CN105648059B - A kind of Schistosoma japonicum kit for detecting nucleic acid and detection method based on RPA - Google Patents
A kind of Schistosoma japonicum kit for detecting nucleic acid and detection method based on RPA Download PDFInfo
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- CN105648059B CN105648059B CN201610056643.7A CN201610056643A CN105648059B CN 105648059 B CN105648059 B CN 105648059B CN 201610056643 A CN201610056643 A CN 201610056643A CN 105648059 B CN105648059 B CN 105648059B
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- 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
Abstract
The invention discloses a kind of Schistosoma japonicum kit for detecting nucleic acid and detection method based on RPA for belonging to field of biotechnology.The kit includes that particle, buffer, magnesium acetate, RPA reaction primer, RPA reaction probe is lyophilized in RPA.The present invention establishes the detection method of Schistosoma japonicum using RPA technology, its sensitivity and specificity are suitable with Q-PCR method, significantly larger than Kato-Katz method, miracidium are incubated for method, ELISA and IHA, the entire reaction time completes in 10-20min, and result judgement only needs to determine by the change of fluorescence curve.The advantages that detection kit has sensitive, and specifically, result judgement is easy, quick.It is applicable not only to bedside diagnosis and can be used for work place study, environmental assessment.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of Schistosoma japonicum kit for detecting nucleic acid based on RPA
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 Japanese schistosomiasis of China's prevalence is to human health damage most serious, and difficulty of prevention and cure is maximum.
Japanese schistosomiasis not only influences personal health, but also influences the economic development in entire Endemic Areas of Schistosomiasis Japonica domain.China in
It is classified as Category B notifiable disease within 2004, is in prevention and treatment position of equal importance with 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 such as west, Guangdong, Fujian share 14,800,000,000 square metres of oncomelania area, add up the infected up to 1160
Ten thousand, compromised population is 100,000,000 or more.By positive prevention and treatment in 60 years, national schistosomiasis epidemic was effectively controlled,
But in recent years since the factors such as biology, nature, social economy, movement of population, policy safeguard change greatly, blood is presented in some places
The situation of fluke disease epidemic situation diffusion sprawling, shows as old Schistosoma japonicum in high endemic areas patient and increases, oncomelania spread is obvious, and new oncomelania area goes out
Existing, infectious oncomelania distribution expands, and part has reached schistosomiasis propagation control and the area of transmission blockage is likely to occur epidemic disease
Feelings are gone up, and various regions Introduced cases snail fever case increases, and illustrate 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 surpasses
Audio clinic etc..Traditional etiological diagnosis method is that Diagnosis of Schistosomiasis is the most reliable, classical, is whether judging snail fever illness
Goldstandard, especially Kato-Katz smear excrement inspection method and miracidium be incubated for method.Kato-Katz method is searched from patient's excrement
Japonice 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 incubate 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 deposits
The problems such as poor with masses' compliance cannot early diagnosed.Immune diagnostic method sensibility with higher and the epidemic-stricken area masses'
The advantages that compliance is higher, it is well-established for numerous professionals and epidemic-stricken area group, and to the extensive of epidemic-stricken area chemotherapy object
Screening and seroepidemiological survey and the evaluation of control efficiency etc. play particularly important effect, have formd skin
Examination, cercaria membrane test, circum oval precipitating test (COPT), indirect hemagglutination test (IHA), a variety of enzyme-linked immunosorbent assay
(ELISA) the methods of and direct immunization detects.COPT, sensibility with higher for Pest- or disease-free area healthy population and lower vacation
Positive rate, but be 72%, the NPV higher of this method to its sensibility of the crowd of the Chemotherapy in epidemic-stricken area, more than 87%, and
The lower 32%-70% of PPV, this method is time-consuming and relative complex, needs microscope.IHA is still the side of community diagnosis' routine
Method, for the screening of chemotherapy crowd, compared with Kato-Katz method and MHT method, sensibility is high, easy, quickly.However anti-
The area that complexification is treated, the PPV of this method is lower, is lower than 37%, sensibility from 69-100%, specificity from 35-94%,
Furthermore this method has the cross reaction of 64-84% with Paragonismus westermani.Which has limited its applications.ELISA method includes
Dot-ELISA, SPA-ELISA etc., sensibility is from 65-100%, but the specificity of most of reports is respectively less than 60%,
The NPV higher of ELISA, more than 88%, but the PPV of most of reports is very low.The ultrasound diagnosis of snail fever is non-damaging
Property 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.?
Scene can check a large amount of crowds in a short time, can be obtained immediately as a result, if being comparable using standardized method.But for morning
Phase Schistosomiasis patients are invalid.
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：1. collecting oncomelania, miracidium microscopy then is incubated for out 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 swims certain time (4 hours/day, totally 2 days) in the waters water surface；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.
It compares, the diagnostic nucleic acid of snail fever is theoretically optimal diagnostic method, it both can effectively make a definite diagnosis blood fluke
Whether patient can include again oncomelania to the infective agent in environment, determine containing blood fluke in epidemic disease water and cow dung, at present
It include the detection methods such as PCR, Q-PCR and LAMP there are many diagnostic nucleic acid method of Schistosoma japonicum.PCR and Q-PCR method
Dependent on thermal cycler instrument, have a higher requirement to operating environment and personnel, and the reaction of LAMP method presently, there are a big problem
It is exactly serious pollution of its product to environment, will lead to negative control and generate positive findings.
Summary of the invention
The purpose of the present invention is to provide a kind of Schistosoma japonicum kit for detecting nucleic acid and detection method based on RPA.
A kind of Schistosoma japonicum kit for detecting nucleic acid based on RPA, the kit include：Particle, buffering is lyophilized in RPA
Liquid, magnesium acetate, RPA react primer, and RPA reacts probe.
The kit also includes distilled water and schistosoma japonicum gene group DNA.
The RPA freeze-drying particle includes bacteriophage recombinase UvsX, confactor UvsY, archaeal dna polymerase, single stranded DNA knot
Hop protein, dNTPS.
The buffer is Rehydration Buffer.
The nucleotide sequence of the RPA reaction primer is as shown in sequence table SEQ ID No.1 and SEQ ID No.2.
The nucleotide sequence of the RPA reaction probe is as shown in sequence table SEQ ID No.3.
A kind of side of the Schistosoma japonicum kit for detecting nucleic acid detection Schistosoma japonicum nucleic acid using above-mentioned based on RPA
Method includes the following steps：
(1) 29.5 μ L Rehydration Buffer, 2.1 10 μM of μ L Primer A are added in RPA freeze-drying particle,
2.1 10 μM of μ L Primer B, 0.6 10 μM of μ L probe, 10.7 μ L distilled waters, 2.5 μ L templates add acetic acid in reaction lid
2.5 μ L of magnesium, the reaction tube that turns upside down are allowed to be centrifuged after mixing well, be placed in TwistDX fluorescence detector, instrument rises automatically
Temperature carries out result interpretation by observation fluorescence curve in 5-20 minutes to 38 DEG C of incubations；
(2) result judgement：Reaction tube fluorescence curve containing schistosoma japonicum gene group DNA rises, and negative control is flat
Sliding straight line；
The sequence of the Primer A is as shown in sequence table SEQ ID No.1；The sequence such as sequence table of the Primer B
Shown in SEQ ID No.2；The sequence of the probe is as shown in sequence table SEQ ID No.3；The template is Schistosoma japonicum base
Because of a group DNA.
Beneficial effects of the present invention：The present invention establishes the detection method of Schistosoma japonicum using RPA technology.This method is same
Existing detection technique is compared, and sensitivity and specificity are suitable with Q-PCR method, and significantly larger than Kato-Katz method, miracidium are incubated for
Method, ELISA and IHA.However Q-PCR needs expensive thermal cycler instrument, higher to environment and personnel requirement, entire reaction needs
90min or more is wanted, lightly portable compared to instrument needed for RPA, the entire reaction time completes in 10-20min, and result judgement only needs
Determined by the change of fluorescence curve.The advantages that detection kit has sensitive, and specifically, result judgement is easy, quick.No
It is only applicable to bedside diagnosis and can be used for work place study, environmental assessment.
Detailed description of the invention
Fig. 1 is that 1 detection Schistosoma japonicum specificity of the experiment of embodiment 2 analyzes result figure；
In figure, 1- Schistosoma japonicum；2- blank control；3- Schistosoma mansoni；4, Schistosoma haematobium.
Fig. 2 is 2 detection Schistosoma japonicum sensitivity analysis result figure of the experiment of embodiment 2；
In figure, 1-9pg/ μ l genomic DNA；2-0.9pg/ μ l genomic DNA；3-0.09pg/ μ l genomic DNA；4-
9fg/ μ l genomic DNA；5-0.9fg/ μ l genomic DNA；6- blank control；
Fig. 3 is the reaction tube fluorescence curve figure containing schistosoma japonicum gene group DNA.
Specific embodiment
The present invention will be further described in the following with reference to the drawings and specific embodiments.
1 template of embodiment, the preparation of primer and probe
Extracting genome DNA：Oncomelania, patient's excrement, blood etc. are utilized respectively corresponding method for extracting nucleic acid or gene
Group extracts kit completes the extraction of genomic DNA.Design of primers：Nucleic acid sequence is downloaded from Genebank, Japanese blood is selected to inhale
Worm guards duplicate SjR2 section, design synthesis RPA primer and probe.RPA reacts primers F 1：
CCAAGTCTCAGTGAAGTTGTGAAGGCTAT；R1：GTTAGTGTTCGAGACCAGTCAGATGGGATT；RPA reacts probe：
CTTAAAGCGAGGGAGAGCGGCAGGACCAGA(dT-FAM)G(THF)A(dT-BHQ1)TGACCCCTGAGATAT[3’-
block].DT (31bp) close to 5 ' ends is marked with FAM, and the dT (33bp) close to 3 ' ends is marked with BHQ1.Primer and spy
Needle is synthesized by the raw work in Shanghai.
2 reaction system of embodiment and identification method
Reaction system：In RPA freeze-drying particle (bacteriophage recombinase UvsX and its confactor UvsY, archaeal dna polymerase,
Single-stranded DNA binding protein (gp32), dNTPS) 29.5 μ L RehydrationBuffer, 2.1 10 μM of μ L Primer A are added
(RPA reacts primers F 1), 2.1 10 μM of μ L Primer B (RPA reacts primer R1), 0.6 10 μM of μ L probe, 10.7 μ L are bis- to be steamed
Water, 2.5 μ L templates add 2.5 μ L of magnesium acetate in reaction lid, carefully cover tightly pipe lid and being centrifuged mixes magnesium acetate with solution,
The reaction tube that turns upside down is allowed to be centrifuged again after mixing well, and is placed in TwistDX fluorescence detector, and instrument automatic heating is extremely
38 DEG C of incubations can carry out result interpretation by observation fluorescence curve in 5-20 minutes.
Result judgement：Reaction tube fluorescence curve containing schistosoma japonicum gene group DNA rises, and negative control is smooth
Straight line.
The analysis of embodiment 3RPA primer specificity
Reaction system：In RPA freeze-drying particle (bacteriophage recombinase UvsX and its confactor UvsY, archaeal dna polymerase,
Single-stranded DNA binding protein (gp32), dNTPS) 29.5 μ L Rehydration Buffer, 2.1 10 μM of μ L Primer A are added
(reaction primers F 1：TCTAATGCTATTGGTTTGAGT), 2.1 μ L, 10 μM of Primer B (reaction primer R1：
TTCCTTATTTTCACAAGGTGA), 0.6 μ L10 μM probe, 10.7 μ L distilled waters, 2.5 μ L templates add vinegar in reaction lid
Sour 2.5 μ L of magnesium carefully covers tightly pipe lid and being centrifuged mixes magnesium acetate with solution, and the reaction tube that turns upside down is allowed to after mixing well again
Secondary centrifugation is placed in TwistDX fluorescence detector, and instrument automatic heating can pass through in 5-20 minutes to 38 DEG C of incubations
It observes fluorescence curve and carries out result interpretation.
Result judgement：Reaction tube fluorescence curve containing schistosoma japonicum gene group DNA does not rise (curve 1), negative
Control is smooth straight line (curve 2) (Fig. 3).
Experiment 1：Referring to above-mentioned reaction system, wherein the concentration of schistosoma japonicum gene group DNA is in 2.5 μ L templates
100pg/ μ l, control have distilled water blank control, and the control containing 100pg/ μ l Schistosoma mansoni genomic DNA contains
The control of 100pg/ μ l Schistosoma haematobium genomic DNA, experimental result is as shown in Figure 1, anti-containing schistosoma japonicum gene group DNA
Should pipe fluorescence curve rise, remaining be smooth straight line, show that this method is feasible.
Experiment 2：Referring to above-mentioned reaction system, wherein the concentration of the DNA of group containing schistosoma japonicum gene is point in 2.5 μ L templates
It is not：9pg/ μ l schistosoma japonicum gene group DNA；0.9pg/ μ l schistosoma japonicum gene group DNA；0.09pg/ μ l Japan's blood is inhaled
Molitor genomic dna；9fg/ μ l schistosoma japonicum gene group DNA；0.9fg/ μ l schistosoma japonicum gene group DNA；Distilled water is sky
White control, experimental result is as shown in Fig. 2, concentration is on the equal curve of schistosoma japonicum gene group DNA profiling of 0.9fg/ μ l or more
It rises, shows that the sensitivity of this method is high.
Claims (5)
1. a kind of Schistosoma japonicum kit for detecting nucleic acid based on RPA, which is characterized in that the kit includes：RPA freeze-drying
Grain, buffer, magnesium acetate, RPA react primer, and RPA reacts probe, the nucleotide sequence such as sequence table of the RPA reaction primer
SEQ ID No.1 and SEQ ID No.2, the nucleotide sequence of the RPA reaction probe is as shown in sequence table SEQ ID No.3.
2. the Schistosoma japonicum kit for detecting nucleic acid based on RPA according to claim 1, which is characterized in that the reagent
Box also includes distilled water and schistosoma japonicum gene group DNA.
3. the Schistosoma japonicum kit for detecting nucleic acid based on RPA according to claim 1, which is characterized in that the RPA freezes
Dry particl includes bacteriophage recombinase UvsX, confactor UvsY, archaeal dna polymerase, single-stranded DNA binding protein, dNTPS.
4. the Schistosoma japonicum kit for detecting nucleic acid based on RPA according to claim 1, which is characterized in that the buffering
Liquid is Rehydration Buffer.
5. a kind of non-disease using the Schistosoma japonicum kit for detecting nucleic acid described in claim 1 based on RPA is diagnosed and is controlled
Treat the application method of purpose, which is characterized in that include the following steps：
(1) 29.5 μ L Rehydration Buffer, 2.1 10 μM of μ L Primer A, 2.1 μ L are added in RPA freeze-drying particle
10 μM of Primer B, 0.6 10 μM of μ L probe, 10.7 μ L distilled waters, 2.5 μ L templates add 2.5 μ of magnesium acetate in reaction lid
L, the reaction tube that turns upside down are allowed to be centrifuged after mixing well, be placed in TwistDX fluorescence detector, instrument automatic heating to 38
It DEG C is incubated for, passes through observation fluorescence curve progress result interpretation in 5-20 minute；
(2) result judgement：Reaction tube fluorescence curve containing schistosoma japonicum gene group DNA rises, and negative control is smooth
Straight line；
The sequence of the Primer A is as shown in sequence table SEQ ID No.1；The sequence such as sequence table SEQ of the Primer B
Shown in ID No.2；The sequence of the probe is as shown in sequence table SEQ ID No.3；The template is schistosoma japonicum gene group
DNA。
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN106701940B (en) * | 2016-12-23 | 2021-08-24 | 江苏省血吸虫病防治研究所 | Method for detecting schistosome DNA by fluorescent probe |
| CN110029152B (en) * | 2019-01-17 | 2021-04-20 | 华中农业大学 | Schistosoma japonicum RPA molecular detection method |
| CN110499359A (en) * | 2019-09-19 | 2019-11-26 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | It is a kind of quickly to identify Schistosoma japonicum, graceful formula blood fluke, the LF-RPA method and its application of the complete fluke in east |
| CN111304340B (en) * | 2020-04-07 | 2023-11-03 | 中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心) | RPA-EC schistosoma japonicum detection kit and detection method |
| CN111304296B (en) * | 2020-04-07 | 2023-11-03 | 中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心) | Electrochemical nucleic acid sensing detection method and kit based on interface RPA amplification |
| CN113151495A (en) * | 2021-04-20 | 2021-07-23 | 中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心) | Primer, probe, kit and method for universal visual detection of schistosoma japonicum and schistosoma mansoni nucleic acid by LFD-RPA |
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| CN101131347A (en) * | 2007-09-04 | 2008-02-27 | 武汉大学 | Fluorescent quantitative PCR reagent kit for fast detecting Japanese blood fluke |
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