CN109666755A

CN109666755A - For detecting the nucleic acid reagent, kit, system and method for invasive fungi

Info

Publication number: CN109666755A
Application number: CN201811594889.5A
Authority: CN
Inventors: 林笑冬; 王雷; 王晓艳; 张志强
Original assignee: Beijing Zhuo Chenghui Biological Polytron Technologies Inc
Current assignee: Beijing Zhuo Chenghui Biological Polytron Technologies Inc
Priority date: 2018-12-25
Filing date: 2018-12-25
Publication date: 2019-04-23

Abstract

This disclosure relates to a kind of for detecting the nucleic acid reagent, kit, system and method for invasive fungi, wherein, the nucleic acid reagent includes storage or mutually probe shown in primer and SEQ ID NO.15-23 shown in the SEQ ID NO.1-10 of any mixed storage independently of one another respectively.The disclosure establishes nucleic acid reagent, kit, the system and method for detection invasive fungi by above-described primer and probe, can be realized quick, comprehensive, sensitive, special, automatic testing result and determines.

Description

For detecting the nucleic acid reagent, kit, system and method for invasive fungi

Technical field

This disclosure relates to field of biotechnology, and in particular, to a kind of nucleic acid reagent for detecting invasive fungi, examination Agent box, system and method.

Background technique

Fungi belongs to eucaryote, is widely present in nature, and majority of fungal is advantageous the mankind, minority sense The dye mankind then will lead to serious disease.According to the difference of infection site, fungal infection is divided into mycotic infection of superficial part and deep is true Bacterium infection, most invasive infections with fungi belong to deep fungal infection.In the past 30 years, with solid organ and Hematopoietic Stem Extensive development, the immune suppression of the technologies such as cell transplantation (hematopoietic stem cell transplantation, HSCT) Preparation, the extensive application of radiotherapy chemotherapy, the extensive development of intubation intervention and indwelling technology keep patient's body's immunity impaired, invade On the disease incidence and the death rate of attacking property fungal infection (invasive fungal infection, IFI) are significant in the world It rises, seriously threatens human health.Epidemiological study shows that Nosocomial fungus infection rate 7.5%, tumour patient disease incidence is 8%, Up to 16%, case fatality rate is up to 55%-70% for serious burn and wounded patient infection rate.In the U.S., yeast bacteremia has leapt to institute The 4th of interior Hematogenous infection, and nosocomial infection the 2nd important composition is had become in Chinese IFI.Inhibition of HIV the infected, device The probability that the impaired patient of the immunologic mechanisms such as official's transplanting infects Pneumocystis greatly increases, and is easy to appear mixed infection.Lung Common fungal infection has candida albicans, Aspergillus, Cryptococcus neoformans, mucor and carinti infections.It should be noted that exhausted There is the case where field planting in human body in most invasive fungi, there is also a large amount of fungal pathogens in healthy human body, Only when the disorder of human immunity mechanism or it is severely damaged when, the fungi of field planting can be converted to have invasion blood vessel, digestion The invasive fungi of the organs such as road jeopardizes patient vitals to cause multi-functional damage.Therefore, the diagnosis of invasive fungi There is biggish clinical value for the patient of immune function depression.

The conventional method of diagnosis studies of invasive fungal infections clinical pathogens mainly has microscopy, culture, histopathological examination, blood It is clear to learn method and molecular biology method.That there is detection cycles is long for classical cultivation, susceptibility is low, technical conditions requirement High, operator's profession requires problem high and that detection target is single, it is difficult to be applied to clinical early diagnosis and guiding treatment.It invades The mycotic non-culture experiment room detection method of attacking property mainly includes fungal antigen detection, fungal antibody Serologic detection and divides Sub- Biological Detection.Fungal antigen detection includes the detection of (1,3)-callose, galactomannan antigen for detection, cryptococcus pod membrane Polysaccharide antigen detection and the detection of Candida mannan antigen etc..Different antigen is for diagnosing different fungal infection.It is anti- The Serologic detection of fungal antibody mainly includes that Histoplasma capsulatum, posadasis spheriforme/pair coccidioides immitis and dermatitis bud are raw The detection of the corresponding serum antibody of bacterium.Serologic detection in special detection can decide whether that there are fungal infections, but not It is able to achieve kind of a horizontal differentiation.

In face of such severe Mortality rate, diagnosis and treatment face the challenge: the emergence of new pathogen, sb.'s illness took a turn for the worse Fastly, institute's sense infection rate and the death rate are high, clinical low with laboratory diagnosis rate cause to be more than that 95% patient's delay is treated；Due to anti- The medicine of fungi is widely used, and the phenomenon that drug resistance occur in some pathogen, and common antifungal drug is not to belonging to or plant Therapeutic effect is different.Therefore it realizes that the diagnosis of early stage fast multi-target is conducive to instruct the rational use of medicines, improves survival, for The variation of monitoring cause of disease spectrum has a very big significance.

Clinical most of pathogen detection method that carries out domestic at present is conventional detection and special detection.Conventional detection is mirror Inspection, culture medium histopathological examination, these types of method positive rate is low, false negative cannot exclude；Serology in special detection Detection can decide whether that there are fungal infections, but can not achieve kind of a horizontal differentiation.In recent years, polymerase chain reaction (Polymerase chain reaction, PCR) technology has become the diagnosis a kind of most common method of invasive fungi body, and Multiple real time fluorescence round pcr (Real-Time PCR, RT-PCR) becomes the parting skill for the invasive fungi being widely used Art.Currently, establishing the method being used for quickly detecting using based on PCR technology to invasive fungi both at home and abroad.For example, Chinese The application for a patent for invention that number of patent application is 201410820825.8, which is disclosed, detects 15 invasions using TAQMAN sonde method The method of fungi: 8 kinds of candidiasis (Candida albicans, Candida glabrata, Candida parapsilosis, Candida kefyr, Candida sake, candida krusei, monilia guilliermondii, candida tropicalis), 4 kinds of Aspergillus (aspergillus nigers, Huang Aspergillus, Aspergillus terreus, aspergillus fumigatus) and cryptococcus, Rhizopus oryzae and volume branch Mucor, this method sensitivity with higher and special Property, it is of great significance for the early diagnosis and therapy of invasive infections with fungi.For another example, Chinese Patent Application No. is 201510586315.3 application for a patent for invention discloses the method using RT-PCR detection aspergillus fumigatus: establishing a kind of for cigarette Aspergillus carries out the fluorescence quantifying PCR method (sonde method) of specific detection, the detection sensitivity aspect of the PCR detection architecture, line The lower limit of property detection range can be to 10²Copies/mL, the upper limit is up to 10⁸Copies/mL, sensibility, specificity and stability are equal Preferably.It should be noted that the extraction of above-mentioned two application for a patent for invention amplifying nucleic acid be realized by manual operation, and for As a result judgement is to carry out artificial judgment by specification, and there are certain subjectivities.

Common PCR detection needs sample process process, cumbersome test procedure and the complicated result of complexity early period to sentence It is disconnected.Wherein the detection scheme of aspergillus has 99% sequence similarity with Penicillium notatum, therefore can not area using Fluorescence PCR assay Divide Penicillium notatum, there are the risks of false positive.In addition, the covered detection target finite of RT-PCR institute, needs through many body system The detection for being just able to achieve multiple target is set, the complexity of operation is increased.

Summary of the invention

Purpose of this disclosure is to provide a kind of nucleic acid reagent, kits, system for fast and accurately detecting invasive fungi And method.

To achieve the goals above, disclosure first aspect: provide it is a kind of for detecting the nucleic acid reagent of invasive fungi, Wherein, the nucleic acid reagent includes shown in the SEQ ID NO.1-10 of storage independent of one another respectively or mutual any mixed storage Probe shown in primer and SEQ ID NO.15-23.

Optionally, primer shown in the SEQ ID NO.1 relative to 1 μM, draws as shown in SEQ ID NO.2-10 respectively The content of object be respectively 0.3-0.5 μM, 0.9-1.1 μM, 0.3-0.5 μM, 0.9-1.1 μM, 0.2-0.4 μM, 0.9-1.1 μM, 0.1-0.3 μM, 0.9-1.1 μM and 0.1-0.3 μM, the content of the probe as shown in SEQ ID NO.15-23 is respectively respectively 0.1-0.3μM、0.1-0.3μM、0.1-0.3μM、0.2-0.3μM、0.1-0.3μM、0.1-0.3μM、0.1-0.3μM、0.1-0.3 μM and 0.1-0.3 μM.

Optionally, the nucleic acid reagent further includes Quality Control and Quality Control in feminine gender in the positive；

Quality Control contains probe shown in primer shown in SEQ ID NO.11-12 and SEQ ID NO.24 in the positive； Quality Control contains probe shown in primer shown in SEQ ID NO.13-14 and SEQ ID NO.25 in the feminine gender.

Optionally, the nucleic acid reagent includes A pipe, B pipe, C pipe and D pipe；A pipe contains to be drawn shown in SEQ ID NO.1-2 Probe shown in object and SEQ ID NO.15-18；B pipe contains primer and SEQ ID NO.19-21 shown in SEQ ID NO.3-8 Shown in probe；C pipe contains probe shown in primer shown in SEQ ID NO.9-10 and SEQ ID NO.22-23；D pipe contains Probe shown in primer shown in SEQ ID NO.11-14 and SEQ ID NO.24-25.

Optionally, SEQ ID NO.15, probe shown in 19,22,24 have the first fluorescent marker；SEQ ID NO.16, Probe shown in 20,23 has the second fluorescent marker；Probe shown in SEQ ID NO.17,21 has third fluorescent marker；SEQ Probe shown in ID NO.18,25 has the 4th fluorescent marker；It is first fluorescent marker, second fluorescent marker, described Third fluorescent marker and the 4th fluorescent marker are different, and are each independently selected from FAM fluorescent marker, JOE fluorescence mark One in note, CY5 fluorescent marker, ROX fluorescent marker, HEX fluorescent marker, VIC fluorescent marker and Quasar670 fluorescent marker Kind.

Optionally, the invasive fungi includes Candida albicans, Candida tropicalis, Candida glabrata, nearly smooth beads At least one of bacterium, Eurotium, mucor, Cryptococcus neoformans, Pneumocystis jiroveci and Pneumocystis carinii.

Disclosure second aspect: providing a kind of for detecting the kit of invasive fungi, which contains the disclosure Nucleic acid reagent described in first aspect, and optionally, the kit also contains reaction system buffer, archaeal dna polymerase, magnesium At least one of ion, dNTP and water.

The disclosure third aspect: it is aggressive true for detecting in preparation to provide nucleic acid reagent described in disclosure first aspect Purposes in the kit of bacterium.

Disclosure fourth aspect: providing a kind of system for detecting invasive fungi, which includes having the detection of A pipe Device, B pipe detector, C pipe detector and D pipe detector PCR instrument, computing device and output device, the A pipe detector, B pipe Detector, C pipe detector and D pipe detector are respectively the nucleic acid reagent tank for being mounted with nucleic acid reagent as described above, The PCR instrument includes the first fluorescence channel, the second fluorescence channel, third fluorescence channel and the 4th fluorescence channel, and described first is glimmering Optical channel, second fluorescence channel, the third fluorescence channel and the 4th fluorescence channel are different, and respectively independent Ground be FAM fluorescence channel, JOE fluorescence channel, CY5 fluorescence channel, ROX fluorescence channel, HEX fluorescence channel, VIC fluorescence channel or Quasar670 fluorescence channel；The computing device includes memory and processor, and computer journey is stored in the memory Sequence, the processor is configured to the computer program stored in the memory is executed, to realize following differentiation:

If it is that have Tm be 57 for 65 DEG C of corresponding dissolution peak curves, the first fluorescence channel of D pipe that the first fluorescence channel of A pipe, which has Tm value, It is 60 DEG C of dissolution peak curve that DEG C corresponding dissolution peak curve, the 4th fluorescence channel of D pipe, which have Tm value, then is determined as Candida albicans It is positive；

If it is that have Tm be 57 for 60 DEG C of corresponding dissolution peak curves, the first fluorescence channel of D pipe that the second fluorescence channel of A pipe, which has Tm value, It is 60 DEG C of dissolution peak curve that DEG C corresponding dissolution peak curve, the 4th fluorescence channel of D pipe, which have Tm value, then is determined as Candida tropicalis It is positive；

If it is that have Tm be 57 for 56 DEG C of corresponding dissolution peak curves, the first fluorescence channel of D pipe that A pipe third fluorescence channel, which has Tm value, It is 60 DEG C of dissolution peak curve that DEG C corresponding dissolution peak curve, the 4th fluorescence channel of D pipe, which have Tm value, then is determined as Candida glabrata It is positive；

If it is that have Tm be 57 for 52 DEG C of corresponding dissolution peak curves, the first fluorescence channel of D pipe that the 4th fluorescence channel of A pipe, which has Tm value, It is 60 DEG C of dissolution peak curve that DEG C corresponding dissolution peak curve, the 4th fluorescence channel of D pipe, which have Tm value, then is determined as nearly smooth beads Bacterium is positive；

If it is that have Tm be 57 for 67 DEG C of corresponding dissolution peak curves, the first fluorescence channel of D pipe that the first fluorescence channel of B pipe, which has Tm value, It is 60 DEG C of dissolution peak curve that DEG C corresponding dissolution peak curve, the 4th fluorescence channel of D pipe, which have Tm value, then is determined as aspergillus sun Property；

If it is that have Tm be 57 for 63 DEG C of corresponding dissolution peak curves, the first fluorescence channel of D pipe that the second fluorescence channel of B pipe, which has Tm value, It is 60 DEG C of dissolution peak curve that DEG C corresponding dissolution peak curve, the 4th fluorescence channel of D pipe, which have Tm value, then is determined as mucor sun Property；

If it is that have Tm be 57 for 56 DEG C of corresponding dissolution peak curves, the first fluorescence channel of D pipe that B pipe third fluorescence channel, which has Tm value, It is 60 DEG C of dissolution peak curve that DEG C corresponding dissolution peak curve, the 4th fluorescence channel of D pipe, which have Tm value, then is determined as Cryptococcus neoformans It is positive；

If it is that have Tm be 57 for 59 DEG C of corresponding dissolution peak curves, the first fluorescence channel of D pipe that the first fluorescence channel of C pipe, which has Tm value, It is 60 DEG C of dissolution peak curve that DEG C corresponding dissolution peak curve, the 4th fluorescence channel of D pipe, which have Tm value, then is determined as Ye Shi lung spore Worm is positive；

If it is that have Tm be 57 for 60 DEG C of corresponding dissolution peak curves, the first fluorescence channel of D pipe that the second fluorescence channel of C pipe, which has Tm value, It is 60 DEG C of dissolution peak curve that DEG C corresponding dissolution peak curve, the 4th fluorescence channel of D pipe, which have Tm value, then is determined as Cattell lung spore Worm is positive.

The 5th aspect of the disclosure: a kind of method for detecting invasive fungi is provided, wherein this method comprises: using Nucleic acid reagent as described above carries out PCR amplification to the DNA of sample to be tested；The PCR instrument for carrying out the PCR amplification includes first Fluorescence channel, the second fluorescence channel, third fluorescence channel and the 4th fluorescence channel；It is first fluorescence channel, described second glimmering Optical channel, the third fluorescence channel and the 4th fluorescence channel are different, and be each independently FAM fluorescence channel, JOE fluorescence channel, CY5 fluorescence channel, ROX fluorescence channel, HEX fluorescence channel, VIC fluorescence channel or Quasar670 fluorescence are logical Road；And carry out following differentiation:

The beneficial effect of the disclosure is:

What the disclosure was established detects invasive fungi Candida albicans by ParaDNA and Hybeacon probe technique (CA), Candida tropicalis (CT), Candida glabrata (CG), Candida parapsilosis (CP), Eurotium (Asp), mucor (Muc), the multiplicated system detection side of Cryptococcus neoformans (Cn), Pneumocystis jiroveci (Pnj) and Pneumocystis carinii (Pnc) Method can be realized morphology, immunology and impossible quickly, comprehensive, sensitive, special, the automatic knot of RT-PCR detection Fruit determines, reaches following detection effect:

(1) higher Multiple detection ability

In recent years about invasive fungi detection report in mostly use the method for RT-PCR to be detected greatly, though this method The detection of multiple target so may be implemented, but limited by fluorescence channel number, multiple amplification system joint inspections is needed just to be able to achieve, Increase the complexity of operation.It is aggressive true that the detection method that the disclosure is established can only need 4 systems disposably to detect Bacterium Candida albicans (CA), Candida tropicalis (CT), Candida glabrata (CG), Candida parapsilosis (CP), Eurotium (Asp), 10 kinds of mucor (Muc), Cryptococcus neoformans (Cn), Pneumocystis jiroveci (Pnj) and Pneumocystis carinii (Pnc) mesh Mark, testing process is simple, as a result automatic interpretation and reliable, saves time, cost of human and material resources.

(2) easy operation link

Clinical sample can be placed directly in the reactor of ParaDNA directly detection by sampler and be can be obtained reliably As a result, avoiding costly and time-consuming sample extraction step, the Emergent detection in addition to Specialty Experiment room is realized.

(3) the detection integration of higher degree

The disclosure is directed to the demand of invasive fungi detection, provides a set of comprehensive, quick, accurate and inspection easy to operate The integrative solution for surveying invasive fungi includes that rapidly extracting, fluorescent PCR amplification and the automation result of nucleic acid is sentenced It is fixed,

(4) specificity is good

Hybeacon probe can recognize the SNP of primer binding zone, so that it has extremely strong identification capability to non-detection target. The detection method specificity that the disclosure is established is also embodied in the specificity of a whole set of primed probe: all primed probes all pass through Blast compares analysis, conservative and specificity with height；It can be good at distinguishing packet by specificity experiments verifying simultaneously Include mycobacterium tuberculosis, Bacterium diphtheriae, cytomegalovirus, haemophilus influenzae, staphylococcus aureus, epidermis grape ball The other pathogens such as bacterium, streptococcus pneumonia, Neisseria meningitidis, Bordetella pertussis, it was demonstrated that detection method has the special of height Property, non-detection target can be accurately distinguished and be come.

(5) minimum detectability

The minimum detectability for the detection method that the disclosure is established can reach 10 copies/reaction.

Other feature and advantage of the disclosure will the following detailed description will be given in the detailed implementation section.

Specific embodiment

The specific embodiment of the disclosure is described in detail below.It should be understood that described herein specific Embodiment is only used for describing and explaining the disclosure, is not limited to the disclosure.

Disclosure first aspect: the nucleic acid reagent for detecting invasive fungi is provided, wherein the nucleic acid reagent includes Storage or mutually primer and SEQ ID NO.15-23 shown in the SEQ ID NO.1-10 of any mixed storage independently of one another respectively Shown in probe.

The disclosure by ParaDNA and Hybeacon probe technique detect invasive fungi, avoid smear, culture and The methods of the RT-PCR detection operating time is long and cumbersome, being capable of quick, accurate, high throughput a variety of invasions of parallel detection Fungi.

For Hybeacon probe technique to the more demanding of probe, the Tm value of probe is particularly important；In addition, probe and primer Combined effect also has important influence to expanding effect.Above-mentioned primer and probe in the design process, has considered not only difference The primer and probe of target gene is in a reaction system the problem of coamplification, i.e. assessment Tm value, the target Tm that corresponds to probe The difference of value, avoids the occurrence of situations such as hairpin structure and dimer at G/C content, and to guarantee alternative primer and probe section point A variety of invasive fungis can not be covered comprehensively, and specificity is good and coverage is high.

Further, primer shown in the SEQ ID NO.1 relative to 1 μM, respectively as shown in SEQ ID NO.2-10 The content of primer respectively can be 0.3-0.5 μM, 0.9-1.1 μM, 0.3-0.5 μM, 0.9-1.1 μM, 0.2-0.4 μM, 0.9-1.1 μM, 0.1-0.3 μM, 0.9-1.1 μM and 0.1-0.3 μM, the content of the probe as shown in SEQ ID NO.15-23 respectively may be used respectively Think 0.1-0.3 μM, 0.1-0.3 μM, 0.1-0.3 μM, 0.2-0.3 μM, 0.1-0.3 μM, 0.1-0.3 μM, 0.1-0.3 μM, 0.1-0.3 μM and 0.1-0.3 μM.

According to the disclosure, to do good quality control, the nucleic acid reagent can also include Quality Control and negative endoplasm in the positive Control.Further, to can be used for detecting fungi general (Fungi) for Quality Control in the positive, can contain SEQ ID NO.11-12 Shown in probe shown in primer and SEQ ID NO.24, Quality Control in the positive.Quality Control is selected human endogenous in the feminine gender Nbccs gene can contain probe shown in primer shown in SEQ ID NO.13-14 and SEQ ID NO.25.This When, primer shown in the SEQ ID NO.1 relative to 1 μM, the content of the primer as shown in SEQ ID NO.11-14 is respectively respectively It can be 0.9-1.1 μM, 0.2-0.4 μM, 0.9-1.1 μM and 0.1-0.3 μM, the probe as shown in SEQ ID NO.24-25 Content can be respectively 0.1-0.3 μM and 0.05-0.15 μM.It, can by the way that Quality Control and Quality Control in the positive in the feminine gender is added Enough precise Identification mycobacterium tuberculosis, avoid the drug resistance result of false positive；Can simultaneously be effectively prompt is because operation is lost False negative testing result caused by the reasons such as mistake, PCR mortifier.

According to the disclosure, in order to enhance the accuracy of testing result, the nucleic acid reagent can be divided into four pipes, i.e., the described core Acid reagent may include A pipe, B pipe, C pipe and D pipe.Wherein, A pipe can contain primer and SEQ shown in SEQ ID NO.1-2 Probe shown in ID NO.15-18；B pipe can be containing shown in primer shown in SEQ ID NO.3-8 and SEQ ID NO.19-21 Probe；C pipe can contain probe shown in primer shown in SEQ ID NO.9-10 and SEQ ID NO.22-23；D pipe can be with Contain probe shown in primer shown in SEQ ID NO.11-14 and SEQ ID NO.24-25.

It is possible to further carry out the permutation and combination of fluorescent marker according to the respective Tm value of probe, so that in same system The amplification of different probe identified respectively.For example, as an implementation, SEQ ID NO.15, shown in 19,22,24 Probe can have the first fluorescent marker；Probe shown in SEQ ID NO.16,20,23 can have the second fluorescent marker；SEQ Probe shown in ID NO.17,21 can have third fluorescent marker；Probe shown in SEQ ID NO.18,25 can have Four fluorescent markers；First fluorescent marker, second fluorescent marker, the third fluorescent marker and the 4th fluorescence mark Remember it is different, and be each independently selected from FAM fluorescent marker, JOE fluorescent marker, CY5 fluorescent marker, ROX fluorescent marker, One of HEX fluorescent marker, VIC fluorescent marker and Quasar670 fluorescent marker.As a kind of particularly preferred embodiment party Formula, SEQ ID NO.15, probe shown in 19,22,24 have FAM fluorescent marker；It is visited shown in SEQ ID NO.16,20,23 Needle set has JOE fluorescent marker；Probe shown in SEQ ID NO.17,21 has CY5 fluorescent marker；SEQ ID NO.18,25 institute The probe shown has ROX fluorescent marker.In order to enhance dissolution peak effect, above-mentioned target-probe can be dual labelled probe.Probe Middle FAM is 6- Fluoresceincarboxylic acid, and JOE 2, the chloro- 6- Fluoresceincarboxylic acid of 7- dimethyl -4,5 two, CY5 is 5H- indoles cyanines, and ROX is 6- carboxy-X-rhodamine.

According to the disclosure, the invasive fungi may include Candida albicans (CA), Candida tropicalis (CT), smooth thought Pearl bacterium (CG), Candida parapsilosis (CP), Eurotium (Asp), mucor (Muc), Cryptococcus neoformans (Cn), Ye Shi lung spore At least one of worm (Pnj) and Pneumocystis carinii (Pnc).

The kit of the disclosure can be realized quick, accurate, sensitive, special, automatic testing result and determine, significantly improve Sensibility, specificity and simplicity that a variety of invasive fungis are detected.

Disclosure fourth aspect: providing a kind of system for detecting invasive fungi, which includes having the detection of A pipe Device, B pipe detector, C pipe detector and D pipe detector PCR instrument, computing device and output device, the A pipe detector, B pipe Detector, C pipe detector and D pipe detector are respectively the core for being mounted with the above-mentioned nucleic acid reagent including A pipe, B pipe, C pipe and D pipe Acid reagent tank, the PCR instrument include the first fluorescence channel, the second fluorescence channel, third fluorescence channel and the 4th fluorescence Channel, first fluorescence channel, second fluorescence channel, the third fluorescence channel and the 4th fluorescence channel are respectively not It is identical, and it is logical to be each independently FAM fluorescence channel, JOE fluorescence channel, CY5 fluorescence channel, ROX fluorescence channel, HEX fluorescence Road, VIC fluorescence channel or Quasar670 fluorescence channel；The computing device includes memory and processor, in the memory It is stored with computer program, the processor is configured to the computer program stored in the memory is executed, to realize such as Under differentiation:

The 5th aspect of the disclosure: a kind of method for detecting invasive fungi is provided, wherein this method comprises: using It is managed as described above including A pipe, B, the nucleic acid reagent of C pipe and D pipe, PCR amplification is carried out to the DNA of sample to be tested；Carry out the PCR The PCR instrument of amplification includes the first fluorescence channel, the second fluorescence channel, third fluorescence channel and the 4th fluorescence channel；Described first Fluorescence channel, second fluorescence channel, the third fluorescence channel and the 4th fluorescence channel are different, and respectively solely It is on the spot FAM fluorescence channel, JOE fluorescence channel, CY5 fluorescence channel, ROX fluorescence channel, HEX fluorescence channel, VIC fluorescence channel Or Quasar670 fluorescence channel；And carry out following differentiation:

Wherein, the sample to be tested can be bronchoalveolar lavage fluid (BALF), and the condition of the PCR amplification can be with are as follows: 45-55 DEG C, 7-10min, 98 DEG C, 60s, (98 DEG C, 10s, 65 DEG C, 10s, 30-40 circulations)；Solubility curve analysis: 98 DEG C, 60s, 35 DEG C, 60s, drop rate is 1.0 DEG C/s；80 DEG C, 5s, liter rate is 0.5 DEG C/s, which collects fluorescence.

Disclosed method can quickly the sensitive system screening for specifically realizing a variety of invasive fungis, testing process be simple Single, as a result automatic interpretation and reliable saves time, manpower and reagent cost.

The disclosure is further elaborated by the following examples, but the disclosure is not therefore by any limit System.

Reagent is commercial products in following embodiment, and primer, probe are synthesized in Biosearch (USA) company.

Embodiment

1, primer, probe synthesis

According to probe sequence shown in primer sequence shown in table 1 and table 2, sequent synthesis is carried out.Y represents degeneracy in sequence Base T/C；R represents degeneracy base A/G.FAM is 6- Fluoresceincarboxylic acid, JOE 2, the chloro- 6- carboxylic of 7- dimethyl -4,5 two in probe Base fluorescein, CY5 are 5H- indoles cyanines, and ROX is 6- carboxy-X-rhodamine.Bracket in the probe sequence of table 2 indicates that bracket is left The t of side has fluorescent marker, the selection of the content representation fluorescent marker in bracket.

Table 1

Table 2

2, sample process

After samplers sample matched with ParaDNA treated bronchoalveolar lavage fluid (BALF), it is placed directly within It is i.e. amplifiable in the reactor of ParaDNA.

3, Hybeacon probe technique detection architecture is constructed

Polymerase Phire Hot Start II DNA Polymerase (article No. F122L), Mg²⁺, dNTPS is purchased from ThermoFisher company；Other biochemical reagents are that import packing or domestic analysis are pure；Fluorescence detector is ParaDNA.

Reaction system is prepared according to following operation: total system 30 μ L, 5 × PCRBuffer 5 μ L, magnesium chloride solution 3-4mM, DNTPS is 1-1.5mM, 0.8-1.0 μM of upstream primer, 0.2-0.5 μM of downstream primer, Hybeacon probe 100-300nM, is reversed Enzyme 1-2 μ L is recorded, specific primer and probe content is shown in Table 3, and residue is supplied with water.

Table 3

SEQ ID NO	Final concentration (μM)	SEQ ID NO	Final concentration (μM)
				1	1	14	0.2
2	0.4	15	0.2
				3	1	16	0.2
4	0.4	17	0.2
				5	1	18	0.25
6	0.3	19	0.2
				7	1	20	0.2
8	0.2	21	0.2
				9	1	22	0.2
10	0.2	23	0.2
				11	1	24	0.2
12	0.3	25	0.1
				13	1

Kit contains 5 × PCRBuffer, Phire Hot Start II DNA Polymerase, 10 × primed probe Mixed liquor A pipe, 10 × primed probe mixed liquid B pipe, 10 × primed probe mixed liquor C are managed, 10 × primed probe mixed liquor D is managed, Pure water.A pipe contains probe shown in SEQ ID NO.15-18 in primer shown in SEQ ID NO.1-2 in upper table 1 and upper table 2； B pipe contains probe shown in SEQ ID NO.19-21 in primer shown in SEQ ID NO.3-8 in upper table 1 and upper table 2；C pipe contains There is in upper table 1 probe shown in SEQ ID NO.22-23 in primer shown in SEQ ID NO.9-10 and upper table 2；D pipe is containing upper Probe shown in SEQ ID NO.24-25 in primer shown in SEQ ID NO.11-14 and upper table 2 in table 1.

PCR pipe is put into fluorescence quantitative PCR instrument, selects FAM, JOE, CY5 and ROX as reporter group, response procedures It is as follows: 50 DEG C, 10min, 98 DEG C, 60s, (98 DEG C, 10s, 65 DEG C, 10s, 35 circulations)；Solubility curve analysis: 98 DEG C, 60s, 35 DEG C, 60s, drop rate is 1.0 DEG C/s；80 DEG C, 5s, liter rate is 0.5 DEG C/s, which collects fluorescence.

Reaction result judgement: yin and yang attribute control is set up, and it is invalid otherwise to regard experimental result；

If it is that have Tm be 57 DEG C for 65 DEG C of corresponding dissolution peak curves, D pipe FAM fluorescence channel that A pipe FAM fluorescence channel, which has Tm value, It is 60 DEG C of dissolution peak curve that corresponding dissolution peak curve, D pipe ROX fluorescence channel, which have Tm value, then is determined as Candida albicans sun Property；

If it is that have Tm be 57 DEG C for 60 DEG C of corresponding dissolution peak curves, D pipe FAM fluorescence channel that A pipe JOE fluorescence channel, which has Tm value, It is 60 DEG C of dissolution peak curve that corresponding dissolution peak curve, D pipe ROX fluorescence channel, which have Tm value, then is determined as Candida tropicalis sun Property；

If it is that have Tm be 57 DEG C for 56 DEG C of corresponding dissolution peak curves, D pipe FAM fluorescence channel that A pipe CY5 fluorescence channel, which has Tm value, It is 60 DEG C of dissolution peak curve that corresponding dissolution peak curve, D pipe ROX fluorescence channel, which have Tm value, then is determined as Candida glabrata sun Property；

If it is that have Tm be 57 DEG C for 52 DEG C of corresponding dissolution peak curves, D pipe FAM fluorescence channel that A pipe ROX fluorescence channel, which has Tm value, It is 60 DEG C of dissolution peak curve that corresponding dissolution peak curve, D pipe ROX fluorescence channel, which have Tm value, then is determined as Candida parapsilosis It is positive；

If it is that have Tm be 57 DEG C for 67 DEG C of corresponding dissolution peak curves, D pipe FAM fluorescence channel that B pipe FAM fluorescence channel, which has Tm value, It is 60 DEG C of dissolution peak curve that corresponding dissolution peak curve, D pipe ROX fluorescence channel, which have Tm value, then is determined as the aspergillus positive；

If it is that have Tm be 57 DEG C for 63 DEG C of corresponding dissolution peak curves, D pipe FAM fluorescence channel that B pipe JOE fluorescence channel, which has Tm value, It is 60 DEG C of dissolution peak curve that corresponding dissolution peak curve, D pipe ROX fluorescence channel, which have Tm value, then is determined as the mucor positive；

If it is that have Tm be 57 DEG C for 56 DEG C of corresponding dissolution peak curves, D pipe FAM fluorescence channel that B pipe CY5 fluorescence channel, which has Tm value, It is 60 DEG C of dissolution peak curve that corresponding dissolution peak curve, D pipe ROX fluorescence channel, which have Tm value, then is determined as Cryptococcus neoformans sun Property；

If it is that have Tm be 57 DEG C for 59 DEG C of corresponding dissolution peak curves, D pipe FAM fluorescence channel that C pipe FAM fluorescence channel, which has Tm value, It is 60 DEG C of dissolution peak curve that corresponding dissolution peak curve, D pipe ROX fluorescence channel, which have Tm value, then is determined as Pneumocystis jiroveci It is positive；

If it is that have Tm be 57 DEG C for 60 DEG C of corresponding dissolution peak curves, D pipe FAM fluorescence channel that C pipe JOE fluorescence channel, which has Tm value, It is 60 DEG C of dissolution peak curve that corresponding dissolution peak curve, D pipe ROX fluorescence channel, which have Tm value, then is determined as Pneumocystis carinii It is positive.

4, specificity verification

Select Penicillium notatum, mycobacterium tuberculosis, Bacterium diphtheriae, cytomegalovirus, haemophilus influenzae, golden yellow Portugal Other cause of diseases such as grape coccus, staphylococcus epidermis, streptococcus pneumonia, Neisseria meningitidis, Bordetella pertussis clinical sample (on Sample standard deviation is stated from Chinese Center for Disease Control and Prevention bacterial disease prevention and control institute) as specificity assessment sample, using system After samplers sample sputum (liquefaction) sample in system detection, is established using early period and the reaction condition of optimization is on ParaDNA It is detected.

As the result is shown under conditions of positive control is set up, dissolution peak of the target to be checked without specificity shows the disclosure Nucleic acid reagent can effective district sorting survey target and non-detection target, there is preferable specificity.

5, minimum detectability is verified

Assessment detection sample: choosing initial concentration is 10⁵Copy/μ L invasive fungi general (Fungi), white are read Pearl bacterium (CA), Candida tropicalis (CT), Candida glabrata (CG), Candida parapsilosis (CP), Eurotium (Asp), mucor (Muc), Cryptococcus neoformans (Cn), Pneumocystis jiroveci (Pnc) and Pneumocystis carinii (Pnj) nucleic acid gradient dilution are 10⁴ Copy/μ L, 10³Copy/μ L, 10²Copy/μ L, 10¹Copy/μ L, 10⁰Copy/μ L, the template as minimum detectability assessment.

The minimum detectability of disclosure kit detection target drug resistant gene can reach 10 copies/reaction as the result is shown.

6, coverage is verified

120 plants of evaluations are selected to use positive bronchoalveolar lavage fluid sample as coverage assessment template.It is anti-as described above System and response procedures is answered to be tested.

All sample standard deviations can be covered as the result is shown and detected.

7, the storage life test of kit

With 100 copies/μ L fungi general (Fungi), Candida albicans (CA), Candida tropicalis (CT), smooth beads Bacterium (CG), Candida parapsilosis (CP), Eurotium (Asp), mucor (Muc), Cryptococcus neoformans (Cn), Pneumocystis jiroveci (Pnj) and Pneumocystis carinii (Pnc) bronchoalveolar lavage fluid is as assessment sample.

At the 0th day, it is distributed into 10 parts and freezes in -70 DEG C of refrigerators.The kit finished will be set up and be placed in -20 DEG C of guarantors It deposits, 0,10,15,30,60,90,120,150,180 and 360 day kit is taken to carry out storage life test respectively.

Disclosure kit is stored in -20 DEG C of refrigerators as the result is shown, is the positive in the detection of different storage lives, shows the examination The storage life of agent box is at least 1 year.

Comparative example

1, primer, probe synthesis

According to primer, probe sequence shown in table 4 and table 5, sequent synthesis is carried out.FAM is 6- Fluoresceincarboxylic acid in probe, JOE is the chloro- 6- Fluoresceincarboxylic acid of 2,7- dimethyl -4,5 two, and CY5 is 5H- indoles cyanines, and ROX is 6- carboxy-X-rhodamine.Table 5 Probe sequence in bracket indicate that the t on the left of bracket has fluorescent marker, the selection of the content representation fluorescent marker in bracket.

Table 4

Table 5

Target designation	Probe identification code	Probe sequence	Tm(℃)	SEQ ID NO
					CA	CA-P1	agc gaaat(fam)gcgat(fam)acgtaatatg	65	48
CT	CT-P1	taagt(joe)gggtggtaaat(joe)tccatctaa	60	49
					CG	CG-P1	ttt(joe)cgctgcgt(joe)tcttcatcgatgcga	56	50
CP	CP-P1	aaattt(rox)atttgtt(rox)aagtaacaagacca	52	51
					Asp	Asp-P1	aagcacggct(fam)tgtgt(fam)gttgg gtcgt	67	52
Muc	Muc-P1	ttataat(joe)tgtaacataagcgt(joe)aaa	63	53
					Cn	Cn-P1	tcaat(cy5)ccct(cy5)cgggttttattacc	56	54
Pnj	Pnj-P1	taggt(fam)atagcact(fam)gagtat ctcg	59	55
					Pnc	Pnc-P1	tgttctt(joe)cgatcct(joe)ccctacgaat	60	56
Fungi	Fu-P1	agaact(fam)gccat(fam)g cacca	57	57
					IAC	RP-P1	atggt(rox)gactt(rox)ccacccacaagg	60	58

2, specificity verification

Specificity verification is carried out according to the method for embodiment.The results show that the reaction result of the primer of comparative example, probe is equal For feminine gender.

3, minimum detectability is verified

Minimum detectability verifying is carried out according to the method for embodiment.The comparison of the minimum detectability of embodiment and comparative example is as follows Table 6.

Table 6

As shown in Table 6, (Fungi) general for the fungi of trace in sample, Candida albicans (CA), Candida tropicalis (CT), Candida glabrata (CG), Candida parapsilosis (CP), Eurotium (Asp), mucor (Muc), Cryptococcus neoformans (Cn), Pneumocystis jiroveci (Pnj) and Pneumocystis carinii (Pnc) nucleic acid nucleic acid, disclosure kit have more than comparative example Strong detectability.

4, coverage is verified

Coverage verifying is carried out according to the method for embodiment.The coverage of embodiment and comparative example comparison such as the following table 7.

Table 7

Detect target	Embodiment	Comparative example
			Candida albicans	10	10
Candida tropicalis	10	10
			Candida glabrata	10	9
Candida parapsilosis	10	9
			Mucor	10	9
Aspergillus	10	8
			Cryptococcus neoformans	10	8
Pneumocystis jiroveci	10	10
			Pneumocystis carinii	10	10
Fungi is general	10	7

As shown in Table 7, the detection coverage of disclosure kit is far longer than the detection coverage of comparative example.

The disclosure can detecte out fungi general (Fungi) it can be seen from the comparison of embodiment and comparative example, white is read Pearl bacterium (CA), Candida tropicalis (CT), Candida glabrata (CG), Candida parapsilosis (CP), Eurotium (Asp), mucor (Muc), Cryptococcus neoformans (Cn), Pneumocystis jiroveci (Pnj) and Pneumocystis carinii (Pnc) nucleic acid, specificity is high, minimum Detection limit is lower, and coverage is wider.

The preferred embodiment of the disclosure is described in detail above, still, during the disclosure is not limited to the above embodiment Detail a variety of simple variants can be carried out to the technical solution of the disclosure in the range of the technology design of the disclosure, this A little simple variants belong to the protection scope of the disclosure.

It is further to note that specific technical features described in the above specific embodiments, in not lance In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the disclosure to it is various can No further explanation will be given for the combination of energy.

In addition, any combination can also be carried out between a variety of different embodiments of the disclosure, as long as it is without prejudice to originally Disclosed thought equally should be considered as disclosure disclosure of that.

Sequence table

<110>biotech inc Beijing Zhuo Cheng Hui Sheng

<120>for detecting the nucleic acid reagent, kit, system and method for invasive fungi

<130> 12634ABT

<160> 58

<170> SIPOSequenceListing 1.0

<210> 1

<211> 23

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<213>artificial sequence (Artificial Sequence)

<400> 1

tctcttggtt ctcgcatcga tga 23

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<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

tgtgcgttca aaaattcgat g 21

<210> 3

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

aacctgcgga aggatcatta c 21

<210> 4

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

ccattgttga aagttttaac tgatt 25

<210> 5

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

tggcctttgc tagttttcta gcgaat 26

<210> 6

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

aagttgtttt ataagttttt tacgct 26

<210> 7

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

atgaatgtaa tcttattata acaat 25

<210> 8

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

ccgacgtcct ttgcaggtcg cggca 25

<210> 9

<211> 3

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

<210> 10

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

acatagtctg attatttatt tt 22

<210> 11

<211> 18

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

caacacgggg aaactcac 18

<210> 12

<211> 16

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

ccagcacgac rgagtt 16

<210> 13

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

cgaggcggtt ctcggtgggg gccgag 26

<210> 14

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

caccaacgga cgtgaagccg gtgag 25

<210> 15

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 15

ctcgcatcga tgaagaacgc ag 22

<210> 16

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

tctttgggtc tggctcttgt ctatgt 26

<210> 17

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 17

gtgtgcgtgg atctctctat tccaaa 26

<210> 18

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 18

aaattatctt ttgataataa gatcgtcg 28

<210> 19

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 19

ccgccggtag acaccacgaa ctctg 25

<210> 20

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 20

cctggtatcc tattaattat t 21

<210> 21

<211> 24

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 21

cagtgaatca tcgaatcttt gaac 24

<210> 22

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 22

atctcagata tttaatctca aaa 23

<210> 23

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 23

tgtagactgt cggcgagaag gtt 23

<210> 24

<211> 17

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 24

agtacggcca tgcacca 17

<210> 25

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 25

ccgccgattc ttccccccga gcggctc 27

<210> 26

<211> 17

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 26

tggcggtggg cccagcc 17

<210> 27

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 27

tgagggagaa acgacgctca 20

<210> 28

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 28

atcaataagc ggaggaaaag aaacca 26

<210> 29

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 29

atctttccat cactgtactt gt 22

<210> 30

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 30

actacacaca gtggagttta cttta 25

<210> 31

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 31

caacacaaga tttcttttag tagaa 25

<210> 32

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 32

cctttagctc ttgctatctg aattatg 27

<210> 33

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 33

attaagtacc attacaggaa ccc 23

<210> 34

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 34

atcgagtctt tgaacgcaca ttgcgc 26

<210> 35

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 35

gacgcggtgc cgccgctgcc tttggg 26

<210> 36

<211> 24

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 36

ttgctagttt tctagcgaat ggtt 24

<210> 37

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 37

ttatcgcact ttgctacgtt cttca 25

<210> 38

<211> 24

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 38

tggcttccac atcgatgaag aacg 24

<210> 39

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 39

acgtcctttg caggtcgcgg caaac 25

<210> 40

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 40

aatcggacta ggatatagct g 21

<210> 41

<211> 24

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 41

tgttctgggc tgtttccctt tcga 24

<210> 42

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 42

gctggttttc tgcgaaactt 20

<210> 43

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 43

tctcgccgac agtctgacaa tt 22

<210> 44

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 44

ggraawctca ccaggtcca 19

<210> 45

<211> 18

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 45

gcyctatccc cagcacga 18

<210> 46

<211> 29

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 46

attggcccga ggcctccgaa gtacatcac 29

<210> 47

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 47

tcccctcctg gtcccctctc tga 23

<210> 48

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 48

agcgaaatgc gatacgtaat atg 23

<210> 49

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 49

taagtgggtg gtaaattcca tctaa 25

<210> 50

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 50

tttcgctgcg ttcttcatcg atgcga 26

<210> 51

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 51

aaatttattt gttaagtaac aagacca 27

<210> 52

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 52

aagcacggct tgtgtgttgg gtcgt 25

<210> 53

<211> 24

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 53

ttataattgt aacataagcg taaa 24

<210> 54

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 54

tcaatccctc gggttttatt acc 23

<210> 55

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 55

taggtatagc actgagtatc tcg 23

<210> 56

<211> 24

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 56

tgttcttcga tcctccctac gaat 24

<210> 57

<211> 17

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 57

agaactgcca tgcacca 17

<210> 58

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 58

atggtgactt ccacccacaa gg 22

Claims

1. a kind of for detecting the nucleic acid reagent of invasive fungi, wherein the nucleic acid reagent includes storage independently of one another respectively Or probe shown in primer shown in the SEQ ID NO.1-10 of mutual any mixed storage and SEQ ID NO.15-23.

2. nucleic acid reagent according to claim 1, wherein primer shown in the SEQ ID NO.1 relative to 1 μM, respectively The content of the primer as shown in SEQ ID NO.2-10 is respectively 0.3-0.5 μM, 0.9-1.1 μM, 0.3-0.5 μM, 0.9-1.1 μ M, 0.2-0.4 μM, 0.9-1.1 μM, 0.1-0.3 μM, 0.9-1.1 μM and 0.1-0.3 μM, respectively by SEQ ID NO.15-23 institute The content of the probe shown is respectively 0.1-0.3 μM, 0.1-0.3 μM, 0.1-0.3 μM, 0.2-0.3 μM, 0.1-0.3 μM, 0.1-0.3 μM, 0.1-0.3 μM, 0.1-0.3 μM and 0.1-0.3 μM.

3. nucleic acid reagent according to claim 1, wherein the nucleic acid reagent further includes Quality Control and negative endoplasm in the positive Control；

Quality Control contains probe shown in primer shown in SEQ ID NO.11-12 and SEQ ID NO.24 in the positive；It is described Quality Control contains probe shown in primer shown in SEQ ID NO.13-14 and SEQ ID NO.25 in feminine gender.

4. nucleic acid reagent according to claim 3, wherein the nucleic acid reagent includes A pipe, B pipe, C pipe and D pipe；A pipe contains There is probe shown in primer shown in SEQ ID NO.1-2 and SEQ ID NO.15-18；B pipe is containing shown in SEQ ID NO.3-8 Primer and SEQ ID NO.19-21 shown in probe；C pipe contains primer and SEQ ID shown in SEQ ID NO.9-10 Probe shown in NO.22-23；D pipe contains spy shown in primer shown in SEQ ID NO.11-14 and SEQ ID NO.24-25 Needle.

5. nucleic acid reagent according to claim 4, wherein SEQ ID NO.15, probe shown in 19,22,24 have the One fluorescent marker；Probe shown in SEQ ID NO.16,20,23 has the second fluorescent marker；Shown in SEQ ID NO.17,21 Probe has third fluorescent marker；Probe shown in SEQ ID NO.18,25 has the 4th fluorescent marker；The first fluorescence mark Note, second fluorescent marker, the third fluorescent marker and the 4th fluorescent marker are different, and select each independently From FAM fluorescent marker, JOE fluorescent marker, CY5 fluorescent marker, ROX fluorescent marker, HEX fluorescent marker, VIC fluorescent marker and One of Quasar670 fluorescent marker.

6. nucleic acid reagent described according to claim 1~any one of 5, wherein the invasive fungi includes that white is read Pearl bacterium, Candida tropicalis, Candida glabrata, Candida parapsilosis, Eurotium, mucor, Cryptococcus neoformans, Ye Shi lung spore At least one of worm and Pneumocystis carinii.

7. a kind of for detecting the kit of invasive fungi, which contains described in any one of claim 1~6 Nucleic acid reagent, and optionally, the kit also contains reaction system buffer, archaeal dna polymerase, magnesium ion, dNTP and water At least one of.

8. nucleic acid reagent described in any one of claim 1~6 is in preparing the kit for detecting invasive fungi Purposes.

9. a kind of system for detecting invasive fungi, which includes with A pipe detector, B pipe detector, the detection of C pipe The PCR instrument of device and D pipe detector, computing device and output device, the A pipe detector, B pipe detector, C pipe detector and D Pipe detector is respectively the nucleic acid reagent tank for being mounted with nucleic acid reagent described in any one of claim 4~6, institute Stating PCR instrument includes the first fluorescence channel, the second fluorescence channel, third fluorescence channel and the 4th fluorescence channel, first fluorescence Channel, second fluorescence channel, the third fluorescence channel and the 4th fluorescence channel are different, and each independently For FAM fluorescence channel, JOE fluorescence channel, CY5 fluorescence channel, ROX fluorescence channel, HEX fluorescence channel, VIC fluorescence channel or Quasar670 fluorescence channel；The computing device includes memory and processor, and computer journey is stored in the memory Sequence, the processor is configured to the computer program stored in the memory is executed, to realize following differentiation:

If it is that have Tm be 57 DEG C right for 65 DEG C of corresponding dissolution peak curves, the first fluorescence channel of D pipe that the first fluorescence channel of A pipe, which has Tm value, It is 60 DEG C of dissolution peak curve that dissolution peak curve, the 4th fluorescence channel of D pipe answered, which have Tm value, then is determined as Candida albicans sun Property；

If it is that have Tm be 57 DEG C right for 60 DEG C of corresponding dissolution peak curves, the first fluorescence channel of D pipe that the second fluorescence channel of A pipe, which has Tm value, It is 60 DEG C of dissolution peak curve that dissolution peak curve, the 4th fluorescence channel of D pipe answered, which have Tm value, then is determined as Candida tropicalis sun Property；

If it is that have Tm be 57 DEG C right for 56 DEG C of corresponding dissolution peak curves, the first fluorescence channel of D pipe that A pipe third fluorescence channel, which has Tm value, It is 60 DEG C of dissolution peak curve that dissolution peak curve, the 4th fluorescence channel of D pipe answered, which have Tm value, then is determined as Candida glabrata sun Property；

If it is that have Tm be 57 DEG C right for 52 DEG C of corresponding dissolution peak curves, the first fluorescence channel of D pipe that the 4th fluorescence channel of A pipe, which has Tm value, It is 60 DEG C of dissolution peak curve that dissolution peak curve, the 4th fluorescence channel of D pipe answered, which have Tm value, then is determined as Candida parapsilosis sun Property；

If it is that have Tm be 57 DEG C right for 67 DEG C of corresponding dissolution peak curves, the first fluorescence channel of D pipe that the first fluorescence channel of B pipe, which has Tm value, It is 60 DEG C of dissolution peak curve that dissolution peak curve, the 4th fluorescence channel of D pipe answered, which have Tm value, then is determined as the aspergillus positive；

If it is that have Tm be 57 DEG C right for 63 DEG C of corresponding dissolution peak curves, the first fluorescence channel of D pipe that the second fluorescence channel of B pipe, which has Tm value, It is 60 DEG C of dissolution peak curve that dissolution peak curve, the 4th fluorescence channel of D pipe answered, which have Tm value, then is determined as the mucor positive；

If it is that have Tm be 57 DEG C right for 56 DEG C of corresponding dissolution peak curves, the first fluorescence channel of D pipe that B pipe third fluorescence channel, which has Tm value, It is 60 DEG C of dissolution peak curve that dissolution peak curve, the 4th fluorescence channel of D pipe answered, which have Tm value, then is determined as Cryptococcus neoformans sun Property；

If it is that have Tm be 57 DEG C right for 59 DEG C of corresponding dissolution peak curves, the first fluorescence channel of D pipe that the first fluorescence channel of C pipe, which has Tm value, It is 60 DEG C of dissolution peak curve that dissolution peak curve, the 4th fluorescence channel of D pipe answered, which have Tm value, then is determined as Pneumocystis jiroveci sun Property；

If it is that have Tm be 57 DEG C right for 60 DEG C of corresponding dissolution peak curves, the first fluorescence channel of D pipe that the second fluorescence channel of C pipe, which has Tm value, It is 60 DEG C of dissolution peak curve that dissolution peak curve, the 4th fluorescence channel of D pipe answered, which have Tm value, then is determined as Pneumocystis carinii sun Property.

10. a kind of method for detecting invasive fungi, wherein this method comprises: using any one in claim 4~6 Nucleic acid reagent described in carries out PCR amplification to the DNA of sample to be tested；The PCR instrument for carrying out the PCR amplification includes first glimmering Optical channel, the second fluorescence channel, third fluorescence channel and the 4th fluorescence channel；First fluorescence channel, second fluorescence Channel, the third fluorescence channel and the 4th fluorescence channel are different, and are each independently FAM fluorescence channel, JOE Fluorescence channel, CY5 fluorescence channel, ROX fluorescence channel, HEX fluorescence channel, VIC fluorescence channel or Quasar670 fluorescence channel； And carry out following differentiation: