CN104561269B - A kind of plasmodium vivax detecting method of rapid sensitive - Google Patents
A kind of plasmodium vivax detecting method of rapid sensitive Download PDFInfo
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- CN104561269B CN104561269B CN201410697136.2A CN201410697136A CN104561269B CN 104561269 B CN104561269 B CN 104561269B CN 201410697136 A CN201410697136 A CN 201410697136A CN 104561269 B CN104561269 B CN 104561269B
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- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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- 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
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- 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
Abstract
The present invention relates to a kind of detection methods of quick, sensitive Plasmodium vivax.The detection method of the present invention incorporates polymerase chain reaction PCR and microarray both powerful Protocols in Molecular Biologies, the PCR probes hybridized is directly anchored in the hybridization cabin in microarray, on the same chip with PCR reative cells；Detection method includes：Sample carries out increasing bacterium processing, the extraction of DNA liquid, and PCR amplification hybridizes, cleaning, as a result interpretation.The present invention can carry out plasmodium vivax the detection of rapid sensitive, the detection efficiency of import-export ports frontline inspection and quarantine personnel can be increased substantially through the invention, not only workload can have been reduced but also can have solved the problems, such as traditional detection method positive missing inspection that may be present to the maximum extent, to prevent the generation of plasmodium epidemic situation to the maximum extent.
Description
Technical field
The present invention relates to technical field of in vitro diagnostic reagents, and in particular to a kind of Plasmodium vivax detection side of rapid sensitive
Method.
Background technology
It is counted according to WHO, global 107 countries and regions have prevalence and the propagation of malaria, 3,200,000,000 people to be threatened by malaria, every year
Have that 300,000,000-5 hundred million human hairs are sick, death toll is up to more than 100 ten thousand.Malaria is one of five big parasites of China's keypoint control.Malaria
(malaria) it is the serious parasitic disease of harm caused by Infected With Plasmodium.The plasmodium of the infection mankind has 4 kinds：Malignant malaria
Protozoon (Plasmodium falciparum, P.f), Plasmodium vivax (Plasmodium vivax, P.v), malariae
(Plasmodium malariae, P.m) and Plasmodium ovale (Plasmodium ovale, P.o).
The pathogen of malaria disease is caused to be mainly tertian fever and plasmodium falciparum in China.Therefore, effectively detection malaria is former
Worm is medical diagnosis and the essential means of malaria control.
Microscopical Method For Detection is still the main means of " three heat " patient monitoring and malaria clinical cases diagnosis at present.As malaria is sent out
The decline of sick rate and the generation of protozoal resistance, density of protozoa in peripheral blood of infected population is relatively low and the atypical feelings of plasmodium morphology
Condition becomes more universal, and time-consuming and laborious using traditional thick blood film microscopy, detection efficiency is low.Seek sensitive special, easy to be fast
The malaria diagnosis method of speed seems extremely important in the current malaria control work.Molecular nucleic acid detection technology is in malaria in recent years
It is applied in disease diagnosis, and shows good development prospect.
It is the technology for being now widely used for plasmodium rapid detection by the molecular biology method of representative of round pcr.
And the real-time fluorescent PCR technology and biochip technology etc. to be grown up by round pcr is to make in plasmodium detection at present
With widest molecular biology method.Real-time fluorescent PCR technology is a kind of new PCR method, is the base in routine PCR reaction
The probe that can be combined with amplification template specificity is increased on plinth, compared with normal PCR, this method is not necessarily to carry out PCR product
Reprocessing is entirely the overall process for completing detection in the closed state, has the characteristics that real-time, accurate and fast, but due to right
Equipment requirement is higher, big to the technical difficulty of operation of staff, so being not suitable for quick, accurately detection.
Genetic chip is the completely new trace analysis to grow up the 1990s, it uses micro Process and micro- electricity
The good genetic fragment of a large amount of engineer is arranged on the carriers such as sheet glass or tunica fibrosa by sub- technology in an orderly manner, to high-density
Obtained from a kind of information detection chip.Genetic chip can fix a large amount of probe molecule simultaneously, theoretically can be primary
All potential originals of causing a disease are detected in experiment, and the various genetic indexes of a certain pathogenic original can also be detected with same chip,
This provides an easily approach for the multiple strains of Parallel testing or with multiple bacterial strains in strain；The sensitivity of detection simultaneously,
Specificity and rapid and convenient are also very high, thus have good development prospect in the analysis and detection of pathogenic factors.
Biochip technology is directed to the specific probe of various microorganisms using the genome sequence design of detected sample, will
Probe (oligonucleotides) point sample designs PCR primer in chip surface, while in probe both sides, during the primer synthesis process to it
5 ' ends carry out fluorescent marker or the dNTP of fluorescent marker are added during PCR, utilize the method for PCR amplification can be obtained in this way
It is marked with the target to be detected of fluorescent dye, then with the target of the micro-array chip containing sample specific probe to be detected and label
Hybridization reaction occurs for mark hybridization, the DNA sequence dna to match on the DNA molecular and chip of fluorescent marker so that the point on chip is in
Reveal fluorescence signal, determines that detection sample whether there is certain spies finally by scanner quantification and analysis of fluorescence distribution pattern
Determine plasmodium.
Be currently used in the genechip detection of plasmodium detection some be applied to research, but these genetic chips
Probe spotting mostly uses manual mode, complicated operation, and increases the chance of many false positives.
The present invention incorporates PCR (PCR) and microarray both powerful Protocols in Molecular Biologies,
The probe of PCR hybridization is directly anchored in the hybridization cabin in microarray, on the same chip with PCR reative cells.PCR reaction knots
Hybridization solution can be directly added into after beam to be hybridized, it is easy to operate, the time is saved, in addition sensitivity can reach tens Copy
DNA molecular.
The detection method of the present invention can operate with the detection that plasmodium vivax is fast and sensitive, as serological test and microscopy
The reliable supplement of method improves the accuracy and timeliness of detection, can greatly reduce the period of detection.
Invention content
Technical problem to be solved by the invention is to provide a kind of detection method of quick, sensitive Plasmodium vivax, this hairs
The bright detection that rapid sensitive can be carried out to Plasmodium vivax gene can increase substantially import-export ports one line inspection through the invention
The detection efficiency of quarantine functionary is tested, can not only reduce workload but also traditional detection method can be solved to the maximum extent is that may be present
Positive missing inspection problem, to prevent the generation of epidemic situation and being passed to for external malaria to the maximum extent.
The present invention is directed to the peculiar gene of Plasmodium vivax, design primer, and design in the primer amplification region can distinguish other
The specific probe of plasmodium and the closer species of affiliation passes through probe specificity verification, probe sensitivity test, method
Specificity experiments etc. prove that this method can be with specific detection Plasmodium vivax, and compared with conventional PCR method, detection sensitivity is apparent
Higher than the detection minimum detectability of conventional method multiplex PCR amplification product, it can not only meet wanting for daily epidemic situation safety detection
It asks, it might even be possible to meet the testing requirements of clinical sample.
Quick, sensitive plasmodium vivax detecting method provided by the present invention, includes the following steps：
(1) blood sample DNA liquid is extracted；
(2) PCR amplification：
A.PCR reaction systems include：PCR reaction buffers, testing gene specificity forward and reverse primer, PCR Grade
H2O, PCR Control, DNA extracting solution；
B. PCR reaction solution is added in the chip containing gene specific probe to be tested, chip is put into reative cell
It carries out amplification reaction；Specific probe is designed according in specific primer amplification region, other species can be distinguished；Specificity is visited
Pin mark enters on chip；.
(3) hybridize：After PCR amplification is complete, chip is added in hybridization reaction solution, hybridization solution and amplification solution is made all to enter hybridization
Hybridization reaction in cabin；
(4) it cleans：Chip after hybridization is rinsed with washing lotion, is directly detected with Fluorescence Scanner；
(5) result interpretation.
Blood sample genome DNA extraction kit can be used to extract DNA for DNA extractions described in step (1).Take 50uL
Whole blood sample extracts DNA according to the extraction step in kit specification；
Specific primer and probe described in step (2) are respectively：
SSUrRNA genes
Pv1-F:AGTTCGTGAATATGATTTGTC (SEQ ID NO 1)
Pv1-R:CTGCGCTTCTGTACTTGGC (SEQ ID NO 2)
Probe:GGGGATTGCAATTATACTTCGTGTCG (SEQ ID NO 3)
Csp genes
Vp2-F:GGTGAGAACCCAGATGACGAGG (SEQ ID NO 4)
Vp2-R:TGGACTCCATGCAGTGTAACCTGT (SEQ ID NO 5)
Probe：GGTGATGGAGCAGCTGTACAG (SEQ ID NO 6)
18SrRNA genes
Lm2-F：GCAACGCTTCTAGCTTAATCCAC (SEQ ID NO 7)
Lm2-R：CAAGCCGAAGCAAAGAAAGTCC (SEQ ID NO 8)
Probe：ACTTTGTGCGCATTTTGCTA (SEQ ID NO 9)
PCR reaction systems described in step (2) are：2X Mater mix 12.5ul, PrimerD 2.5ul, PCR
Grade H2O 4ul,PCR Control 1ul,Sample 5ul；
The reaction condition of PCR amplification described in step (2) is：95 DEG C of pre-degeneration 15min；95 DEG C of denaturation 15s, 60 DEG C of annealing
40s, 72 DEG C of extension 30s, 45 cycles, 72 DEG C of extension 1min.
Hybridization reaction condition described in step (3) is：The hybridization solution of injection is 14.5ul, reaction time 30min.Hybridization
Chip afterwards is rinsed with washing lotion 3000rpm 2min, is directly detected with Fluorescence Scanner.
PCR amplification described in step (2) is completed with hybridizing described in step (3) on chip instrument.
The amplification process and hybridization detection process that DNA hybridization detects is concentrated to by the present invention compared with existing detection method
It is detected on one chip, time and the complexity of detection is greatly reduced, detection device is simply easy to carry about with one.Detecting system can be with
Quantitative detection substantially increases current detectability down to the plasmodium of 78copy DNA.And it is total from sample collection to detection
Time can complete in 3 hours completely, be beneficial to fast and accurately detect Plasmodium vivax.
Description of the drawings
1 plasmodium vivax microscopic examination positive result of Fig. 1 embodiments.
1 Plasmodium vivax chip of Fig. 2 embodiments detection specificity.
Specific implementation mode
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, people in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
The specificity and sensitivity of the detection of 1 Plasmodium vivax of embodiment
One, experimental procedure
1. the acquisition of blood sample
Take the whole blood sample 1.0ml of port detection patient.
2. the Preliminary Identification of blood sample
Primarily determine whether the patient infects Plasmodium vivax using colloidal gold method.
3. the extraction of genomic DNA
Use blood sample genome DNA extraction kit (centrifugal column type, Qiagen companies) extraction tertian fever DNA.It takes
50uL whole blood samples extract DNA according to the extraction step in kit specification.
The measurement of 4.DNA concentration
Plasmodium DNA concentration is quantified by the method for real-time quantitative PCR using the real-time fluorescence detector of ABI.
5. the amplification of target gene
The design of 5.1 primer and probes
For the peculiar gene of Plasmodium vivax, design primer, design in the primer amplification region can distinguish other bacterial strains
Specific probe.The present embodiment is devised for the peculiar gene SSUrRNA genes of Plasmodium vivax, csp genes, 18sRNA genes
Three pairs of primer and probes are respectively：
SSUrRNA genes
Pv1-F:AGTTCGTGAATATGATTTGTC (SEQ ID NO 1)
Pv1-R:CTGCGCTTCTGTACTTGGC (SEQ ID NO 2)
Probe:GGGGATTGCAATTATACTTCGTGTCG (SEQ ID NO 3)
Csp genes
Vp2-F:GGTGAGAACCCAGATGACGAGG (SEQ ID NO 4)
Vp2-R:TGGACTCCATGCAGTGTAACCTGT (SEQ ID NO 5)
Probe：GGTGATGGAGCAGCTGTACAG (SEQ ID NO 6)
18SrRNA genes
Lm2-F：GCAACGCTTCTAGCTTAATCCAC (SEQ ID NO 7)
Lm2-R：CAAGCCGAAGCAAAGAAAGTCC (SEQ ID NO 8)
Probe：ACTTTGTGCGCATTTTGCTA (SEQ ID NO 9)
Specific probe is clicked and entered on chip.
5.2 reaction platforms
PCR amplification and hybridization reaction are carried out on the chip instrument of Witter.To prevent the interaction between primer, it is divided to two reactions
Room carries out PCR reactions, and two reative cells can be connected to hybridization chamber, and the amplification liquid after PCR can pass through injection pressure mode
It enables its flow into hybridization chamber and carries out hybridization reaction.
PCR reaction systems are：2X Mater mix 12.5ul, PrimerD 2.5ul, PCR Grade H2O 4ul,PCR
Control 1ul,Sample 5ul.
Reaction condition is identical：95 DEG C of pre-degeneration 15min；95 DEG C denaturation 15s, 60 DEG C annealing 40s, 72 DEG C extension 30s, 45
Cycle, 72 DEG C of extension 1min.
6. hybridization reaction
PCR reative cell 14.5ul hybrid nights are injected separately into, so that hybrid night and amplification liquid is all entered in hybridization cabin, reacts
30min.Chip after hybridization is rinsed with washing buffer 3000rpm 2min, directly Witter Fluorescence Scanner is used to detect.
7. the sensitivity of detection architecture and specificity
Microscopical Method For Detection determines the positive of sample：
System sensitivity test：DNA extracting solutions are diluted to 10-1、10-2、10-3、10-4, using each dilution as template,
By the method for Real-time PCR, the concentration of DNA is judged.The DNA of each bacterium is diluted to about 10 according to DNA concentration,
100,1000copy/ul.It takes each gradient DNA to be added in chip as template to be detected.
8. field sample specificity test
In Yunnan malaria epidemic area, microscopy of learning from else's experience is confirmed as 10 parts of the sample of plasmodium falciparum, 10 parts of vivax malaria sample, malaria
10 parts of protozoon negative sample extracts DNA, carries out chip measurement.
Two, result
1. microscopic examination result shows positive findings, Fig. 1 is seen
2. the detectability of chip
Array experiment has been carried out with the plasmodium vivax positive sample that port detects, has as a result seen Fig. 2.
Fig. 2 is shown, by array experiment, will appear apparent positive signal in the corresponding position of chip.Positive sample position
It sets and apparent hybridization signal occurs, hybridization signal does not occur in negative sample.This shows that the detection performance of chip is good.
3. the sensitivity of chip
Determine that DNA template concentration is by the method for real-time PCR：
The detection of various concentration DNA sample chip shows that the chip Monitoring lower-cut is 78copyDNA molecules.
Chip detects after the dilution of 1 real-time quantitative fluorescence PCR detection template of table
| Plasmodium SSUrDNA | Quantitative (copies/ul) | 101Dilution | 102Dilution | 103Dilution | 104Dilution |
| Tertian fever (Pv) | 7.8*103 | + | + | + | - |
4. sample specificity test：
To Yunnan on-site sample chip test result, 30 parts of samples are shown, 20 parts without detection signal, tertian fever positive sample
Originally 10 parts are detected, and catalogue number(Cat.No.) is consistent with microscopic examination result.
Claims (1)
1. the primer and probe for rapid sensitive detection Plasmodium vivax, which is characterized in that the primer and probe is three groups：
According to SSUrRNA genes design positive and negative primer and probe be
Pv1-F:AGTTCGTGAATATGATTTGTC(SEQ ID NO 1)
Pv1-R:CTGCGCTTCTGTACTTGGC(SEQ ID NO 2)
Probe:GGGGATTGCAATTATACTTCGTGTCG(SEQ ID NO 3)
According to csp genes design positive and negative primer and probe be
Vp2-F:GGTGAGAACCCAGATGACGAGG(SEQ ID NO 4)
Vp2-R:TGGACTCCATGCAGTGTAACCTGT(SEQ ID NO 5)
Probe：GGTGATGGAGCAGCTGTACAG(SEQ ID NO 6)
According to 18SrRNA genes design positive and negative primer and probe be
Lm2-F：GCAACGCTTCTAGCTTAATCCAC(SEQ ID NO 7)
Lm2-R：CAAGCCGAAGCAAAGAAAGTCC(SEQ ID NO 8)
Probe：ACTTTGTGCGCATTTTGCTA(SEQ ID NO 9).
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220419A (en) * | 2011-04-26 | 2011-10-19 | 广东出入境检验检疫局检验检疫技术中心 | Nested PCR (Polymerase Chain Reaction) detection and fluorescence PCR detection kit for universal type malaria and subtypes thereof |
| CN103898208A (en) * | 2013-11-27 | 2014-07-02 | 中华人民共和国上海出入境检验检疫局 | Quick high-throughput intestines source pathogenic bacterium detection method |
| CN103911447A (en) * | 2014-04-03 | 2014-07-09 | 河北国际旅行卫生保健中心 | Primers, probes and method for detecting plasmodium |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220419A (en) * | 2011-04-26 | 2011-10-19 | 广东出入境检验检疫局检验检疫技术中心 | Nested PCR (Polymerase Chain Reaction) detection and fluorescence PCR detection kit for universal type malaria and subtypes thereof |
| CN103898208A (en) * | 2013-11-27 | 2014-07-02 | 中华人民共和国上海出入境检验检疫局 | Quick high-throughput intestines source pathogenic bacterium detection method |
| CN103911447A (en) * | 2014-04-03 | 2014-07-09 | 河北国际旅行卫生保健中心 | Primers, probes and method for detecting plasmodium |
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