CN105483278A - Histoplasma capsulatum infectious molecular diagnosis reagent kit based on recombinase and polymerase amplification technological principle and application thereof - Google Patents
Histoplasma capsulatum infectious molecular diagnosis reagent kit based on recombinase and polymerase amplification technological principle and application thereof Download PDFInfo
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Abstract
The invention relates to a histoplasma capsulatum infectious molecular diagnosis reagent kit based on the recombinase and polymerase amplification technological principle and application thereof. A primer is designed for an M antigen gene of an rDNA area of a histoplasma capsulatum capsule variation distributed according to the most common advantages of histoplasma capsulatum in clinical in China, the primer reacts with DNA of 30 histoplasma capsulatum standard strains, clinic and environment separated strains and 50 strains in relevant species according to an RPA system, and it is proved that the primer has excellent specificity. It is also proved that the primer has very high sensitivity through sensitivity testing. Accordingly, the primer can be used for early clinical diagnosis of histoplasma capsulatum infection, the problems that in clinical, histoplasma capsulatum infection confirmed diagnosis period is long and requirements for detection staff skills and laboratory platform conditions are high are solved, the primer can be prepared into the reagent kit, and convenience is provided for early diagnosis and timely treatment of clinical histoplasma capsulatum infection.
Description
Technical field
The present invention relates to field of molecular detection, specifically, relate to a kind of based on recombinase polymeric enzymatic amplification know-why, can early diagnosis histoplasma capsulatum infect molecule diagnosis kit and application.
Background technology
Histoplasma is that a diphasic fungi being common in birds and bat ight soil belongs to, and is Hyphal form in its natural state, and is converted into Yeast Phase in animal or human's body.Histoplasma comprises three species, i.e. Histoplasma capsulatum (Histoplasmacapsulatum), Histoplasma capsulatum Du Shi mutation (Histoplasmavar.duboisii) and farcy Histoplasma capsulatum (Histoplasmacapsulatumvar.farciminosum).Wherein, farcy Histoplasma capsulatum main infection animal, causes lymphangitis epizootic in the animals such as horse; And histoplasmosis (Histoplasmosis, HP) be caused by biphasic or bipolar type histoplasma capsulatum be distributed widely in the whole world mycosis, it mainly divides for the american type HP caused by Histoplasma capsulatum and the African type HP caused by Histoplasma capsulatum Du Shi mutation.
The mankind often infect because sucking by the fungal spore in the earth of birds or bat fecal pollution or dust.Rate of clinical misdiagnosis, mortality ratio are up to 80%.U.s.a. military affairs pathologist Taylor Sai Miaoer in 1906 reaches woods and reports the first, and research at that time shows the most generally Ohio and river valley, Mississippi.Research in the last hundred years thinks that this disease is that a kind of endemic conditions is sick mostly, diverse clinical manifestations, totally be divided into asymptomatic, acute pulmonary type, disseminated and chronic pulmonary type, Major Epidemic in America, Africa, the area such as Asia, Europe is relatively rare, therefore previously this disease does not cause enough attention in China, but relevant report is in rising trend over nearly 20 years, the epidemiology surveys such as such as osmanthus Skien show the crowd that China exists histoplasma capsulatum's infection, and have areal variation, region of Southeast infection rate is higher than the northwestward; Pulmonary Disease patients's infection rate, higher than normal people, is height with lunger especially.It should be noted that, due to Histoplasma capsulatum, morphologically mould Buddhist nun's Penicillium notatum cannot reliably be distinguished with Ma Er, clarifies a diagnosis and needs to cultivate and serum blood test, waste time and energy, high to laboratory qualification requirement, therefore the diagnostics state of the art of this disease far can not meet clinical practice needs.
And, HP infect prognosis with can clarify a diagnosis in early days closely related, therefore how promoting the research of HP diagnosis, develop quick, the special and diagnostic techniques method of easy handling as early as possible, is that current clinical HP infects technique for detection and studies major issue in the urgent need to address.At present, domestic and international clinical fungi laboratory serological test once used the combination of latex agglutination, immunodiffusion(ID), complement, immunofluorescence and radioimmunoassay antibody, but morbidity early diagnostic rate is low.Histoplasmin skin test may contribute to the diagnosis of chronic patients, but reaction does not often appear in immune deficiency person, and therefore tuerculoderma is only mainly used in epidemiological survey.Recently also often detect histoplasma capsulatum's polysaccharide antigen of Urine in Patients, serum, hydrothorax or cerebrospinal fluid abroad, wherein urine positive rate is the highest, and points out Active infection, can make early diagnosis foundation.Though have the limitations such as length consuming time with the pathogenic fungi Morphologic Diagnosis method of cultivating, pathologic finding is representative, but still be the gold standard of studies of invasive fungal infections diagnosis clinically, histoplasmosis is no exception.Due to its thalline size and form similar to Leishmania amastigotes, the two is often confused, and the latter exists tiny shaft-like the kinetoplast of color depth, and the former akinetoplastic, this is key form being distinguished two kinds of pathogenic agent, but but requires quite high to pathology blood slide examiners.The most reliable etiological diagnosis is fungus culture or animal inoculation pvaccination, and marrow, pathological tissues puncture thing, blood, phlegm all can be cultivated, but there is the defects such as higher, the consuming time length of false negative rate (incubation time at least 2 week).In recent years, non-cultivation diagnostic techniques becomes the focus of domestic and international invasive fungi disease early diagnosis technical study gradually, and its technological line is mainly based on the serological method of immunology principle with take nucleic acid detection technique as the molecular biology method of representative.In general, this two large class technological method respectively has superiority, although part serological technique method through improvement relative maturity, susceptibility and specificity still can, the build-in attribute based on immunology principle also causes evading the problem such as false positive, false negative; Though and take nucleic acid detection technique as the Protocols in Molecular Biology method not yet fully matured of representative, there is the technical superiority of the drawback making up current serological method well, there is higher development potentiality and clinical value.
HP early diagnosis technology based on detection of nucleic acids principle mainly comprises DNA bar code graphical spectrum technology and DNA sequence analysis etc.Theoretically, DNA sequence analysis is the gold standard of appraisement organization endochylema bacterium.But round pcr needs PCR instrument device, to Infrastructure and personnel requirement higher, often because strict subregion, experimenter can not lacking experience and add highly sensitive, easily pollute and make result present false negative, false positive etc. in domestic laboratory.Recombinase polymeric enzymatic amplification (RecombinasePolymeraseAmplification, RPA), is known as and can substitutes the nucleic acid detection technique of PCR, is developed by UK corporation TwistDxInc.RPA technology depends on three kinds of enzymes: can in conjunction with the recombinase of single-chain nucleic acid (Oligonucleolide primers), single-stranded DNA binding protein (SSB) and strand displacement archaeal dna polymerase.The mixture of these three kinds of enzymes also has activity at normal temperatures, and optimal reaction temperature is at about 37 DEG C.Based on this
the monomolecular nucleic acid that nucleic acid amplification products can be carried out under normal temperature in 15 minutes detects.This technology is very low to the requirement of hardware device, is particularly suitable for the fields such as in-vitro diagnosis, animal doctor, food safety, Biosafety, agricultural.Current RPA technology is widely used in the detection of the common pathogens such as clostridium difficile, tubercule bacillus, methicillin-resistant staphylococcus aureus MRSA, gonococcus (Neisseriagonorrhoeae), Salmonellas, escherichia coli STEC.
In sum, based on the sequential analysis for each DNA fragmentation of histoplasma capsulatum's strain gene group, filter out suitable target gene, research and develop the technological method that a kind of histoplasma capsulatum based on RPA know-why infects Rapid&Early diagnosis, make a definite diagnosis time cycle length for solving histoplasma capsulatum's infection clinically, the problem tools such as testing staff's technology and lab platform conditional request height are of great significance, will for early diagnosis and timely treatment provide facility clinically.
Summary of the invention
The object of the invention is for deficiency of the prior art, provide a kind of can early diagnosis histoplasma capsulatum infect primer pair.
Of the present invention again one object be that the purposes of described primer pair is provided.
Another object of the present invention is, provide a kind of can the test kit that infects of early diagnosis histoplasma capsulatum.
For realizing above-mentioned first object, the technical scheme that the present invention takes is:
Can early diagnosis histoplasma capsulatum infect a primer pair, two primers that described primer pair is respectively SEQNO.1 and SEQNO.2 by nucleotide sequence form.
For realizing above-mentioned second object, the technical scheme that the present invention takes is:
The purposes of primer pair as above in the reagent of preparation early diagnosis histoplasma capsulatum infection.
Primer pair as above detects the purposes in the reagent that in sample, histoplasma capsulatum exists in preparation.
Primer pair as above is preparing the purposes in reagent, and described reagent is used for from posadasis spheriforme (Coccidioidesimmitis), Sporothrix schenckii (Sporothrixschenckii), penicillium Marneffei (Penicilliummarneffei), capsule Penicillium notatum (Penicilliumcapsulatum), Aspergillus fumigatus (Aspergillusfumigatus), Niger's aspergillus niger (Aspergillusniger), aspergillus versicolor (Aspergillusversicolor), Apophysomyces elegans (Apophysomyceselegans), arthroderma benhamiae (Arthrodermabenhamiae), Blastomyces dermatitidis (Blastomycesdermatitidis), Paracoccidioides brasiliensis (Coccidioidesposadasii), chrysosporium tropicum (Chrysosporiumtropicum), chrysosporium keratinophilum (Chrysosporiumkeratinophilum), saw shape actinomycetes (Ctenomycesserratus), acrothesium floccosum (Epidermophytonfloccosum), pasteur gold pityrosporion ovale (Emmonsiapasteurian), emmonsia parva (Emmonsiaparva), Fusarium oxysporum (Fusariumoxysporum), Fusarinm solani (Fusariumsolani), in western naked capsule bacterium (Gymnoascusreessii), Sabouraudites lanosus (Microsporumcanis), microsporon gypseum (Microsporumgypseum), strephopodia sporidiole bacteria (Microsporumequinum), volume branch Mucor (Mucorcircinelloides), Mucor indicus (Mucorindicus), thermophilic fungus destroyed wire (Myceliophthorathermophila), Ah Sa's trichosporon (Trichosporonasahii), rind gall trichosporon (Trichosporoninkin), trichophyton (Trichophytonrubrum), Rhizopus oryzae (Rhizopusoryzae), Malassezia furfur (Malasseziafurfur), Candida albicans (Candidaalbicans), Candida glabrata (Candidaglabrata), candida tropicalis (Candidatropicalis), Candida catenulata bacterium (Candidacatenulata), candida krusei (Candidakrusei), Cryptococcus neoformans (Cryptococcusneoformans), histoplasma capsulatum is identified in the special cryptococcus (CryptococcusGattii) of lattice.
For realizing above-mentioned 3rd object, the technical scheme that the present invention takes is:
Can early diagnosis histoplasma capsulatum infect a test kit, described test kit comprises primer pair as above:
Described test kit also comprises further
rPARehydrationBuffer, magnesiumacetate.
The invention has the advantages that:
The present invention is directed to the M antigen gene (Genbank:AF026268) in the rDNA region of the mutation histoplasma capsulatum pod membrane mutation of the clinical modal advantage distribution of China of histoplasma capsulatum, devise primer.Based on histoplasma capsulatum's phylogenetic systematics correlative study in early stage, to 30 strain histoplasma capsulatum type strains with clinically to verify with the DNA of environment separation bacterial strain and 50 strain related species bacterial strains, result is presented in 30 minute reaction times, all histoplasma capsulatum DNA can be detected, and not close with 38 kinds of other phylogenetic systematics relations kind bacterial strain generation cross reaction, indicate primer of the present invention and possess excellent specificity.The present invention also tests the sensitivity of this primer, and result shows that this primer possesses very high sensitivity, reaches the excellent level of PRA reaction sensitivity.Therefore, primer of the present invention can be used for the clinical early diagnosis that histoplasma capsulatum infects, and possess quick, easy, strong operability, result interpretation advantage easily, solve histoplasma capsulatum's infection clinically and make a definite diagnosis time cycle length, to problems such as testing staff's technology and lab platform conditional request height.This primer can be prepared into test kit, for the early diagnosis that histoplasma capsulatum infects clinically provides convenient with treatment in time.
Accompanying drawing explanation
Accompanying drawing 1 is RPA technical schematic diagram.Recombinase is combined the Protein-DNA mixtures formed with primer, scanning double-stranded DNA finds homologous sequence.Once primer located homologous sequence, Exchange reaction of chain will be there is and formed and start DNA synthesis, exponential amplification is carried out to the target area in template.The DNA chain be replaced and single-stranded DNA binding protein (SSB) combine, and prevent further replacement.In this individual system, by two relative initial compound events of primer.
Accompanying drawing 2 is pair of primers different strains DNARPA amplification rear electrophoresis figure.M represents Marker from left to right, and 1 ~ 6 is Histoplasma: CBS114.388, CBS215.53, CBS136.72, CBS633.91, CBS205.35, CBS537.84; 7 ~ 16 is close genus kind: CBS144.34, ATCC10268, ATCC201013, CBS134186, CBS113.61, ATCC64704, CBS113838, CBS130793, CBS221.64, CBS204.48, ATCC38033, ATCC36031.
Accompanying drawing 3 is second couple of primer different strains DNARPA amplification rear electrophoresis figure.
Accompanying drawing 4 is different concns HcDNARPA amplification rear electrophoresis figure.
Embodiment
Below in conjunction with accompanying drawing, embodiment provided by the invention is elaborated.
The screening of the primer that embodiment 1 infects for early diagnosis histoplasma capsulatum and specificity thereof and susceptibility checking
One, major experimental material and equipment
1 bacterial strain
26 strain histoplasma capsulatum reference cultures, 4 strains are clinical in environment separation bacterial strain and 50 strain related species bacterial strains.Histoplasma capsulatum's reference culture is purchased from the international fungal diversity research centre of Dutch CBS, is clinically pod membrane mutation with environment separation bacterial strain.
2 equipment
Bechtop: CA-1390-1 vertical laminar flow clean bench, the clean treating plant company limited in upper sea; Biohazard Safety Equipment: NUAIREBiologicalSafefyCabinetClass II; Water isolation type electro-heating standing-temperature cultivator: HeraeusB5060EKCO
2; Water purification machine: HITECHwaterpurificationsystem; Micropipette rifle: 0.5 ~ 10 μ l (FINNpipette), 20 ~ 200 μ l (EppendorfResearch), 1000 μ l (EppendorfResearch); Autoclave sterilizer: SANYOAutoCLAVEMLS-3020; Colibri: Shanghai Da Mai biotechnology company; Supercentrifuge: Eppendorfcentrifuge5810R, EppendorfNorthAmerica, Inc.; Ice-making machine: ScotsmanAF100; Constant water bath box: Shanghai precision instrument; Electrophoresis apparatus: TANETS300, domestic; Labworks image acquisition and analysis software: FR-980 biology electrophoresis of multiple day image analysis system; EppendorfBiophotometer spectrophotometer.
3 reagent and substratum
Basic medium is Sabouraud substratum (SDA), corn culture medium (CMA), brain heart leaching extraction blood meida (BHIB): OXOIDUK, Mei Liai biphasic culture (MLI): BioM é rieuxFrance.
M antigen gene (Genbank:AF026268) primer: synthesize by Sheng Gong Shanghai bio tech ltd.
DNAMarker (DL2000): upper Hypon biotechnology company limited (TAKARA).
basickitRPA detection kit: TwistDx company of Britain.
Analytical pure Benzyl Chloride: Sheng Gong Shanghai bio tech ltd.
Fungal DNA extract: 1mL1MTris.Hcl, 4mL500mMEDTA, adds ddH
2o is settled to 10mL.
AR level 3MNaAc, 20%SDS solution, aqueous isopropanol, 70% ethanolic soln.
4 other consumptive materials
12CM culture dish: Zhuo Kang bio tech ltd, Shanghai; Micropipette tip: Zhuo Kang bio tech ltd, Shanghai; EP manages (1.5ml, 5ml): Zhuo Kang bio tech ltd, Shanghai.
Two, experimental technique
(1) target sequence is determined and design of primers
1 target sequence M antigen gene (Genbank:AF026268)
The corresponding sequence of the histoplasma capsulatum's pod membrane mutation be confirmed is obtained from (http://www.ncbi.nlm.nih.gov/nuccore/) U.S. NCBI nucleic acid database.
2 design of primers
Design and have selected two pairs of primers, sequence is as follows:
First pair
ForwardPrimer1：5’-TTGATCCCCAACGCGGCGTGAACATGACCTATT-3’(SEQIDNO.1)；
ReversePrimer：5’-TCACCGACGGCACCAACGGGCTTTCCATA-3’(SEQIDNO.2)；
Second pair
ForwardPrimer2：5’-TATTCCGGCGCCGACGGCTCG”ATCTTCG-3’(SEQIDNO.3)；
ReversePrimer：5’-TCACCGACGGCACCAACGGGCTTTCCATA-3’(SEQIDNO.2)。
Above primer and target sequence tie be merged into ring structural points as Fig. 1 (for pair of primers).
3 primer screenings
By the primer designed respectively with template DNA (histoplasma capsulatum) according to RPA reaction system application of sample, be placed in thermostat water bath and react, default temperature of reaction is 38 DEG C, and the reaction times is 30min.
RPA reaction system is as follows: cumulative volume 50 μ L, comprises 2.4 μ LPrimerF (10 μm of ol/L), 2.4 μ LPrimerR (10 μm of ol/L), 29.5 μ LRehydrationBuffer, 2 μ LDNA templates, 11.2 μ LddH
2o and 2.5 μ l280mMmagnesiumacetate.
Reaction terminates rear observation and often overlaps primer reaction amplification curve, and carries out specificity and sensitivity experiments to each cover primer.
(2) bacterial strain complete genome DNA to be verified extracting
The recovery of 1 bacterial strain and cultivation
Take out the bacterial strain of-20 DEG C of preservations, recover 24 hours at being placed in 20 DEG C, make it recover breeding vigor, turn bacterium to prepared media multiplication culture.Get bacterial strain specimen inoculation in MLI, 25 DEG C of cultivations, obtain transferred species SDA, each 2 parts of CMA, BHIB after single bacterium colony, respectively at 25 DEG C and 35 DEG C of cultivations.HC is biphasic or bipolar type deep fungal, and Time in Vitro is longer, usually needs about 2 weeks.Yeast Phase is slower than mould-growth, and high to the nutritional requirement of substratum.
The each bacterial strain complete genome DNA of 2 Benzyl chloride method extracting
1. 500 μ L fungal DNA extracts are added in EP pipe, the large small volume bacterium colony of picking match end from SDA substratum, rear concuss 1min;
2. in EP pipe, add 50 μ L20%SDS and 300 μ L Benzyl Chloride solution, concuss is emulsus to solution, EP pipe is placed in 50 DEG C of water-baths 1 hour, concussion mixing in every 10 minutes 1 time;
3. water-bath terminates, and adds 300 μ L3MNaAc (sodium-acetate) ice-water bath 15 minutes;
4. the EP pipe after ice-water bath is placed in supercentrifuge, select 6000rpm rotating speed, after 15min, careful sucking-off supernatant is also transferred in clean EP pipe;
5. in the supernatant migrated out, add the Virahol of equal-volume precooling, precipitate 20min, recentrifuge under room temperature, rotating speed is 10000rpm, 15min;
6. take out EP pipe, carefully abandon supernatant, and in precipitation, add 70% ethanol of equal-volume precooling, 10000rpm, 10min are centrifugal again;
7., after centrifugal end, supernatant is abandoned, air-dry under carefully precipitation being placed in room temperature, and dissolve with 50 μ LTE.Above-mentioned DNA is placed in-20 DEG C of refrigerator cold-storages to preserve.
3 spectrophotometer detect each strain gene group DNA and concentration thereof
Utilize EppendorfBiophotometer spectrophotometer, by the concentration of operational manual operation detection genomic dna.
(3) specificity experiments
1 amplified reaction
By primer and the off-the-shelf each bacterial strain DNA of above-mentioned steps of design according to RPA system, 38 DEG C, 30min reacts, observation amplified reaction situation.Set up histoplasma capsulatum as positive control, distilled water group is as negative control.
2RPA gel electrophoresis checks
1. 1.5% sepharose of 12cm × 6cm is recorded with the encapsulating die that HE120 electrophoresis apparatus provides: in beaker, add 1.5g agarose, 1MTAE, fully mix after being settled to 100mL, heated and boiled, immediately glue is added in encapsulating plate, avoid producing a large amount of bubble, room temperature is placed to gel sets;
2. in the electrophoresis chamber that 1 × TAE damping fluid (40mMTris-Aceticacid, pH8.0,1mMEDTA) is housed, carefully keep flat into agar gel, make its submergence;
3. extract 5 μ L in each RPA reaction system, after fully mixing with 1 μ L5xLadderBuffer respectively, be splined on the agar gel of 1.5%;
4. 120 volts of electrophoresis 30min;
5. agar gel is immersed EB solution 15min;
6. 10min in distilled water is immersed after careful taking-up;
7. take out agar gel to take pictures through FR-980 biology electrophoresis of multiple day image analysis system.
(4) sensitivity experiments
1 amplification curve
By histoplasma capsulatum's complete genome DNA concentration ten times of step dilutions several respectively, DNA content is made to be respectively 53ng, 5.3ng, 530pg, 53pg, 5.3pg, 530fg, 53fg, ddH
2o.By the DNA of above-mentioned different concns gradient respectively according to above-mentioned RPA system, 38 DEG C, 30min reacts, and terminates rear observation amplification situation.Set up and do not contain DNA group for negative control.
The electrophoretic examinations of 2RPA product gel
1. 1.5% sepharose of 12cm × 6cm is recorded with the encapsulating die that HE120 electrophoresis apparatus provides: in beaker, add 1.5g agarose, 1MTAE, fully mix after being settled to 100mL, heated and boiled, immediately glue is added in encapsulating plate, avoid producing a large amount of bubble, room temperature is placed to gel sets;
2. in the electrophoresis chamber that 1 × TAE damping fluid (40mMTris-Aceticacid, pH8.0,1mMEDTA) is housed, carefully keep flat into agar gel, make its submergence;
3. extract 5 μ L in each RPA reaction system, after fully mixing with 1 μ L5xLadderBuffer respectively, be splined on the agar gel of 1.5%;
4. 120 volts of electrophoresis 30min;
5. agar gel is immersed EB solution 15min;
6. 10min in distilled water is immersed after careful taking-up;
7. take out agar gel to take pictures through FR-980 biology electrophoresis of multiple day image analysis system.
Three, experimental result
(1) primer checking and screening
Between 250 ~ 500bp, occur assorted band (Fig. 3) according to second pair of reacted gel electrophoresis figure of primer in two pairs of primers that M antigen sequence is designed, specificity is not high.And pair of primers reacted gel electrophoresis figure display band is clear, without assorted band (Fig. 2), is extremely convenient to the judgement of result, interference can not be produced, the accuracy of result can be significantly improved further.
(2) specificity experiments
The pair of primers filtered out reacts according to RPA system with each bacterial strain DNA by 1 respectively, observes point other amplified reaction situation.Shown in table 1 specific as follows.
Table 1 specificity experiments result
Note: * P (Positive) represents positive reaction, N (Negative) represents negative reaction.
2 for the primer designed, and amplified production Gel electrophoresis results as shown in Figure 2.It can thus be appreciated that: the primer based on the design of histoplasma capsulatum M antigen sequence has good specificity in RPA reaction system, except histoplasmosis bacteria strain all occurs positive findings, other bacterial strains comprise posadasis spheriforme (Coccidioidesimmitis), Sporothrix schenckii (Sporothrixschenckii), penicillium Marneffei (Penicilliummarneffei), capsule Penicillium notatum (Penicilliumcapsulatum), Aspergillus fumigatus (Aspergillusfumigatus), Niger's aspergillus niger (Aspergillusniger), aspergillus versicolor (Aspergillusversicolor), Apophysomyces elegans (Apophysomyceselegans), arthroderma benhamiae (Arthrodermabenhamiae), Blastomyces dermatitidis (Blastomycesdermatitidis), Paracoccidioides brasiliensis (Coccidioidesposadasii), chrysosporium tropicum (Chrysosporiumtropicum), chrysosporium keratinophilum (Chrysosporiumkeratinophilum), saw shape actinomycetes (Ctenomycesserratus), acrothesium floccosum (Epidermophytonfloccosum), pasteur gold pityrosporion ovale (Emmonsiapasteurian), emmonsia parva (Emmonsiaparva), Fusarium oxysporum (Fusariumoxysporum), Fusarinm solani (Fusariumsolani), in western naked capsule bacterium (Gymnoascusreessii), Sabouraudites lanosus (Microsporumcanis), microsporon gypseum (Microsporumgypseum), strephopodia sporidiole bacteria (Microsporumequinum), volume branch Mucor (Mucorcircinelloides), Mucor indicus (Mucorindicus), thermophilic fungus destroyed wire (Myceliophthorathermophila), Ah Sa's trichosporon (Trichosporonasahii), rind gall trichosporon (Trichosporoninkin), trichophyton (Trichophytonrubrum), Rhizopus oryzae (Rhizopusoryzae), Malassezia furfur (Malasseziafurfur), Candida albicans (Candidaalbicans), Candida glabrata (Candidaglabrata), candida tropicalis (Candidatropicalis), Candida catenulata bacterium (Candidacatenulata), candida krusei (Candidakrusei), Cryptococcus neoformans (Cryptococcusneoformans), all there is not amplified reaction in the special cryptococcus (CryptococcusGattii) of lattice.Preliminary identification has a good specificity based on the RPA primer of M antigen sequence design.
(3) sensitivity experiments
For the primer of design, after the DNA of different concns gradient and primer react, amplification curve as shown in Figure 4.530pg can be reached based on the RPA primer sensitivity of intergenic region M antigen sequence in histoplasma capsulatum's rrna.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the inventive method; can also make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.
Claims (6)
1. can early diagnosis histoplasma capsulatum infect a primer pair, it is characterized in that, two primers that described primer pair is respectively SEQNO.1 and SEQNO.2 by nucleotide sequence form.
2. the purposes of primer pair according to claim 1 in the reagent of preparation early diagnosis histoplasma capsulatum infection.
3. primer pair according to claim 1 detects the purposes in the reagent that in sample, histoplasma capsulatum exists in preparation.
4. primer pair according to claim 1 is preparing the purposes in reagent, it is characterized in that, described reagent is used for from posadasis spheriforme, Sporothrix schenckii, penicillium Marneffei, capsule Penicillium notatum, Aspergillus fumigatus, Niger's aspergillus niger, aspergillus versicolor, Apophysomyces elegans, arthroderma benhamiae, Blastomyces dermatitidis, Paracoccidioides brasiliensis, chrysosporium tropicum, chrysosporium keratinophilum, saw shape actinomycetes, acrothesium floccosum, pasteur gold pityrosporion ovale, emmonsia parva, Fusarium oxysporum, Fusarinm solani, in western naked capsule bacterium, Sabouraudites lanosus, microsporon gypseum, strephopodia sporidiole bacteria, volume branch Mucor, Mucor indicus, thermophilic fungus destroyed wire, Ah Sa's trichosporon, rind gall trichosporon, trichophyton, Rhizopus oryzae, Malassezia furfur, Candida albicans, Candida glabrata, candida tropicalis, Candida catenulata bacterium, candida krusei, Cryptococcus neoformans, histoplasma capsulatum is identified in the special cryptococcus of lattice.
5. can early diagnosis histoplasma capsulatum infect a test kit, described test kit comprises primer pair according to claim 1.
6. primer pair according to claim 5, described test kit also comprises further
rPARehydrationBuffer, magnesiumacetate.
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| CN114075564A (en) * | 2022-01-17 | 2022-02-22 | 广东华美众源生物科技有限公司 | Malassezia detection composition, kit and detection method thereof |
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| CN109825628A (en) * | 2019-03-28 | 2019-05-31 | 南京林业大学 | A kind of primer and probe combination and its application based on RPA- Sidestream chromatography technology detection Fusarium solani |
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| CN113755620B (en) * | 2021-08-30 | 2023-12-19 | 江苏省血液中心 | Nucleic acid detection kit for rapidly detecting two leishmanias and application thereof |
| CN114075564A (en) * | 2022-01-17 | 2022-02-22 | 广东华美众源生物科技有限公司 | Malassezia detection composition, kit and detection method thereof |
| CN114075564B (en) * | 2022-01-17 | 2022-04-22 | 广东华美众源生物科技有限公司 | Malassezia detection composition, kit and detection method thereof |
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