CN106801097A - Ring mediated isothermal amplification method based on the exclusive gene of plasmodium falciparum ring bodies - Google Patents
Ring mediated isothermal amplification method based on the exclusive gene of plasmodium falciparum ring bodies Download PDFInfo
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Abstract
The invention belongs to plasmodium falciparum detection technique field, and in particular to the ring mediated isothermal amplification method based on the exclusive gene of plasmodium falciparum ring bodies, comprise the following steps：Step one, bioinformatic analysis, search PlasmoDB databases, screening is only in the expression high of plasmodium falciparum erythrocyte stage ring bodies stage (preceding 20%) and this kind of peculiar gene of plasmodium, its distribution situation between eucaryote different genera is verified using OrthoMCL database, meanwhile, screen only in Erythrocytic Stages of Plasmodivm Falciparum trophozoite or schizont phase expression high (preceding 20%) and its each one of peculiar gene, as parallel control；Step 2, LAMP primer design；The peculiar gene screened to step one using LAMP Photographing On-line software PrimerExplorerV4 carries out LAMP primer design, and parameter is set to default value, selection fraction ranking the first two to the LAMP primer of first five, then carry out LAMP primer synthesis.It is prepared by step 3, masterplate DNA；Step 4, primer is prepared；Step 5, the foundation of LAMP methods and sensitiveness, Evaluation on specificity.
Description
Technical field
The invention belongs to plasmodium falciparum detection technique field, and in particular to based on the exclusive gene of plasmodium falciparum ring bodies
Ring mediated isothermal amplification method.
Background technology
Malaria is one of global three big public health problems of serious harm human health, can cause the 5 of human body malaria
Plant in plasmodium, the malignant malaria caused by plasmodium falciparum is the most serious, often with chilly, heating, headache as onset symptoms, concurrently
Disease is more, if treating easy threat to life not in time.The disease is popular universal in Sub-Saharan Africa area, morbidity and mortality
High, particularly pregnant woman and less than five years old children deeply hurts.
After 20 century 70s, as malaria declines, low parasitemia patient proportion increase, mixed infection
Case increases.Additionally, check after patient's drug therapy or being in the requirement to microscopy such as chronic infection stage and improving.It is conventional
Blood film smear staining Microscopical Method For Detection is likely to result in mistaken diagnosis or fails to pinpoint a disease in diagnosis, and the microscopy accordingly, as goldstandard has been difficult in adapt to epidemic situation
Detection demand after change.Further, since microscopy needs observation for a long time, specialty to differentiate and check, it is difficult to extensive use.Together
When, immunological method it is unstable, time-consuming for PCR, it is high to spend so that is all difficult to find that a kind of practicality all the time and is applicable
The plasmodium falciparum detection method that property has both.World malaria report display in 2016, it is new that the whole world in 2015 there are about 2.12 hundred million malaria
Increase case, wherein death about 429000.At present, China's malaria is changed into Introduced cases via based on local case
Based on case, and introduced cases ratio rises year by year.Particularly in recent years, with domestic and international economic and trade contact frequently, go abroad through
Business, travel, study abroad and spend a holiday increasing, cause China's input-response relation epidemic situation more and more to protrude.Therefore, a kind of operation is found
Simply, rapidly and efficiently, high specific and highly sensitive detection method it is extremely urgent.Traditional malaria detection method is thickness blood
The dyed rear microscopy of film smear.Its advantage is simple to operate, low cost, can carry out species identified；It is disadvantageous in that its expense
When, it is laborious, the specialty of proofer is required high, particularly detect easy to cause missed diagnosis during low parasitemia sample.Therefore, researcher builds
The malaria immune quick diagnosis side based on EUSA (ELISA) and indirect immunofluorescence assay (IFA) is found
Method.These methods can realize detecting the purpose of Infected With Plasmodium, and sensitiveness can match in excellence or beauty with professional microscopy, but its detection efficiency
And it is unstable.Then, researcher develops the Protocols in Molecular Biology based on DNA cloning, and the most frequently used has PCR, nest-type PRC
And real-time quantitative PCR.Compared with microscopy, molecular biology method has sensitivity higher (minimum to can detect 1~5 malaria original
Worm/μ l) and it is preferably specific；But the technology exist it is costly, by professional high-tech human users, and can only must match somebody with somebody in facility
Preparing full laboratory is carried out, so as to limit its application in the relatively poor area of basic hospital and medical and health conditions, and
These areas are generally again that malaria prevalence is widest in area, endanger the areas of most serious.Therefore, in the urgent need to researching and developing a kind of new malaria
Disease diagnostic techniques, to make up the deficiency of microscopy, immunology and conventional molecular biological method.
Ring mediated isothermal amplification method (Loop-mediated isothermal amplification, LAMP) is 2000
A kind of new constant temperature nucleic acid amplification technology for developing.It is characterized in that designing 4 kinds for 6 regions of target gene specifically draws
Thing, incubating 30~60min under isothermy (63 DEG C or so) using a kind of strand displacement archaeal dna polymerase can complete DNA cloning
Reaction.Compared with regular-PCR, LAMP technology do not need template thermal denaturation, complicated temperature cycles, long-time electrophoresis and gel into
As etc. process, the characteristics of with simple, quick, High sensitivity, high specificity, these unique advantages make it more can be numerous doctors
The author that works is received.At present, the LAMP skills that CSP GFPs and 18S rRNA are encoded based on plasmodium falciparum have been established
Art, and it is widely used in field investigation.However, such highly conserved gene is all deposited extensively in Plasmodium or even Plasmodiidae
, LAMP methods are a kind of hypersensitivity and specific technology in addition, easily cause false positive, so as to cause worm kind judge by accident.
The content of the invention
Based on above-mentioned technical problem, the invention provides the ring mediated isothermal based on the exclusive gene of plasmodium falciparum ring bodies
TRAP, is used to solve the problems, such as that existing plasmodium falciparum detection method is many in the presence of erroneous judgement etc..
To realize above-mentioned technical purpose, the technical solution adopted by the present invention is as follows：
Based on the ring mediated isothermal amplification method of the exclusive gene of plasmodium falciparum ring bodies, comprise the following steps：
Step one, bioinformatic analysis：Search PlasmoDB databases, screen only in plasmodium falciparum erythrocyte stage
(preceding 20%) and this kind of peculiar gene of plasmodium, verify it in eucaryon using OrthoMCL database for ring stage expression high
Distribution situation between biological different genera, meanwhile, screen only in Erythrocytic Stages of Plasmodivm Falciparum trophozoite or schizont phase table high
Up to (preceding 20%) and its each one of peculiar gene, as parallel control；
Step 2, LAMP primer design：Step one is sieved using LAMP Photographing On-line softwares PrimerExplorerV4
The peculiar gene chosen carries out LAMP primer design, and parameter is set to default value, selection fraction ranking the first two to the LAMP of first five
Primer, then carries out LAMP primer synthesis；
It is prepared by step 3, masterplate DNA：Using poba gene group DNA extraction kit extract respectively containing plasmodium falciparum,
Plasmodium vivax, the Whole Blood Genomic DNA of Plasmodium yoelii；Carried using blood/cell/tissue genome DNA extracting reagent kit
Take the mouse ascites DNA of toxoplasma gondii infection；The template DNA that the genomic DNA that mentioned reagent box is extracted as LAMP is used；
Step 4, primer is prepared：
LAMP primer deposits liquid storage is prepared：The LAMP primer dry powder for being placed in 1.5ml centrifuge tubes that step 2 is obtained is existed
1min is centrifuged under 12000r/min, so that the powder for being bonded at lid and tube wall all concentrates on ttom of pipe.
Prepare mother liquor：Dry powder is dissolved in 1.5ml centrifuge tubes according to primer dry powder molal quantity, inner primer (FIP/BIP) is prepared
The concentration of mother liquor is 200pmol/ μ l, and the concentration of outer primer (F3/B3) mother liquor is 100pmol/ μ l, the inner primer that will be prepared
(FIP/BIP) mother liquor and outer primer (F3/B3) mother liquor room temperature place 5min, and centrifuge is put into after vibration is well mixed,
1min is centrifuged under 12000r/min；
It is prepared by LAMP primer working solution PM：Take each 4 μ l of FIP mother liquors and BIP mother liquors of 200pmol/ μ l, 100pmol/ μ l
F3 mother liquors and each 1 μ l of B3 mother liquors, being eventually adding 10 μ l ultra-pure waters, to be prepared into 20 μ l working solutions standby；
Step 5, the foundation of LAMP methods and sensitiveness, Evaluation on specificity：
A.LAMP reaction systems include 12.5 μ l 2 × RM reaction solutions, the LAMP primer working solution PM that 1 μ l step 4 is obtained,
The DNA profiling of 1 μ l and corresponding gene, 1 μ l Bst DNA Polymerase, plus ultra-pure water is to 25 μ l, is added after being well mixed
12.5 μ l sealing fluids are centrifuged again, finally drop in the middle of PCR pipe lid 1 μ l nitrite ions, lid are gently covered tightly, in constant-temperature metal bath
Reaction, overturns PCR pipe for several times or 1000~16000r/min centrifugation 30sec~3min after terminating reaction, make nitrite ion with reaction
Mixed liquor is fully mixed, observing response liquid color change after of short duration centrifugation, is positive (having nucleic acid amplification) if green is presented, if
It is then negative (free nucleic acid amplification) that brownish red is presented；
B. the whole blood DNA containing P. falciparum genes group DNA that step 3 is obtained is carried out into doubling dilution with ultra-pure water,
Then detected according to the LAMP decoration methods described in A in step 5, evaluation detects the sensitiveness of gene；
C. the whole blood DNA containing P. falciparum genes group DNA that step 3 is obtained, Plasmodium vivax genome is contained
The whole blood DNA of DNA, the whole blood DNA containing Plasmodium yoelii genomic DNA, the ascites DNA containing toxoplasma cdna group DNA；Press
According to the LAMP decoration methods detection described in A in step 5, the LAMP primer using 18S rRNA designs is carried out as positive control primers
Evaluation on specificity, while setting up blank and negative control.
Used as preferred, the peculiar gene of plasmodium falciparum is PF3D7_1253300, PF3D7_ in the step one
0202200,PF3D7_0702300,PF3D7_1001900,PF3D7_1002000,PF3D7_1148900,PF3D7_
1240100, PF3D7_1301700 and PF3D7_1334700, such design can be preferably more preferable to plasmodium falciparum
Difference, is preferably detected.
As preferred, Erythrocytic Stages of Plasmodivm Falciparum trophozoite stage expression high (the preceding peculiar base of 20%) in the step one
Because PF3D7_0220300, such design is comparative stronger.
As preferred, Erythrocytic Stages of Plasmodivm Falciparum schizont phase expression high (the preceding peculiar base of 20%) in the step one
Because PF3D7_1401600, such design, with reference to, it is comparative stronger.
Used as preferred, the extracting method of blood/cell/tissue genome DNA extracting reagent kit is in the step 3
Plasmodium filter paper blood cake DNA is extracted, and the plasmodium filter paper blood cake DNA methods are：A little filter paper, the filter paper are taken with card punch
Containing blood sample, 1.5ml centrifuge tubes are placed in, first add 1.0ml sterilized waters immersion 2h, 5min then is centrifuged under 16000r/min, abandoned
Supernatant liquor；Again plus 160 μ l 5%chelex-100 suspensions, it is denatured 5 after 56~65 DEG C of 1~2h of heating at 95~98 DEG C~
10min, vibrates 30s~3min, then takes 3~5min of centrifugation under 12000~16000r/min, draws the μ l of supernatant 150 ± 10,
Preserved at -20 DEG C.
Used as preferred, the card punch takes 3mm*3mm filter paper, and the filter paper contains 10 ± 0.5 μ l blood samples, such to set
Meter, can obtain enough blood samples.
Used as preferred, the parasitemia densities of the whole blood containing plasmodium falciparum are 50000/μ l in the step 3, this
The design of sample, can enable preferably to be detected when detection.
Used as preferred, the temperature in the step 5 in constant-temperature metal bath is 60~65 DEG C, and such design can make
Detection results become apparent from.
Used as preferred, the reaction time in the step 5 in constant-temperature metal bath is 30~90min, such design,
Accuracy of detection can be made higher.
Used as preferred, plasmodium falciparum DNA ultra-pure waters are according to 10 in the step 5-1、10-2、10-3、10-4With 10-5Doubling dilution, such design can more preferably detect contrast.
Beneficial effects of the present invention：
1st, ring mediated isothermal amplification method is more quick to the genetic test of plasmodium falciparum ring bodies, efficient；
2nd, the present invention can detect the peculiar gene of plasmodium falciparum ring bodies, so that other other plasmodiums；
3rd, the present invention is relative with other detection methods, and the detection to plasmodium falciparum is more accurate, judges by accident lower.
Brief description of the drawings
The nonlimiting examples that the present invention can be given by accompanying drawing are further illustrated；
Fig. 1 is the LAMP of ring mediated isothermal amplification method embodiment of the present invention based on the exclusive gene of plasmodium falciparum ring bodies
Detection architecture Quality Control schematic diagram；
Fig. 2 is the foundation of ring mediated isothermal amplification method embodiment of the present invention based on the exclusive gene of plasmodium falciparum ring bodies
With sensitivity assessment schematic diagram；
Fig. 3 is the special of ring mediated isothermal amplification method embodiment of the present invention based on the exclusive gene of plasmodium falciparum ring bodies
Property evaluate schematic diagram；
Fig. 4 is the clinic of ring mediated isothermal amplification method embodiment of the present invention based on the exclusive gene of plasmodium falciparum ring bodies
Pattern detection evaluates schematic diagram.
Specific embodiment
In order that those skilled in the art may be better understood the present invention, with reference to the accompanying drawings and examples to this hair
Bright technical scheme is further illustrated.
Embodiment
Step one, with reference to plasmodium falciparum full-length genome and transcript profile database, screens altogether from 5403 genes
1799 genes of 20% expression high before ring stage, accounting 33.30%；Analyzed by comparing, finally in 1799 genes
In have selected nine peculiar genes as candidate gene for LAMP study；In nine genes, PF3D7_1253300 is false base
Cause, remaining eight PF3D7_0202200, PF3D7_0702300, PF3D7_1001900, PF3D7_1002000, PF3D7_
1148900, PF3D7_1240100, PF3D7_1301700 and PF3D7_1334700 are encoding egg white genes；According to OrthoMCL
Database analysis result shows, PF3D7_0702300, PF3D7_1001900, PF3D7_1253300 and PF3D7_1301700 tetra-
Individual gene only exists in plasmodium falciparum, is found not in other plasmodiums；20% expression high is only before choosing trophozoite simultaneously
20% exclusive gene PF3D7_1401600 is as parallel control before having gene PF3D7_0220300 and the expression high of schizont phase；
Step 2, screening and the design of LAMP primer：
Nine genes in step one are had altogether using LAMP Photographing On-line software PrimerExplorerV4 has screened 31
Set LAMP primer is used for follow-up study, specially PF3D7_0202200, PF3D7_0702300, PF3D7_1001900,
PF3D7_1002000, PF3D7_1148900, PF3D7_1240100, PF3D7_1253300, PF3D7_1301700 and PF3D7_
1334700 have chosen 5,3,2,4,2,3,5,2, and 5 sets of LAMP primers (being shown in Table 1) respectively.Choose 20% high before trophozoite simultaneously
20% exclusive gene PF3D7_1401600 is screened respectively before expressing exclusive gene PF3D7_0220300 and the expression high of schizont phase
Pair of primers (is shown in Table 1).
The exclusive gene LAMP primer of the plasmodium falciparum of table 1
Step 3, the preparation of template DNA：Extract former containing malignant malaria respectively using poba gene group DNA extraction kit
Worm, Plasmodium vivax, the whole blood DNA of Plasmodium yoelii；Bow is extracted using blood/cell/tissue genome DNA extracting reagent kit
Shape worm mouse ascites DNA, DNA masterplates are fabricated to by the DNA of extraction respectively；The extraction side of poba gene group DNA extraction kit
Method is extracted for plasmodium filter paper blood cake DNA, and the plasmodium filter paper blood cake DNA methods are：3mm × 3mm filter paper is taken with card punch,
10 ± 0.5 μ l blood samples, are placed in 1.5ml centrifuge tubes, first add 1.0ml sterilized waters immersion 2h, are then centrifuged under 16000r/min
5min, abandons supernatant liquor；Again plus 160 μ l 5%chelex-100 suspensions, 8min, vibration are denatured at 95 DEG C after 56 DEG C of heating 2h
30s, then takes and 5min is centrifuged under 16000r/min, draws the μ l of supernatant 150 ± 10, is preserved at -20 DEG C.
Step 4, primer is prepared：
LAMP primer deposits liquid storage is prepared：31 sets of LAMP primers that step 2 is obtained are made dry powder, then load dry powder
1.5ml centrifuge tubes, 1min is centrifuged under 12000r/min；
Prepare mother liquor：According to 31 sets of LAMP primer dry powder molal quantity dissolving dry powder, prepare draw in each LAMP primer respectively
The concentration of thing (FIP/BIP) mother liquor and ring primer (LPF/LPB) mother liquor is 200pmol/ μ l, outer primer (F3/B3) mother liquor
Concentration is 100pmol/ μ l, the inner primer that will be prepared (FIP/BIP) mother liquor, ring primer (LPF/LPB) mother liquor and is drawn outward
Thing (F3/B3) mother liquor room temperature places 5min, and centrifuge is put into after vibration is well mixed, is centrifuged under 12000r/min
1min；
It is prepared by LAMP primer working solution PM：Take the FIP mother liquors and each 4 μ l of BIP mother liquors and phase of the 200pmol/ μ l for preparing
The F3 mother liquors and each 1 μ l of B3 mother liquors of corresponding 100pmol/ μ l, being eventually adding 10 μ l ultra-pure waters, to be prepared into 20 μ l working solutions standby.
Step 5, the foundation of LAMP methods and sensitiveness, Evaluation on specificity：
LAMP reaction systems include 12.5 μ l 2 × RM reaction solutions, LAMP primer working solution PM, 1 μ that 1 μ l step 4 is obtained
The DNA profiling that l step 3 is obtained, 1 μ l Bst DNA Polymerase, plus ultra-pure water is to 25 μ l, and 12.5 are added after being well mixed
μ l sealing fluids are centrifuged again, finally drop in the middle of PCR pipe lid 1 μ l nitrite ions, gently cover tightly lid.65 DEG C in constant-temperature metal bath
Reaction 60min, PCR pipe is overturned for several times after terminating reaction, nitrite ion is fully mixed with reaction mixture, is observed after of short duration centrifugation
Reaction solution color change, is positive (having nucleic acid amplification) if green is presented, and is negative (free nucleic acid expansion if brownish red is presented
Increase), the data for obtaining are shown in Table two；
The exclusive gene LAMP primer sensitivity assessment result of the plasmodium falciparum of table two
Remarks：P, W and N are respectively positive, weakly positive and feminine gender.1 to 0.00001 is plasmodium blood in 10 times of doubling dilutions
Concentration after being diluted with pure water after the plasmodium falciparum DNA extractions of 50000 protozoons of disease/μ l.
By table two, we are it can be found that gene PF3D7_0112300 (18S rRNA) and PF3D7_0220300 (are nourished
Body phase gene), PF3D7_1401600 (schizont phase gene) respectively as positive and parallel control, in original concentration
(50000parasite/ μ l), PF3D7_0112300, PF3D7_0220300 and PF3D7_1401600LAMP testing result difference
It is positive, weakly positive and feminine gender (Fig. 1).
P, B, N are respectively positive, the negative and blank that kit is carried in Fig. 1；C, T, S represent PF3D7_ respectively
0112300, PF3D7_0220300 and PF3D7_1401600LAMP, tri- genes.
LAMP testing results for nine exclusive genes show, have and are from 8 the 23 of gene sets of LAMP testing results
The positive, 4 the 5 of gene sets of primer detection results are weakly positive, and 2 the 3 of gene sets of primer detection results are feminine gender.Specially come
The result detected from the LAMP primer of PF3D7_1148900 is feminine gender；From PF3D7_0702300, PF3D7_1001900,
There is a set of primer positive respectively with PF3D7_1301700 genes LAMP detection displays；PF3D7_1240100 has three sets of LAMP primers
Detection display is positive；Tri- genes of PF3D7_1002000, PF3D7_0202200, and PF3D7_1334700 have four sets to draw respectively
Thing display is positive；Particularly five sets of LAMP testing results of PF3D7_1253300 are the positive.
Then, the sensitiveness of exclusive gene is evaluated using the DNA of doubling dilution.In control, PF3D7_
0112300 10-1,10-2Two gradient displays are positive, 10-3~10-5Three gradient displays are negative；PF3D7_0220300 and
PF3D7_1401600 is 10-1To 10-5Five gradients show negative (table 2).In exclusive genetic test, 10-1Have during dilution
It is positive from 6 the 13 of gene sets of LAMP primers displays, from 8 the 11 of gene sets of LAMP primers display weakly positives, from 5
7 sets of LAMP primers display of gene is negative；10-2Have positive from 6 the 8 of gene sets of LAMP primers displays during dilution, from 5
10 sets of LAMP primers display weakly positive of gene, it is negative from 7 the 13 of gene sets of LAMP primers displays；10-3Have during dilution and
It is negative from 8 the 20 of gene sets of LAMP primers displays from 7 the 11 of gene sets of LAMP primers display weakly positives；10-4With 10-5
Have during dilution from 3 the 5 of gene sets of LAMP primers display weakly positives, remaining is negative (Fig. 2, table 2).
In Evaluation on specificity, coming from 5 the 6 of gene sets of primers has carried out LAMP detections, recycle plasmodium falciparum,
Plasmodium vivax, Plasmodium yoelii and toxoplasma gondii DNA carry out showing PF3D7_0202200-3, PF3D7_ when LAMP is detected
1334700-1 and PF3D7_1334700-2 are feminine gender, in the absence of cross reaction；PF3D7_0702300-3,PF3D7_
1001900-2 and PF3D7_1002000-1 shows weakly positive when Plasmodium yoelii and toxoplasma gondii is detected, exists certain
Cross reaction.Used as control, PF3D7_0112300 (18S rRNA) shows strong positive when Plasmodium vivax is detected, in detection
Weakly positive is shown when Plasmodium yoelii and toxoplasma gondii, Fig. 3 is seen in the presence of certain cross reaction.
The clinical evaluation of exclusive gene LAMP detections
In clinical detection evaluation, 2 the 3 of gene sets of primers PF3D7_0202200-3, PF3D7_1334700-1 are selected
LAMP detections have been carried out with PF3D7_1334700-2.30 parts of the clinical sample of different parasitemias is randomly selected, has been divided into minuent
Parasitemia (≤5000parasite/ μ l), moderate parasitemia (5000-50000parasite/ μ l) and severe parasitemia
Three groups, every group 10 parts of (>=50000parasite/ μ l).In three groups of samples, the positive rate difference of PF3D7_0112300
It is 60%, 70% and 100%.Testing result shows that 2 the 3 of gene sets of primers can effectively detect that 30 points of clinical samples are shown in figure
4A.Control group PF3D7_0112300 and detection group PF3D7_0202200-3, PF3D7_1334700-1 and PF3D7_1334700-
No difference of science of statistics (χ 2=1.847, P=0.397) between 2.
In low parasitemia group, PF3D7_0202200-3, PF3D7_1334700-1 and PF3D7_1334700-2 sun
Property recall rate is respectively 10%, 30% and 20%；See Fig. 4 B.In moderate parasitemia group, PF3D7_0202200-3,
PF3D7_1334700-1 and PF3D7_1334700-2 positive rates are respectively 70%, 100% and 70%；See Fig. 4 C.In weight
In degree parasitemia group, PF3D7_0202200-3, PF3D7_1334700-1 and PF3D7_1334700-2 positive rate are
90%；See Fig. 4 D.In, in severe parasitemia group, its detection sensitivity and PF3D7_0112300 no difference of science of statistics (P=
0.096th, P=0.287, P=1.0).
Plasmodium falciparum clinical sample parasitemia
Low parasitemia (≤5000parasite/ μ l), moderate parasitemia (5000- are divided into based on parasitemia
50000parasite/ μ l) and three groups, every group 10 parts of severe parasitemia (>=50000parasite/ μ l)；Then according to above-mentioned
Method is detected.More accurate data can be obtained.
The merely exemplary explanation principle of the invention of above-described embodiment and its effect, not for the limitation present invention.It is any ripe
The personage for knowing this technology all can carry out modifications and changes under without prejudice to spirit and scope of the invention to above-described embodiment.Cause
This, all those of ordinary skill in the art are completed under without departing from disclosed spiritual and technological thought
All equivalent modifications or change, should be covered by claim of the invention.
Claims (10)
1. the ring mediated isothermal amplification method of the exclusive gene of plasmodium falciparum ring bodies is based on, it is characterised in that comprised the following steps：
Step one, bioinformatic analysis：Search PlasmoDB databases, screen only in plasmodium falciparum erythrocyte stage ring-type
(preceding 20%) and this kind of peculiar gene of plasmodium, verify it in eucaryote to the expression high of body phase using OrthoMCL database
Distribution situation between different genera, meanwhile, screen only (preceding in Erythrocytic Stages of Plasmodivm Falciparum trophozoite or schizont phase expression high
20%) and its each one of peculiar gene, as parallel control；
Step 2, LAMP primer design：Step one is screened using LAMP Photographing On-line softwares PrimerExplorerV4
Peculiar gene carry out LAMP primer design, parameter is set to default value, selection fraction ranking the first two to the LAMP primer of first five,
Then LAMP primer synthesis is carried out；
It is prepared by step 3, masterplate DNA：Extracted respectively using poba gene group DNA extraction kit and contain plasmodium falciparum, every other day
Plasmodium, the Whole Blood Genomic DNA of Plasmodium yoelii；Sense is extracted using blood/cell/tissue genome DNA extracting reagent kit
Contaminate the mouse ascites DNA of Infection of Toxoplasma Gondii；The template DNA that the genomic DNA that mentioned reagent box is extracted as LAMP is used；
Step 4, primer is prepared：
LAMP primer deposits liquid storage is prepared：The LAMP primer dry powder for being placed in 1.5ml centrifuge tubes that step 2 is obtained is in 12000r/
1min is centrifuged under min, so that the powder for being bonded at lid and tube wall all concentrates on ttom of pipe.
Prepare mother liquor：Dry powder is dissolved in 1.5ml centrifuge tubes according to primer dry powder molal quantity, inner primer (FIP/BIP) mother liquor is prepared
Concentration be 200pmol/ μ l, the concentration of outer primer (F3/B3) mother liquor is 100pmol/ μ l, the inner primer (FIP/ that will be prepared
BIP) mother liquor and outer primer (F3/B3) mother liquor room temperature place 5min, centrifuge are put into after vibration is well mixed, in 12000r/min
Lower centrifugation 1min；
It is prepared by LAMP primer working solution PM：Take each 4 μ l of FIP mother liquors and BIP mother liquors, the F3 mothers of 100pmol/ μ l of 200pmol/ μ l
Liquid and each 1 μ l of B3 mother liquors, being eventually adding 10 μ l ultra-pure waters, to be prepared into 20 μ l working solutions standby；
Step 5, the foundation of LAMP methods and sensitiveness, Evaluation on specificity：
A.LAMP reaction systems include 12.5 μ l 2 × RM reaction solutions, LAMP primer working solution PM, 1 μ l that 1 μ l step 4 is obtained
With the DNA profiling of corresponding gene, 1 μ l Bst DNA Polymerase, plus ultra-pure water is to 25 μ l, is added after being well mixed
12.5 μ l sealing fluids are centrifuged again, finally drop in the middle of PCR pipe lid 1 μ l nitrite ions, lid are gently covered tightly, in constant-temperature metal bath
Reaction, overturns PCR pipe for several times or 1000~16000r/min centrifugation 30sec~3min after terminating reaction, make nitrite ion with reaction
Mixed liquor is fully mixed, observing response liquid color change after of short duration centrifugation, is positive (having nucleic acid amplification) if green is presented, if
It is then negative (free nucleic acid amplification) that brownish red is presented；
B. the whole blood DNA containing P. falciparum genes group DNA that step 3 is obtained is carried out into doubling dilution with ultra-pure water, then
Detected according to the LAMP decoration methods described in A in step 5, evaluation detects the sensitiveness of gene；
C. the whole blood DNA containing P. falciparum genes group DNA that step 3 is obtained, Plasmodium vivax genomic DNA is contained
Whole blood DNA, the whole blood DNA containing Plasmodium yoelii genomic DNA, the ascites DNA containing toxoplasma cdna group DNA；According to
LAMP decoration methods detection in step 5 described in A, the LAMP primer using 18S rRNA designs carries out spy as positive control primers
The opposite sex is evaluated, while setting up blank and negative control.
2. the ring mediated isothermal amplification method based on the exclusive gene of plasmodium falciparum ring bodies according to claim 1, it is special
Levy and be：The peculiar gene of plasmodium falciparum is PF3D7_1253300, PF3D7_0202200, PF3D7_ in the step one
0702300,PF3D7_1001900,PF3D7_1002000,PF3D7_1148900,PF3D7_1240100,PF3D7_1301700
With PF3D7_1334700.
3. the ring mediated isothermal amplification method based on the exclusive gene of plasmodium falciparum ring bodies according to claim 2, it is special
Levy and be：(the preceding peculiar gene of 20%) is PF3D7_ to the expression high of Erythrocytic Stages of Plasmodivm Falciparum trophozoite stage in the step one
0220300。
4. the ring mediated isothermal amplification method based on the exclusive gene of plasmodium falciparum ring bodies according to claim 3, it is special
Levy and be：(the preceding peculiar gene of 20%) is PF3D7_ to the expression high of Erythrocytic Stages of Plasmodivm Falciparum schizont phase in the step one
1401600。
5. the ring mediated isothermal amplification method based on the exclusive gene of plasmodium falciparum ring bodies according to claim 4, it is special
Levy and be：The extracting method of blood/cell/tissue genome DNA extracting reagent kit is plasmodium filter paper blood in the step 3
Spot DNA is extracted, and the plasmodium filter paper blood cake DNA methods are：A little filter paper is taken with card punch, the filter paper contains blood sample, is placed in
1.5ml centrifuge tubes, first add 1.0ml sterilized waters immersion 2h, and 5min then is centrifuged under 16000r/min, abandon supernatant liquor；Again
Plus 160 μ l 5%chelex-100 suspensions, 5~10min are denatured at 95~98 DEG C after 56~65 DEG C of 1~2h of heating, vibrate 30s
~3min, then takes 3~5min of centrifugation under 12000~16000r/min, draws the μ l of supernatant 150 ± 10, is protected at -20 DEG C
Deposit.
6. the ring mediated isothermal amplification method based on the exclusive gene of plasmodium falciparum ring bodies according to claim 5, it is special
Levy and be：The card punch takes 3mm*3mm filter paper, and the filter paper contains 10 ± 0.5 μ l blood samples.
7. the ring mediated isothermal amplification method based on the exclusive gene of plasmodium falciparum ring bodies according to claim 6, it is special
Levy and be：The parasitemia densities of the whole blood containing plasmodium falciparum are 50000/μ l in the step 3.
8. the ring mediated isothermal amplification method based on the exclusive gene of plasmodium falciparum ring bodies according to claim 7, it is special
Levy and be：Temperature in the step 5 in constant-temperature metal bath is 60~65 DEG C.
9. the ring mediated isothermal amplification method based on the exclusive gene of plasmodium falciparum ring bodies according to claim 8, it is special
Levy and be：Reaction time in the step 5 in constant-temperature metal bath is 30~90min.
10. the ring mediated isothermal amplification method based on the exclusive gene of plasmodium falciparum ring bodies according to claim 9, it is special
Levy and be：Plasmodium falciparum DNA ultra-pure waters are according to 10 in the step 5-1、10-2、10-3、10-4With 10-5Doubling dilution.
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