CN105385686B - A kind of Schistosoma japonicum kit for detecting nucleic acid and detection method based on improvement LAMP method - Google Patents
A kind of Schistosoma japonicum kit for detecting nucleic acid and detection method based on improvement LAMP method Download PDFInfo
- Publication number
- CN105385686B CN105385686B CN201510931300.6A CN201510931300A CN105385686B CN 105385686 B CN105385686 B CN 105385686B CN 201510931300 A CN201510931300 A CN 201510931300A CN 105385686 B CN105385686 B CN 105385686B
- Authority
- CN
- China
- Prior art keywords
- schistosoma japonicum
- primer sets
- sequence
- sample
- dna
- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Analytical Chemistry (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Physics & Mathematics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of Schistosoma japonicum kit for detecting nucleic acid and detection method based on improvement LAMP method.The invention discloses a kind of for detecting the primer sets and its application of Schistosoma japonicum.Present invention firstly provides a primer sets, single strand dna (B3) shown in the sequence 2 of single strand dna (F3), sequence table shown in the sequence 1 by sequence table, sequence table sequence 3 shown in single strand dna (BIP) shown in single strand dna (FIP) and the sequence 4 of sequence table form；The function of the primer sets is for following (a) or (b): (a) identifying or assist identification Schistosoma japonicum；(b) detect in sample to be tested whether contain Schistosoma japonicum.The present invention has great application value for bilharzial generation monitoring and prevention and control.
Description
Technical field
The invention belongs to field of biotechnology, are related to a kind of Schistosoma japonicum nucleic acid detection reagent based on improvement LAMP method
Box and detection method.
Background technique
Snail fever is the second largest parasitic disease in the world, is a kind of parasitic disease of infecting both domestic animals and human, is popular in Asia, non-
Continent and Hispanic 76 countries and regions, are one of public health problems important in the world.Now it is primarily present 6 kinds of parasitisms
In the blood fluke of human body, wherein the blood fluke of China's prevalence is Schistosoma japonicum.Schistosoma japonicum not only influences personal health, and
And influence the economic development in entire Endemic Areas of Schistosomiasis Japonica domain.Snail fever was classified as Category B notifiable disease in 2004 by China,
Prevention and treatment position of equal importance is in severe acute respiratory syndrome, AIDS.
The snail fever in China is distributed in Jiangsu, Zhejiang, Shanghai, Anhui, Jiangxi, Hunan, Hubei, Sichuan, Yunnan, wide
433 counties (city, area) of 12 provinces (city) such as west, Guangdong, Fujian share 14,800,000,000 square metres of oncomelania area, and accumulative the infected reaches
11600000, compromised population is 100,000,000 or more.By positive prevention and treatment in 60 years, national schistosomiasis epidemic was obtained effectively
Control, but in recent years since the factors such as biology, nature, social economy, movement of population, policy safeguard change greatly, some places
The situation that schistosomiasis epidemic diffusion sprawling is presented shows as old Schistosoma japonicum in high endemic areas patient and increases, and oncomelania spread is obvious, new to follow closely
Luo Qu occurs, and infectious oncomelania distribution expands, and part has reached schistosomiasis propagation control and the area of transmission blockage may
There is Re-emergence, various regions Introduced cases snail fever case increases, and illustrates that the schistosomiasis control in China is also shouldered heavy responsibilities.
Country pays much attention to the prevention and treatment of snail fever, to carry out a large amount of fund for blood fluke patient and epidemic disease water every year
Investment is used for bilharzial prevention and control.
Method currently used for blood fluke diagnosis mainly has etiological diagnosis, immunology diagnosis and ultrasound diagnosis etc..Tradition
Etiological diagnosis method is that Diagnosis of Schistosomiasis is the most reliable, classical, is goldstandard whether judging snail fever illness, especially
Kato-Katz smear excrement examines method and miracidium is incubated for method (MHT).Kato-Katz method is the lookup blood fluke worm from patient's excrement
Ovum, it is more time-consuming, laborious, and epidemic-stricken area masses' compliance declines, and easily causes missing inspection.MHT method include Nylon Bag, push bench process,
Collection incubates method and rush incubates method etc., and this method recall rate is higher than conventional method, but the used time is too long, heavy workload, and equally existing cannot
The problems such as early diagnosis and masses' compliance are poor.Immune diagnostic method sensibility with higher and the compliance of the epidemic-stricken area masses
The advantages that higher, for numerous professionals and epidemic-stricken area group it is well-established, and to epidemic-stricken area chemotherapy object extensive screening and
Seroepidemiological survey and the evaluation of control efficiency etc. play particularly important effect, have formd skin test, tail
Larva of a tapeworm or the cercaria of a schistosome film test, circum oval precipitating test (COPT), indirect hemagglutination test (IHA), a variety of enzyme-linked immunosorbent assay (ELISA) and straight
Connect the methods of immune detection.COPT, sensibility with higher for Pest- or disease-free area healthy population and lower false positive rate, but it is right
Its sensibility of the crowd of the Chemotherapy in epidemic-stricken area is 72%, the NPV higher (more than 87%) of this method, and PPV is lower
(32%-70%), this method is time-consuming and relative complex, needs microscope.IHA is still the method for community diagnosis' routine, is used for
The screening of chemotherapy crowd, compared with Kato-Katz method and MHT method, sensibility is high, easy, quick, however in Chemotherapy
Area, the PPV of this method lower (being lower than 37%), sensibility 69-100%, specificity is 35-94%, furthermore this side
Method has the cross reaction of 64-84% with Paragonismus westermani, and which has limited its applications.ELISA method includes Dot-ELISA,
SPA-ELISA etc., sensibility from 65-100%, but the specificity of most of report be respectively less than the NPV of 60%, ELISA compared with
High (more than 88%), but the PPV of most of reports is very low.The ultrasound diagnosis of snail fever is non-damage diagnostic method,
It is easy to operate, it can accurately find the pathological change of liver snail fever, the severity of the state of an illness can be assessed.In a short time may be used at the scene
It checks a large amount of crowds, can be obtained immediately as a result, if being comparable using standardized method.But for early stage Schistosomiasis patients
In vain.
Epidemic-stricken area will carry out large-scale epidemic disease water identification, the spiral shell that goes out work every year.Currently, identification work master of the China to epidemic disease water
If by: oncomelania is 1. collected, is then incubated for out miracidium microscopy in the case where illumination condition abundance；2. using doing for " whistle mouse "
Method determines whether the water-bath has cercaria；3. acquiring cow dung " excrement inspection " japonice ovum.Miracidium is incubated for from oncomelania to the item of oncomelania
Part requires height, if oncomelania saves, the improper born of the same parents' larva of a tapeworm or the cercaria of a schistosome for leading to its endobiosis is dead or vigor declines, and will lead to false negative
It generates；On the other hand, microscopy miracidium is also required to certain professional technique, more demanding to reviewer, otherwise also results in leakage
Inspection etc.." whistle mouse " be even more it is a kind of time-consuming and the lower method of inspection of sensibility, this method first choice selectes waters, then allows little Bai
Mouse is swum certain time (4 hours/day, 2 days) totally in the waters water surface, and the small white mouse swum passes through the culture in or so 6 weeks
After, detect whether the small white mouse infects blood fluke, thus come identify the waters whether epidemic disease water, " whistle mouse " method determines epidemic disease water
Work have a kind of serious lagging feeling in time, often epidemic situation be already expired and qualification result there are no come out.
Summary of the invention
The object of the present invention is to provide a kind of for detecting the primer sets and its application of Schistosoma japonicum.
Present invention firstly provides a primer sets, the single strand dna as shown in the sequence 1 of sequence table (F3), sequence
Single strand dna (FIP) shown in the sequence 3 of single strand dna (B3), sequence table shown in the sequence 2 of table and sequence table
Single strand dna shown in sequence 4 (BIP) composition；The function of the primer sets is for following (a) or (b): (a) identifying or assists
Identify Schistosoma japonicum；(b) detect in sample to be tested whether contain Schistosoma japonicum.
The present invention also protects application of the primer sets in reagent preparation box；The function of the kit is following (a)
Or (b): (a) identifying or assist identification Schistosoma japonicum；(b) detect in sample to be tested whether contain Schistosoma japonicum.
Kit containing the primer sets also belongs to protection scope of the present invention；The function of the kit is as follows
(a) or (b): (a) identifying or assist identification Schistosoma japonicum；(b) detect in sample to be tested whether contain Schistosoma japonicum.
It may also include the conventional reagent of other loop-mediated isothermal amplifications in the kit.
It may also include calcein and Bst archaeal dna polymerase in the kit.It may also include UNG in the kit
Enzyme.It may also include bromophenol blue in the kit.LAMP reaction presently, there are a big problem be exactly its product to the tight of environment
Heavily contaminated will lead to the generation of false positive.UNG enzyme reaction system is introduced into traditional LAMP reaction system in the present invention,
Antipollution and LAMP reaction are carried out in same reaction system, greatly reduce influence of the environmental pollution to subsequent detection, utmostly
The generation for reducing false positive.Color developing agent calcein is used in combination with auxiliary aobvious agent bromophenol blue in the present invention, yin and yang attribute knot
The visual color of fruit is better than the simple visual color for using color developing agent calcein at present, so that result judgement more succinctly may be used
It leans on.
It may also include that dNTPs, Mg ion, glycine betaine, manganese ion and Bst Buffer in the kit.
The kit specifically may include LAMP reaction solution, Bst archaeal dna polymerase and UNG enzyme.
The preparation method (22.5 μ L) of the LAMP reaction solution is concretely: by 3 μ L 10mmol dNTPs, 0.5 μ L, 10 μ
mol/L F3、0.5μL 10μmol/L B3、1μL 40μmol/L FIP、1μL 40μmol/L BIP、1.5μL 100mM
MgSO4Aqueous solution, 1 μ L 5M aqueous solutions of betaine, 2.5 μ L calceins and manganese chloride mixed liquor (calcein containing 1.5mM and
100mM MnCl2；Solvent is water), 2.5 μ L Bst Buffer, 1 μ L, 10 μM of bromophenol blue solution and 8 μ L distilled waters.
The present invention also protects the preparation method of the kit, independent including carrying out each primer in the primer sets
The step of packaging.
The present invention also protects a kind of method identified or auxiliary identifies Schistosoma japonicum, includes the following steps:
(1) bilharzial genomic DNA to be measured is extracted；
(2) genomic DNA extracted using step (1) carries out ring mediated isothermal amplification using the primer sets as template；Such as
Primer sets described in fruit may be implemented using the genomic DNA as the specific amplification of template, and blood fluke to be measured is or candidate is day
Japonicum；If the primer sets can not achieve using the genomic DNA as the specific amplification of template, blood fluke to be measured is
Or candidate is non-Schistosoma japonicum.
Calcein and Bst archaeal dna polymerase can be contained in the reaction system of the ring mediated isothermal amplification.The ring is situated between
UNG enzyme can also be contained by leading in the reaction system of isothermal duplication.Bromine can also be contained in the reaction system of the ring mediated isothermal amplification
Phenol is blue.In the initial reaction system of the ring mediated isothermal amplification, the concentration of calcein can polymerize for 0.15mM, Bst DNA
The concentration of enzyme can be 0.32U/ μ L, UNG enzyme concentration can be 0.04U/ μ L, bromophenol blue concentration can be 0.4 μM.The ring mediates
The reaction system concrete composition of isothermal duplication is as follows: LAMP reaction solution described in 22.5 μ L, 1 μ L Bst archaeal dna polymerase (8U), 0.5
μ L UNG enzyme (1U) and 1 μ L template.The reaction system concrete composition of the ring mediated isothermal amplification is as follows: LAMP described in 22.5 μ L
Reaction solution, 1 μ L Bst archaeal dna polymerase, 0.5 μ L UNG enzyme and 1 μ L template.
The present invention also protect in a kind of detection sample to be tested whether the method containing Schistosoma japonicum, include the following steps:
(1) total DNA of sample to be tested is extracted；
(2) total DNA extracted using step (1) carries out ring mediated isothermal amplification using the primer sets as template；If institute
Stating primer sets may be implemented using the total DNA as the specific amplification of template, the biological sample to be measured contain or it is doubtful containing
Schistosoma japonicum；If the primer sets can not achieve using the total DNA as the specific amplification of template, the biological sample to be measured
Originally it does not contain or doubtful without containing Schistosoma japonicum.
Calcein and Bst archaeal dna polymerase can be contained in the reaction system of the ring mediated isothermal amplification.The ring is situated between
UNG enzyme can also be contained by leading in the reaction system of isothermal duplication.Bromine can also be contained in the reaction system of the ring mediated isothermal amplification
Phenol is blue.In the initial reaction system of the ring mediated isothermal amplification, the concentration of calcein can polymerize for 0.15mM, Bst DNA
The concentration of enzyme can be 0.32U/ μ L, UNG enzyme concentration can be 0.04U/ μ L, bromophenol blue concentration can be 0.4 μM.The ring mediates
The reaction system concrete composition of isothermal duplication is as follows: LAMP reaction solution described in 22.5 μ L, 1 μ L Bst archaeal dna polymerase (8U), 0.5
μ L UNG enzyme (1U) and 1 μ L template.The reaction system concrete composition of the ring mediated isothermal amplification is as follows: LAMP described in 22.5 μ L
Reaction solution, 1 μ L Bst archaeal dna polymerase, 0.5 μ L UNG enzyme and 1 μ L template.
The sample to be tested is in vitro biological sample or water sample.The in vitro biological sample is blood, muscle etc..Institute
Stating in vitro biological sample is the in vitro sample of rabbit, in vitro sample of oncomelania etc..
The reaction condition of any description above ring mediated isothermal amplification is specific as follows: 37 DEG C of 20min, then 65 DEG C of 60min.
Any description above Schistosoma japonicum can be adult, larva or worm's ovum.
The diagnostic nucleic acid of snail fever is theoretically optimal diagnostic method, it both can effectively make a definite diagnosis Patients with Schistosomiasis Japonica
Whether can be determined again containing blood fluke in the infective agent (including oncomelania, epidemic disease water and cow dung) in environment.
Schistosoma japonicum is detected using primer sets provided by the invention or kit, high with sensitivity, special strong, operation
The simple and efficient, used time short (1.5h), do not depend on specific apparatus (only needing thermostat water bath), result judgement succinctly (only need meat
Eye observation), detect the advantages that whole reaction tube is covered, pollution-free, be very suitable in Japanese blood fluke epidemic source carrying out
Environmental assessment and hospital carry out clinical diagnosis.The present invention has great application for bilharzial generation monitoring and prevention and control
Value.
Detailed description of the invention
Fig. 1 is the result of specificity.
Fig. 2 is the result of sensitivity.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.Bst archaeal dna polymerase: New England biolab；Article No.: M0537L.UNG enzyme: Takara；Article No.: 2820.
DNTPs be dATP, dUTP, dCTP and dGTP press 1:1:1:1 mixing mixture, 10mmol dNTPs refer to dATP, dUTP,
The concentration of dCTP and dGTP is 2.5mmol.
Embodiment 1, primer sets design and prepare
By a large amount of sequence analyses, design of primers, primer screening and compliance test result, one group is obtained for identifying that Japanese blood is inhaled
The LAMP primer group of worm is as follows:
F3 (sequence 1 of sequence table): 5 '-TGGGCCAATAGTCTGTGGT-3 '；
B3 (sequence 2 of sequence table): 5 '-GCTCGCAACTTGGAAAGT-3 '；
FIP (sequence 3 of sequence table): 5 '-CGTAACGCCCAATGACTCGCGCACCTGACCCGTCTTGAAAC-3 '；
BIP (sequence 4 of sequence table): 5 '-AACCCAAAGGCGAAGTGAAGGTTGAGCAAGGCAACAGGATC-3 '.
The target sequence of primer sets is to guard duplicate 18s section in schistosoma japonicum gene group.
It is respectively synthesized above-mentioned each primer.
The preparation method of the LAMP reaction solution of 22.5 μ L: by 3 μ L 10mmol dNTPs, 0.5 μ L 10 μm of ol/L F3,0.5
μL 10μmol/L B3、1μL 40μmol/L FIP、1μL 40μmol/L BIP、1.5μL 100mM MgSO4Aqueous solution, 1 μ L
5M aqueous solutions of betaine, 2.5 μ L calceins and manganese chloride mixed liquor (calcein containing 1.5mM and 100mM MnCl2；Solvent
For water), 2.5 μ L Bst Buffer, 1 μ L, 10 μM of bromophenol blue solution and 8 μ L distilled waters mixing.
The application of embodiment 2, primer sets
The oncomelania (sample B), that sample to be tested is respectively as follows: Schistosoma japonicum (sample A), has confirmed that infection Schistosoma japonicum
The whole blood (sample C) of the rabbit of confirmation infection Schistosoma japonicum has confirmed that the water sample (sample that the waters of Schistosoma japonicum occurs
D), have confirmed that and be uninfected by the oncomelania (sample E) of Schistosoma japonicum, have confirmed that the whole blood (sample for being uninfected by the rabbit of Schistosoma japonicum
This F), have confirmed that the water sample (sample G) that the waters of Schistosoma japonicum does not occur.
1, the total DNA of sample to be tested is extracted.
2, the total DNA extracted using step 1 carries out ring mediated isothermal amplification using the primer sets that embodiment 1 designs as template.
Ring mediated isothermal amplification carries out in reaction tube.
The reaction system of ring mediated isothermal amplification: LAMP reaction solution, the 1 μ L Bst DNA of 22.5 μ L embodiments 1 preparation are poly-
Synthase (8U), 0.5 μ L UNG enzyme (1U) and 1 μ L template.
The reaction condition of ring mediated isothermal amplification: 37 DEG C of 20min, then 65 DEG C of 60min.
3, after completing step 2, reaction tube is taken out, (bright green represents the positive, and navy blue represents yin for visual color variation
Property).
Sample A, sample B, sample C and sample D reaction tube in be illustrated as bright green, sample E, sample F and sample G's
Navy blue is illustrated as in reaction tube.
Embodiment 3, specific test
Sample to be tested is respectively as follows: Schistosoma japonicum, Schistosoma mansoni and the bacillus coli DH 5 obtained from Schistosoma mansoni excrement
α。
1, the genomic DNA of sample to be tested is extracted.
2, the genomic DNA extracted using step 1 carries out ring mediated isothermal using the primer sets that embodiment 1 designs as template
Amplification.Ring mediated isothermal amplification carries out in reaction tube.The reaction system of ring mediated isothermal amplification and ring mediated isothermal amplification
Reaction condition is the same as embodiment 2.The blank control that template is replaced with isometric sterile water is set.
3, after completing step 2, reaction tube is taken out, (bright green represents the positive, and navy blue represents yin for visual color variation
Property).
The result is shown in Figure 1.Be shown as bright green in the reaction tube of Schistosoma japonicum, Schistosoma mansoni, bacillus coli DH 5 alpha and
Navy blue is illustrated as in the reaction tube of blank control.
Embodiment 4, sensitivity test
Sample to be tested are as follows: Schistosoma japonicum.
1, the genomic DNA of sample to be tested is extracted.
2, the genomic DNA for taking step 1 to obtain carries out 10 times of gradient dilutions using sterile water, obtains every microlitre and contain 100
The target gene of a copy, 101The target gene of a copy, 102The target gene of a copy, 103The target gene of a copy, 104A copy
Target gene, 105The target gene of a copy and 106Each dilution of the target gene of a copy.
3, each dilution obtained using step 2 carries out ring mediated isothermal using the primer sets that embodiment 1 designs as template
Amplification.Ring mediated isothermal amplification carries out in reaction tube.The reaction system of ring mediated isothermal amplification and ring mediated isothermal amplification
Reaction condition is the same as embodiment 2.The blank control that template is replaced with isometric sterile water is set.
4, after completing step 2, reaction tube is taken out, (bright green represents the positive, and navy blue represents yin for visual color variation
Property).
As a result see Fig. 2.In Fig. 2,1 to 8 successively are as follows: blank control, every microlitre contain 100The target gene of a copy, 101It is a
The target gene of copy, 102The target gene of a copy, 103The target gene of a copy, 104The target gene of a copy, 105A copy
Target gene and 106The dilution of the target gene of a copy.Every microlitre contains 103The target gene of a copy, every microlitre contain 104It is a
The dilution of the target gene of copy, every microlitre contain 105The dilution of the target gene of a copy and every microlitre contain 106A copy
Target gene dilution reaction tube in be illustrated as bright green.Blank control, every microlitre contain 100The target base of a copy
Because, every microlitre contain 101The dilution of the target gene of a copy and every microlitre contain 102The dilution of the target gene of a copy
Navy blue is illustrated as in reaction tube.The result shows that sensitivity can detecte to 103The target gene of a copy.
Embodiment 5, comparative test
One, one is tested
Under same experimental enviroment, 10 Schistosoma japonicum and 10 distilled water are subjected to continuous detection in 10 days (daily
Each sample respectively detects once according to test method and contrast method).
The method of test method, that is, embodiment 2.
The difference of contrast method and test method, which is only that, replaces UNG enzyme with isometric distilled water.
On day 1 to the 10th day, 10 Schistosoma japonicum are positive findings using test method and contrast method.
On day 1 to the 10th day, 10 distilled water are negative findings using test method.
Since the 3rd day, 10 distilled water were positive findings using contrast method.Show to have occurred since the 3rd day
Pollution.
Two, two are tested
Under same experimental enviroment, using Schistosoma japonicum or Schistosoma mansoni as sample to be tested, test method is respectively adopted
It is detected with contrast method.
The method of test method, that is, embodiment 3.
The difference of contrast method and test method, which is only that, replaces bromophenol blue solution with isometric distilled water.
When using test method, the corresponding reaction tube of Schistosoma japonicum is shown as bright green, the corresponding examination of Schistosoma mansoni
It tests pipe and is shown as navy blue, color is distinguished clearly, judged very easy.
When using contrast method, the corresponding reaction tube of Schistosoma japonicum is shown as light green, the corresponding examination of Schistosoma mansoni
It tests pipe and is shown as faint yellow, color distinguishes unobvious, bad judgement.
Claims (5)
1. a kind of kit, including primer sets, calcein, Bst DNA polymerase, UNG enzyme and bromophenol blue；
Primer sets single strand dna as shown in the sequence 1 of sequence table, sequence table sequence 2 shown in single stranded DNA point
Single strand dna composition shown in single strand dna shown in sub, sequence table sequence 3 and the sequence of sequence table 4.
2. the preparation method of kit described in claim 1, including each primer in the primer sets is carried out independent packaging
The step of.
3. a kind of method of the identification or auxiliary identification Schistosoma japonicum of non-diagnostic purpose, includes the following steps:
(1) bilharzial genomic DNA to be measured is extracted；
(2) genomic DNA extracted using step (1) carries out ring mediation etc. using the primer sets described in claim 1 as template
Temperature amplification；If primer sets described in claim 1 may be implemented using the genomic DNA as the specific amplification of template, to be measured
Blood fluke is Schistosoma japonicum；If primer sets described in claim 1 can not achieve using the genomic DNA as the special of template
Property amplification, blood fluke to be measured be non-Schistosoma japonicum；
Contain calcein, Bst DNA polymerase, UNG enzyme and bromophenol blue in the reaction system of the ring mediated isothermal amplification.
4. in a kind of detection sample to be tested of non-diagnostic purpose whether the method containing Schistosoma japonicum, include the following steps:
(1) total DNA of sample to be tested is extracted；
(2) total DNA extracted using step (1) carries out ring mediated isothermal expansion using the primer sets described in claim 1 as template
Increase；If primer sets described in claim 1 may be implemented using the total DNA as the specific amplification of template, the sample to be tested
Contain Schistosoma japonicum；If primer sets described in claim 1 can not achieve using the total DNA as the specific amplification of template,
The sample to be tested does not contain Schistosoma japonicum；
Contain calcein, Bst DNA polymerase, UNG enzyme and bromophenol blue in the reaction system of the ring mediated isothermal amplification.
5. method as claimed in claim 4, it is characterised in that: the sample to be tested is water sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510931300.6A CN105385686B (en) | 2015-12-15 | 2015-12-15 | A kind of Schistosoma japonicum kit for detecting nucleic acid and detection method based on improvement LAMP method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510931300.6A CN105385686B (en) | 2015-12-15 | 2015-12-15 | A kind of Schistosoma japonicum kit for detecting nucleic acid and detection method based on improvement LAMP method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105385686A CN105385686A (en) | 2016-03-09 |
| CN105385686B true CN105385686B (en) | 2019-03-22 |
Family
ID=55418466
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510931300.6A Active CN105385686B (en) | 2015-12-15 | 2015-12-15 | A kind of Schistosoma japonicum kit for detecting nucleic acid and detection method based on improvement LAMP method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105385686B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636439A (en) * | 2017-02-17 | 2017-05-10 | 湖北医药学院 | Loop-mediated isothermal amplification (LAMP) method based on cercaria-stage gene of schistosoma japonicum katsurada |
| CN107058595B (en) * | 2017-06-19 | 2020-05-26 | 南京市疾病预防控制中心 | Method for detecting schistosome ovum and kit and primer used by same |
Citations (4)
| 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 |
| CN102286633A (en) * | 2010-01-08 | 2011-12-21 | 江苏省家禽科学研究所 | Avian infectious bronchitis virus quick detection kit based on loop-mediated isothermal amplification technology and application method thereof |
| CN102559928A (en) * | 2011-12-28 | 2012-07-11 | 中国海洋大学 | Specific primer group, kit comprising the primer group, use method and detection method |
| CN102608306A (en) * | 2011-12-20 | 2012-07-25 | 江苏省血吸虫病防治研究所 | Method for detecting insecticide resistance of schistosoma japonicum eggs to praziquantel in vitro |
-
2015
- 2015-12-15 CN CN201510931300.6A patent/CN105385686B/en active Active
Patent Citations (4)
| 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 |
| CN102286633A (en) * | 2010-01-08 | 2011-12-21 | 江苏省家禽科学研究所 | Avian infectious bronchitis virus quick detection kit based on loop-mediated isothermal amplification technology and application method thereof |
| CN102608306A (en) * | 2011-12-20 | 2012-07-25 | 江苏省血吸虫病防治研究所 | Method for detecting insecticide resistance of schistosoma japonicum eggs to praziquantel in vitro |
| CN102559928A (en) * | 2011-12-28 | 2012-07-11 | 中国海洋大学 | Specific primer group, kit comprising the primer group, use method and detection method |
Non-Patent Citations (2)
| Title |
|---|
| 几种水产养殖动物病原快速检测方法的研究;丁文超;《全国优秀硕士论文全文数据库》;20120315;D052-43 * |
| 增加环介导等温扩增灵敏度与特异性的两种实用方法;王德国;《许昌学院学报》;20140930;第33卷(第5期);65-69 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105385686A (en) | 2016-03-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105648059B (en) | A kind of Schistosoma japonicum kit for detecting nucleic acid and detection method based on RPA | |
| CN106916906A (en) | A kind of Primer composition and its kit for detecting infectious diarrhea pathogen | |
| CN104726566B (en) | Multiplex PCR detection kit and detection method for newly-emerged intestinal protozoa | |
| CN105695631A (en) | Fast joint inspection kit for human immunodeficiency viruses, hepatitis B viruses and hepatitis C viruses and preparation and application thereof | |
| Pillay et al. | Laboratory-confirmed case of yaws in a 10-year-old boy from the Republic of the Congo | |
| CN106566892A (en) | Method for detecting banana bunchy top virus by utilizing IC-LAMP | |
| CN106434979A (en) | Kit for detecting gastric cancer susceptibility and SNP marker thereof | |
| CN107881261A (en) | Detect LAMP primer group, kit and the application of the type of pig circular ring virus 3 | |
| CN105385686B (en) | A kind of Schistosoma japonicum kit for detecting nucleic acid and detection method based on improvement LAMP method | |
| Tian et al. | A LAMP-based microfluidic module for rapid detection of pathogen in cryptococcal meningitis | |
| CN107619885A (en) | A kind of fluorescence RT RAA primers, probe and detection method for being used to detect dengue virus | |
| Feau et al. | Improved detection and identification of the sudden oak death pathogen Phytophthora ramorum and the Port Orford cedar root pathogen Phytophthora lateralis | |
| CN104388592B (en) | A kind of Porcine epidemic diarrhea virus S gene RT LAMP detection kits and detection method | |
| CN107058628A (en) | A kind of LAMP primer composition thing and its related application for being used to detect the related pathogen of hand-foot-and-mouth disease | |
| CN112522378A (en) | Kit for detecting MCR gene, detection method and application thereof | |
| Hashemzadeh et al. | Development of dual TaqMan based one-step rRT-PCR assay panel for rapid and accurate diagnostic test of MERS-CoV: a novel human coronavirus, ahead of Hajj pilgrimage | |
| CN101368204B (en) | Fast detection primer and reagent kit for enterobacter sakazakii hymenial veil mediated isothermality amplification technique | |
| CN103074428B (en) | A kind of mycobacterium tuberculosis TB detection kits | |
| CN103060452A (en) | Kit for detecting chlamydia trachomatis (CT) | |
| Sun et al. | Rapid and sensitive detection of hepatitis C virus in clinical blood samples using reverse transcriptase polymerase spiral reaction | |
| CN107630097A (en) | A kind of primer sets and kit of quick detection urogenital infections common causative | |
| CN106319054B (en) | Detect the primer and detection method of brucella | |
| Zhang et al. | Duplex droplet digital PCR method for the detection of Enterocytozoon hepatopenaei and Vibrio parahaemolyticus acute hepatopancreatic necrosis disease | |
| Zhang et al. | Development of a loop-mediated isothermal amplification assay for the detection and quantification of epizootic epitheliotropic disease virus (salmonid herpesvirus-3) | |
| Dai et al. | Rapid and sensitive detection of Nosema bombycis using loop-mediated isothermal amplification and colorimetric nanogold. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |