked, the method is proved to be applicable to early identification of the oncomelania infected Japanese schistosome, the DNA extraction method is simple and practical, the steps are few, the time is short, the on-site rapid detection can be realized within 1 hour by matching with the RAA method, the practical application value is realized, and the method has important significance for prevention and control of schistosomiasis.
Example 2
The first step: sample material preparation
350 negative oncomelania which are not infected by schistosoma japonicum cercaria, 24 positive oncomelania which are infected by schistosoma japonicum cercaria, and the samples are provided by culture of schistosomiasis prevention and control research institute of Jiangsu province.
And a second step of: preparation of samples
Sample No. 1: 50 negative oncomelania were used as negative controls;
sample No. 2: mixing 1 Japanese blood fluke cercaria-infected positive oncomelania into 50 negative oncomelania;
sample No. 3: mixing 2 negative oncomelania with oncomelania positive oncomelania infected by Japanese blood fluke cercaria;
sample No. 4: mixing 3 negative oncomelania with oncomelania positive oncomelania infected by Japanese blood fluke cercaria;
sample No. 5: mixing 4 negative oncomelania with oncomelania positive oncomelania infected by Japanese blood fluke cercaria;
sample No. 6: mixing 5 negative oncomelania with oncomelania positive oncomelania infected by Japanese blood fluke cercaria;
sample No. 7: 50 negative oncomelania were mixed with 10 positive oncomelania infected with schistosoma japonicum cercaria.
And a third step of: cleaning solution A, lysate B and Buffer C are prepared according to the method of the invention.
1) Cleaning solution A was prepared as follows
NaCL 0.9％
Na3C6H5O7 1.5％
2) The cracking liquid B is prepared according to the following components
3) Buffer C was formulated as follows
CH 3 COOH 1.2mol/L
Fourth step: extracting DNA, and respectively extracting the prepared sample DNA for standby according to the following steps;
1) Respectively placing the sample oncomelania soft bodies selected from the oncomelania shell into a clean centrifuge tube, adding a sufficient amount of cleaning liquid A to overflow the oncomelania soft bodies, vibrating and mixing uniformly, centrifuging at 12000rpm for 2min, and pouring out the supernatant;
2) Fully grinding the oncomelania tissue by using a grinding rod;
3) Adding 500 mu L of lysate B into the centrifuge tube, shaking and mixing uniformly, standing for 10min in a water bath kettle at 80 ℃ and fully performing pyrolysis;
4) Placing each centrifuge tube subjected to water bath pyrolysis into a centrifuge, centrifuging at 12000rpm for 5min, taking 400 mu L of supernatant into a new centrifuge tube, adding 160 mu L of Buffer C, shaking and mixing uniformly, centrifuging at 12000rpm for 5min, transferring the supernatant into a new centrifuge tube, and preserving at-20 ℃ for later use to finish DNA extraction;
fifth step: identification of extracted DNA Using RAA technology
1) The following schistosoma japonica primers and probes were designed and commissioned for synthesis by the division of bioengineering (Shanghai) Co., ltd:
forward primer sequence: 5'-TCATCTCTGATCTGTATGCCTTTCCATTAG-3' the number of the individual pieces of the plastic,
reverse primer sequence: '-AGTTAACTAAAAGTCAGTCAGTCACTTACAACG-3';
the fluorescent probe sequence is as follows:
5’-TACTCTGTTACTACCTCCTCTACTCTGGGATCTGGTCCGAAAATTT-3’。
the synthesized forward and reverse primers were formulated at a concentration of 0.05mM and the probe concentration was 0.03mM for use.
Detecting by using a detection instrument RAA-F1620 manufactured by the scientific and technological company of Xidetian science and technology; the RAA-F1620 is powered on for preheating, the reaction parameters are set, the temperature is set to 39 ℃, and the reaction time is as follows: 20min.
4) Taking RAA isothermal amplification reagent with the commodity number of F00001A (purchased from Sonchi gene biotechnology Co., ltd.) and preparing a reaction system: absorbing 43.5 mu L of reaction buffer solution, adding 2 mu L of forward primer, 2 mu L of reverse primer and 0.5 mu L of probe to prepare premix, adding the premix into RAA basic fluorescent universal reaction reagent to redissolve freeze-dried powder, adding 2 mu L of sample DNA extracted in the first step, fully and uniformly mixing, and then placing the mixture into a prepared detection instrument RAA-F1620 for detecting fluorescent signals.
5) Whether an exponential amplification signal appears or not is observed to judge positive, or positive can be judged according to a positive judging method in an RAA-F1620 detecting instrument, and negative can be judged when the slope value K is more than or equal to 20 and the slope value K is less than 20; the amplification condition is shown in fig. 2, no peak is found in the sample No. 1, no peak is found in the sample No. 2, 3, 4, 5, 6 and 7, the identification of the sensitivity of the positive oncomelania infected with schistosoma japonicum cercaria can reach that of the positive oncomelania mixed with 1 positive oncomelania, and the identification proves that the method can be applied to early identification of the oncomelania japonica infected with schistosoma japonicum.
The protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the present invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.
Claims (5)
1. A method for rapidly identifying schistosoma japonicum infected oncomelania is characterized by comprising the following steps of:
comprises a rapid, nontoxic and harmless positive oncomelania DNA extraction method without cleaning and purifying and a RAA rapid detection positive oncomelania DNA method;
the rapid, nontoxic and harmless positive oncomelania DNA extraction method without cleaning and purifying comprises the following steps:
1) Grinding Oncomelania Hupensis Gredler, removing shell, placing into centrifuge tube, adding sufficient amount of cleaning liquid A overflowed Oncomelania Hupensis Gredler, shaking, mixing, centrifuging at 12000rpm for 2min, and removing supernatant;
the cleaning liquid A comprises NaCl and Na 3 C 6 H 5 O 7 ；
Wherein the mass percentage concentration of NaCl is 0.1-1.5%; na (Na) 3 C 6 H 5 O 7 The mass percentage concentration of (2) is 1-2.5%.
2) Fully grinding the oncomelania tissue by using a grinding rod;
3) Adding 500 mu L of lysate B into the centrifuge tube, shaking and mixing uniformly, and standing in a water bath kettle at 80 ℃ for 5-10min;
the components of the lysate B are NaOH, triton X-100, SDS and EDTA with pH of 8.0; wherein the volume molar concentration of NaOH is 0.1-1mol/L, the mass percentage concentration of Triton X-100 is 0.2-2%, the mass percentage concentration of SDS is 0.1-0.4%, and the volume molar concentration of EDTA is 0.1-0.5mmol/L;
4) Placing the centrifuge tube after water bath in a centrifuge, centrifuging at 12000rpm for 5min, collecting 400 μL of supernatant in a new centrifuge tube, adding 160 μL of Buffer C, shaking and mixing, centrifuging at 12000rpm for 5min, transferring the supernatant into a new centrifuge tube, and preserving at-20deg.C for standby, to complete DNA extraction, wherein the Buffer C comprises CH 3 COOH，CH 3 The volume molar concentration of COOH is 1-2mol/L;
the RAA rapid detection method for positive oncomelania DNA comprises a specific primer and a fluorescent probe;
specific forward primer sequence:
5’-TCATCTCTGATCTGTATGCCTTTCCATTAG-3’,
specific reverse primer sequence:
5’-AGTTAACTAAAAGTCAGTCAGTCACTTACAACG-3’；
the fluorescent probe sequence is as follows:
5’-TACTCTGTTACTACCTCCTCTACTCTGGGATCTGGTCCGAAAATTT-3’
the fluorescent probe is respectively marked with a report fluorescent group and a quenching fluorescent group, and the report fluorescent group is a FAM fluorescent group; the quenching fluorescent group is BHQ; the probe modified by the fluorescent group is as follows:
5’-TACTCTGTTACTACCTCCTCTACTCTGGGA/i6FAMdT//THF//iBHQdTGGTCCGAAAATTT-3’；
the isothermal amplification temperature is 35-42 ℃;
isothermal amplification time is 5-20 min;
the volume of the amplification system is 25-100 mu L;
the volume of the DNA sample of the schistosome sample is 1-5 mu L;
the concentration of the forward primer is 0.02-0.1 mM, and the concentration of the reverse primer is 0.02-0.1 mM;
the concentration of the probe is 0.02 to 0.07mM.
2. A method for rapid identification of an infection of schistosoma japonicum oncomelania according to claim 1, wherein: the mass percentage concentration of NaCl in the cleaning solution A is 0.9 percent, and Na is 3 C 6 H 5 O 7 The mass percentage concentration of (2) is 1.5%.
3. A method for rapid identification of an infection of schistosoma japonicum oncomelania according to claim 1, wherein: the volume molar concentration of NaOH in the lysate B is 0.3mol/L, the mass percentage concentration of Triton X-100 is 1%, the mass percentage concentration of SDS is 0.2%, and the volume molar concentration of EDTA is 0.1mmol/L.
4. A method for rapid identification of an infection of schistosoma japonicum oncomelania according to claim 1, wherein: CH in Buffer C 3 The molar concentration of COOH by volume was 1.2mol/L.
5. A method for rapid identification of an infection of schistosoma japonicum oncomelania according to claim 1, wherein: the isothermal amplification temperature is 39 ℃; the isothermal amplification time is 20min;
the volume of the amplification system was 50 μl;
the volume of the DNA sample of the schistosome is 2 mu L;
the forward and reverse primer concentrations were 0.05mM, and the volumes of the forward and reverse primers were added to each 2. Mu.L;
the concentration of the probe was 0.03mM; the volume used was 0.5. Mu.L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811342007.6A CN111172244B (en) | 2018-11-12 | 2018-11-12 | Method for rapidly identifying schistosoma japonicum infected oncomelania |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811342007.6A CN111172244B (en) | 2018-11-12 | 2018-11-12 | Method for rapidly identifying schistosoma japonicum infected oncomelania |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111172244A CN111172244A (en) | 2020-05-19 |
| CN111172244B true CN111172244B (en) | 2023-08-08 |
Family
ID=70651665
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811342007.6A Active CN111172244B (en) | 2018-11-12 | 2018-11-12 | Method for rapidly identifying schistosoma japonicum infected oncomelania |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111172244B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111961737B (en) * | 2020-09-23 | 2022-03-29 | 江苏省血吸虫病防治研究所 | DNA kit for detecting schistosoma japonicum and using method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101457258A (en) * | 2008-12-26 | 2009-06-17 | 江苏省血吸虫病防治研究所 | Schistosome infectious oncomelania detection kit and detection method thereof |
| CN102816756A (en) * | 2012-09-07 | 2012-12-12 | 江苏奇天基因生物科技有限公司 | Isothermal nucleic acid amplification reaction reagent and isothermal nucleic acid amplification method |
| CN104651489A (en) * | 2014-12-16 | 2015-05-27 | 江苏省血吸虫病防治研究所 | Q-PCR primer, identification method and kit for identifying schistosoma japonicum infected oncomelania |
| CN106480223A (en) * | 2016-12-23 | 2017-03-08 | 江苏省血吸虫病防治研究所 | A kind of method of detection schistosomicide DNA |
| CN106701940A (en) * | 2016-12-23 | 2017-05-24 | 江苏省血吸虫病防治研究所 | Method for detecting DNA of schistosoma through fluorescent probe |
-
2018
- 2018-11-12 CN CN201811342007.6A patent/CN111172244B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101457258A (en) * | 2008-12-26 | 2009-06-17 | 江苏省血吸虫病防治研究所 | Schistosome infectious oncomelania detection kit and detection method thereof |
| CN102816756A (en) * | 2012-09-07 | 2012-12-12 | 江苏奇天基因生物科技有限公司 | Isothermal nucleic acid amplification reaction reagent and isothermal nucleic acid amplification method |
| CN104651489A (en) * | 2014-12-16 | 2015-05-27 | 江苏省血吸虫病防治研究所 | Q-PCR primer, identification method and kit for identifying schistosoma japonicum infected oncomelania |
| CN106480223A (en) * | 2016-12-23 | 2017-03-08 | 江苏省血吸虫病防治研究所 | A kind of method of detection schistosomicide DNA |
| CN106701940A (en) * | 2016-12-23 | 2017-05-24 | 江苏省血吸虫病防治研究所 | Method for detecting DNA of schistosoma through fluorescent probe |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111172244A (en) | 2020-05-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107630109A (en) | A kind of fluorescence quantification PCR primer and kit for detecting Novel pig acute diarrhea syndrome coronavirus | |
| CN112080584A (en) | Primer, probe, kit and method for detecting largemouth black bass virus | |
| CN103725796A (en) | BVDV (bovine viral diarrhea virus) internal control typing fluorescent PCR (polymerase chain reaction) detection kit and preparation thereof | |
| CN113278718A (en) | Primer pair, amplification reagent, amplification kit, detection method and application for detecting litopenaeus vannamei liver enterocytozoon | |
| CN104032038A (en) | Detection kit and detection method for siniperca chuatsi rhabdoviruses | |
| CN113637778A (en) | Kit and method for detecting brucella | |
| CN111172244B (en) | Method for rapidly identifying schistosoma japonicum infected oncomelania | |
| CN109439801B (en) | Real-time fluorescence RT-PCR detection kit and detection method for israel acute paralysis virus of bees | |
| CN101381767B (en) | Universal real time fluorescent PCR detection method of trichinella | |
| CN108676917B (en) | Dual nano PCR detection method for identifying CCoVI and CCoVII | |
| CN102108414B (en) | Real-time fluorescence transcription-polymerase chain reaction (RT-PCR) detection method and kit for pike fry rhabdovirus (PFRV) | |
| CN103484534A (en) | Set of primers and probe used for real-time fluorescence PCR detection of neisseria gonorrhoeae and applications of set of primers and probe | |
| CN103215386B (en) | Isothermal amplification method for enterovirus EV nucleic acid | |
| CN115786587A (en) | Primer group for multiplex PCR (polymerase chain reaction) for simultaneously detecting 4 pathogens as well as detection method and kit thereof | |
| CN103397106A (en) | Hybrid snakehead rhabdovirus fluorescent quantitative PCR (polymerase chain reaction) detection kit and detection method thereof | |
| CN112094854A (en) | Specific primer, probe and kit for detecting pelodiscus sinensis flavivirus | |
| CN107988429B (en) | Reagent for detecting rabies virus and application thereof | |
| CN106521038A (en) | High-sensitivity BHV-2 (bovine herpes virus 2) quantitative real-time PCR (polymerase chain reaction) detection method and kit | |
| Zhu et al. | Onsite and visual detection of ranavirus in largemouth bass (Micropterus salmoides) by an isothermal recombinase polymerase amplification method | |
| CN106701961B (en) | Reverse transcription loop-mediated isothermal amplification primer, kit and detection method for visually detecting Hobi-like pestivirus | |
| Song et al. | Histopathology and detection of cyprinid herpesvirus infection in two koi farms in Tianjin City (China) | |
| CN104328219B (en) | Sturgeon irido virus TaqMan real-time fluorescence quantitative PCR kit and application | |
| CN103882150A (en) | Primer, probe and real-time fluorescent PCR (polymerase chain reaction) method for detecting TTSuV II (torque teno sus virus II) | |
| CN103882151A (en) | Primer, probe and real-time fluorescence polymerase chain reaction (PCR) detection method for detecting I-type torque teno sus virus | |
| CN117737069B (en) | BVDV specific crRNA based on CRISPR-Cas12a, and related kit and detection method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |