CN107354221A - The PCR detection kit of Ye Shi lung pityrosporion ovales - Google Patents
The PCR detection kit of Ye Shi lung pityrosporion ovales Download PDFInfo
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- CN107354221A CN107354221A CN201710750749.1A CN201710750749A CN107354221A CN 107354221 A CN107354221 A CN 107354221A CN 201710750749 A CN201710750749 A CN 201710750749A CN 107354221 A CN107354221 A CN 107354221A
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The present invention proposes a kind of PCR detection kit of Ye Shi lungs pityrosporion ovale, including nucleic acid extraction liquid, PCR reaction solutions, enzyme mixation, positive control and negative control, the PCR reaction solutions include the pair for amplification primer and a specific amplification probe designed for the target sequence of Ye Shi lung pityrosporion ovales, pair for amplification primer is made up of positive amplimer and direction amplimer, the positive amplimer has the nucleotide sequence shown in SEQ ID NO.1, the reversely amplimer has the nucleotide sequence shown in SEQ ID NO.2, the specific amplification probe is made up of annulus sequence and two short handle sequences, 5 ' ends of specific amplification probe are marked with fluorescent reporter group, the end of specific amplification probe 3 ' is marked with fluorescent quenching group；The sequence of the SEQ ID NO.1 is 5 ' GGAGTCGAGAGGGAAACAGC 3 '；The sequence of the SEQ ID NO.2 is 5 ' GCTCCCTTCCTGAGATCATTACAC 3 '.The kit susceptibility is high, detection is quick and stability is good, prevents from polluting.
Description
Technical field
The invention belongs to lung pityrosporion ovale detection technique field, and in particular to a kind of PCR detection kit of Ye Shi lungs pityrosporion ovale.
Background technology
Lung pityrosporion ovale is to parasitize a kind of opportunistic pathomycete that is extracellular, having host specificity, can be caused serious
Pneumocystis pneumonia (pneumocystispneumonia, PCP), AIDS patient's fatal rate to immunologic hypofunction is
10%~20%.However, some non-AIDS such as rheumatic arthritis, blood pain, tumour etc. is complicated by infection the increasing of PCP quantity in recent years
More, fatal rate is high by 50%.Moreover, chronic obstructive pulmonary disease (chronic obstructive pulmonary disease,
COPD) relevant with PCP, especially settling down (colonization) with bacterium colony has substantial connection.
Pneumocystis pneumonia pathogen has been considered as protozoon always since 1909, but confirmed in 1988 be it is a kind of between
Eucaryote between ascus (true) bacterium subphylum and load (true) bacterium subphylum, it is a member of fungi family.Frenkel will within 1999
Parasitizing human body causes mankind PCP lung pityrosporion ovale to be named as Ye Shi lungs pityrosporion ovale (Pneumoeystisjiroveci, PJ).Face
Bed diagnosis PCP cases rely on clinical manifestation, make a definite diagnosis the trophozoite and/or packing for needing to find PJ.However, because PJ can not be external
Culture, typically takes colouring method to be diagnosed, but this method positive rate is low, cumbersome time-consuming, constrains PCP aetology
Diagnosis, easily causes mistaken diagnosis and fails to pinpoint a disease in diagnosis.
In recent years, external multinomial research is thought, polymerase chain reaction (PCR) method can significantly improve the quick of PCP diagnosis
Perception and specificity, and just can discover whether to infect such fungi in disease early stage, one of goldstandard of diagnosis can be turned into.So
And because the bacterium is between fungi and protozoon, form needs to extract the core of the bacterium similar to protozoon using PCR
Sour DNA, to destroy such fungi technically has certain, and the domestic PCR detection method about the bacterium is seldom reported.Patent
Application No. 201110086848.7 describes adds gel electrophoresis to detect lung pityrosporion ovale with PCR, but this method operation is numerous
Trivial, open operation is also easy to pollute, and is not suitable for clinically applying.
The content of the invention
The present invention proposes a kind of PCR detection kit of Ye Shi lungs pityrosporion ovale, the kit susceptibility is high, detection is quick and
Stability is good, and PCR amplifications start all to be afterwards hands-off operation, can effectively prevent from polluting, the addition of UNG enzymes, can more prevent two
The pollution of secondary amplification.
The technical proposal of the invention is realized in this way：
A kind of PCR detection kit of Ye Shi lungs pityrosporion ovale, including nucleic acid extraction liquid, PCR reaction solutions, enzyme mixation, sun
Property control and negative control, the positive control be the specific sequence of artificial synthesized Ye Shi lung pityrosporion ovales, the feminine gender is right
According to for aqua sterilisa, archaeal dna polymerase containing Taq and UNG enzymes in the enzyme mixation；The PCR reaction solutions include being directed to Ye Shi lung spores
The pair for amplification primer and a specific amplification probe of the target sequence design of daughter bacteria, pair for amplification primer is by positive amplimer
Formed with direction amplimer, the positive amplimer has the nucleotide sequence shown in SEQ ID NO.1, described reversely to expand
Increasing primer has the nucleotide sequence shown in SEQ ID NO.2, and the specific amplification probe is by annulus sequence and two short handles
Sequence forms, and two short handle sequences are located at the both ends of the annulus sequence respectively, and the annulus sequence has SEQ ID NO.4 institutes
The nucleotide sequence shown, short handle sequence are rearranged by 4~8 GC bases mixing, and 5 ' ends of specific amplification probe are marked with
Fluorescent reporter group, the end of specific amplification probe 3 ' are marked with fluorescent quenching group；
The sequence of the SEQ ID NO.1 is 5 '-GGAGTCGAGAGGGAAACAGC-3 '；
The sequence of the SEQ ID NO.2 is 5 '-GCTCCCTTCCTGAGATCATTACAC-3 '；
The sequence of the SEQ ID NO.4 is 5 '-GATGGCTGTTTCCAAGCCCACTTC-3 '.
Preferably, the short handle that the specific amplification probe 5 ' is held has the nucleotide sequence described in SEQ ID NO.5, institute
The short handle for stating the end of specific amplification probe 3 ' has nucleotide sequence described in SEQ ID NO.6；The sequence of the SEQ ID NO.5
It is classified as 5 '-GGCGGC-3 '；The sequence of the SEQ ID NO.6 is 5 '-GCCGCC-3 '.
Preferably, the specific amplification probe has the nucleotide sequence shown in SEQ ID NO.3, the SEQ ID
NO.3 sequence is 5 '-GGCGGCGATGGCTGTTTCCAAGCCCACTTCGCCGCC-3 '；The 5 ' of the specific amplification probe
It is FAM that end, which is marked with fluorescent reporter group, and it is DABCYL that 3 ' ends, which are marked with fluorescent quenching group,.
Preferably, the nucleic acid extraction liquid is made up of cleaning fluid and lysate, and the lysate is by component A and B component group
Into the component A is mainly enzyme and solution made of surfactant, and the B component is Sha's graceful and lauryl sodium sulfate of ancient India
Ethanol solution, one or more of the enzyme in lysozyme, cellulase and glusulase, the surfactant is
Buddhist Sha is graceful.The nucleic acid extraction of the more common nucleic acid extraction liquid of the nucleic acid extraction liquid is more efficient, than used in 201110086848.7
Nucleic acid extraction liquid it is more preferable.
Preferably, the concentration of lysozyme is 1~15mg/mL in the component A, 1~15mg/mL of cellulase, glusulase 1
~15mg/mL, Sha's graceful concentration of ancient India is 0.001wt%~0.02wt%；In the B component Sha's graceful concentration of ancient India for 0.002wt%~
0.5wt%, lauryl sodium sulfate 0.002wt%~0.5wt%.
Preferably, there is the specific spy of internal standard of the interior label primer and humanized of a pair of humanizeds in the PCR reaction solutions
Pin, the interior label primer of a pair of humanizeds are made up of positive interior label primer and reverse interior label primer, and positive interior label primer has SEQ
Nucleotide sequence described in ID NO.7, reverse interior label primer have the nucleotide sequence described in SEQ ID NO.8, humanized's
Internal standard specific probe has the nucleotide sequence described in SEQ ID NO.9,5 ' end marks of the internal standard specific probe of humanized
Note has fluorescent reporter group, and the end of internal standard specific probe 3 ' of humanized is marked with fluorescent quenching group；
The sequence of the SEQ ID NO.7 is 5 '-CTGAGGAGAAGTCTGCCGTTAC-3 '；
The sequence of the SEQ ID NO.8 is 5 '-CACATGCCCAGTTTCTATT GG-3 '；
The sequence of the SEQ ID NO.9 is 5 '-GCAAGGTGAACGTGGATGAAG-3 '.
Preferably, the internal standard specific probe of humanized 5 ' end be marked with fluorescent reporter group be selected from HEX, JOE, ROX,
TET, Texas Red, CY3 or CY5, the end of internal standard specific probe 3 ' of humanized are marked with fluorescent quenching group and are selected from
Tamra, BHQ1 or BHQ2.
Preferably, the target sequence of the Ye Shi lungs pityrosporion ovale is selected from Ye Shi lung pityrosporion ovale mitochondria large subunit rRNA genes
Conserved sequence, the conserved sequence of ITS genes, the conserved sequence of HSP70 genes, DHPS bases on Ye Shi lung pityrosporion ovale Matrix attachment regions
The conserved sequence of cause or the conserved sequence of DHFR genes.
Beneficial effects of the present invention：
1st, the patent of invention of the present invention contrast patent No. 201110086848.7, the method for detection is different, reagent of the present invention
The susceptibility of box is high, and detection is quick, and stability is good.
2nd, the present invention is easy to operate, and PCR amplifications start all to be afterwards hands-off operation, can effectively prevent from polluting, UNG enzymes
Add, can more prevent the pollution of secondary amplification.
3rd, kit of the present invention introduces the control of complete series monitoring, is extracted from sample, and augmentation detection to the end is each
Step can policer operation and experimental result quality, so as to ensure the accuracy of clinical effectiveness and reliability.
4th, optimization design has been done in terms of nucleic acid extraction of the present invention, nucleic acid extraction is more efficient.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the required accompanying drawing used in technology description to be briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, on the premise of not paying creative work, can be with
Other accompanying drawings are obtained according to these accompanying drawings.
Fig. 1 is the sample negative findings schematic diagram of kit of the present invention detection；
Fig. 2 is a kind of positive findings schematic diagram of sample of kit of the present invention detection；
Fig. 3 is another this positive findings schematic diagram of sample of kit of the present invention detection.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation describes, it is clear that described embodiment is only part of the embodiment of the present invention, rather than whole embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other under the premise of creative work is not made
Embodiment, belong to the scope of protection of the invention.
The target spot of embodiment 1 selects
By taking Ye Shi lung pityrosporion ovale mitochondrial genomes sequences JX499145.1 as an example, choose big sub- in the mitochondrial genomes
Base rRNA (mtLSUrRNA) sequence be template be used as reference sequences, preferably the sequence be because of mitochondria copy number with respect to the bacterium
Will more several times for genome copy numbers, therefore, with regard to more, the bacterium for the DNA of unit extraction template amount Relative gene group
Extracting genome DNA is inherently highly difficult, so, the gene on mitochondria is chosen as reference gene, can undoubtedly be greatly improved
The amplification efficiency of follow-up template.Large subunit rRNA (mtLSUrRNA) sequence is as follows in mitochondrial genomes：
AAAGAAGTTAATAAGGATAACTAGCTAGTATATTTTAGGAGGTTTTATACCTAATTCATGATTATACAT
ATGTTATCATGTAACTCCGAGAAGGAAACTTTATTAAGATTACGAAGTGAACTGAAACATCTTAGTAACTTTAGGAA
TAGAAATCAACCGAGATTTTGTGAGTAGTGGTGAGCGAAAGCAAAGTAGCCTATGCATTTATTGATAGTAATATTGT
CATTATTAATCTGACTCTATGAGTTGGAATCTTATAACTGAGATTTAATTTCATGATAAAAAGTTGGAAACCTTTCA
ATAGAATAGATAGAATAGTAATGTTCTTGATATGTGGAAGCGAAAGATGGTGAAAGCCCATGATCCATGAAGATAAA
TGTTATTTAAGTAGAACGAGGTTAATCTTGTTTGAATACAGGGGATCTATCCTCTAAGGCTAAATATAGTATACTAA
GCGATAGTGAAGAGTACCGTGAGGGAAAGTTGAAAAGAATATAATAAGGAGTATTATCTTTATTAAGTAAGTGAAAT
AGATCTTGAATTATTAACTTTATGAGCAGCCGGAGGACTTAGGTCTGACGGTGTACCTTTTGCATAATGGGTCAGCG
AGTTAATATTAAATGTAAGTAGTGATACCTAAAGAAATTGATTCTGATAAGGAGTATAATATTTAATATTAGACCCG
AAACCTAGTGATCTTACTATGATCAGATGAAAGATTGAACTGGTGTACGTTGCAAAGTACTCAGATGAATTGTGGTA
AGGAGTGAAATACAAATCGGACTAGGATATAGCTGGTTTTCTGCGAAATCTATTTTGGTAGATGACTTGTTATTATT
TGTAGTGGGTATAGCACTGAATATCTAACTTATGTTAGAAGGGAGTATGAAGGTACTTACTTTGGATATTTAATCTC
AGAATAGCTATTAATATATGATGAGTTATCAGACTTCTTGCGATAAGGTGAGGAGTCGAGAGGGAAACAGCCCAGAA
TAATATATAAAGTTCCAAAATTGTTATTGAGTGAATTAAAAGAAGTTTTCTTTCGTAGACAGTCAAGAAGTGGGCTT
GGAAACAGCCATCTTATAATAAACACGTAATAGTGTAATGATCTCAGGAAGGGAGCGCTTAAAATATACGGATCTAA
ATAACATACTGAAATATTATTATATTATTATTAATCAGTGTATGCTGGATACTTGTAAGTGACTACGGGGTTTTTGT
ATATAGGTTTAGTTGTAAAGAGTATTAATAATGATATGGGTAGCAGAACATTTAGTAAATAGATGAAGAACAGTATT
TTATTGTTTGGATATAACTAAAGAGAGAATGCTGACATGAGTAACGTTAAAGTGGGTGAGAATCCTGCTCGCCGGAA
ACAGAAGGGTTTTATGGTAAAGCTTAACTACCATAAGTGAGTGCGGTCTCTAACAGTATCTCGAAGGAGTAATGGAT
GAGTAGAATATAAGTAAAAATTATGGTTGTGATGGGTTATTATTCATTATAATTGTAATTCGGGAAAAGGTATATTT
ATAAACCGTACTAAAACCGACACAGGTCTGTGGATATTAATGTATTAAGGCGTATGAGAGAATTATTGCGAAGGAAC
TCGGCAAAATAATCTCGTAATTTAGAGACAAGAGGTGCCTATTAGTGGGAATTCCTATATGTATATATATGTTATGT
ATATGTAGGAACCTTCTTAAGGTGTCACTAAAAAATGTTGTACAACTGTTTACTTAAAACACAGTACTTTGCAAAGA
TGAGAATCTTAGTATAAAGTATGAGATCTGCCCAATGCTAGGGAACGAACAGTTTGATTTTGAGTGAATCTTGAAAT
CGATTGAAGTTCTAGTGAATGGCGGCCTTAACTATAAGGGTCCTAAGGTAGCGAAATTCCTTGGCCGTTAAATGCGG
TCCCGCATGAATGGTTTAATGATACAACAACTGTCTCCGCAATAAACTTAGTGAAATTGGAATAGCCGTGCAGATAC
GGTTTATGTGTAGAAAGACGGGAAGACCCTGTGCAGCTTTACTGTAGTTAGTTATTGTTATTAACTCTCACGTTGGT
AGTATATAAGGTAGTTGATTAGACATAAATGAAATACCTTTATCGTGGGCTGCGATGACTTACTGATATATCAGGAC
ATTGATTAACAGACAGTTTATGTGGGGCGCATGTCTCAAAAAGAGTATCTGGGATGTCCTAAGGTGAAGTAAAATTG
GATGGTAACCAATCAAGTTTGAAATATTAGTTATGGTGTCTAATTGTTAGGATTCTAAATTAGAATGGGTGTCAGTA
TTGTGAGATATTGAGATCAATAGATATTTGTAATACTAATGTTAATGAATTCTTGGAAGAAGCATAATGGTATAACT
TTGCTTAACAGTGAGATTGACAGATCGATCTGACACGTAAGTGGGGCATAATGACCCTCGTATTCATAATGGAATGG
TACGAGAACAACGAATCAAAGCTACGCCAGGGATAACAGGGTTATTTCGTGCGAGAGATCCTATCGACCACGAAGTT
TGCCACCTCGATGTCGACTCAACCTATCCTCCAGAGGTAAAAGATTGGAAGGGTTTGGCTGTTCGCCAATTAAAAGG
TTACGTGAGTTGGGTTAAAAACGTTGTGAAACAGTTTGGTTCCTATCTTCTATGTATAAAAAGTTAACGAAGAATTT
ATTCCTAGTACGCAAGGATCGGAATATCTTAATCCCTGGTTTAATTGTTGTGAAAGCATGGCAATAAAGCTAAATTA
GGTAGGAATAAAATTTGAAAGCATCTAAAAATTGAAATCCCTCTTCAGAAACTTGATATTTTCGTTAAAGATGATGA
CGTGGATAGGCTTTAGCTGTAAGAATAGTAATGTTCGAAGGTATAAAGTACTAATAAAAGATGAAACTGAAATAT
Embodiment 2 is directed to shot design specific primer and probe
Nucleotide polymorphisms in 85,248,288 3 sites easily be present in document report, the sequence, thus design primer and
During probe, these sites are avoided as far as possible, then select remaining region than more conservative sequence to be designed, during design, as far as possible
Ensure selected region G/C content between 40%-60%, amplification length is between 100bp-150bp, and primer Tm is at 50 DEG C -65
Between DEG C, probe Tm is higher than primer 0 DEG C -5 DEG C.With PrimerBLAST inside Primer5 and NCBI, Double Selection is carried out, it is final to obtain
Proper to following pair for amplification primer and specific amplification probe, positive amplimer has shown in SEQ ID NO.1
Nucleotide sequence, the reversely amplimer have the nucleotide sequence shown in SEQ ID NO.2, and specific amplification probe has
Nucleotide sequence shown in SEQ ID NO.3.
The sequence of the SEQ ID NO.1 is 5 '-GGAGTCGAGAGGGAAACAGC-3 '；
The sequence of the SEQ ID NO.2 is 5 '-GCTCCCTTCCTGAGATCATTACAC-3 '；
The sequence of the SEQ ID NO.3 is
5′-GGCGGCGATGGCTGTTTCCAAGCCCACTTCGCCGCC-3′。
The preparation of the nucleic acid extraction liquid of embodiment 3
Nucleic acid extraction liquid is made up of cleaning fluid and lysate, and cleaning fluid is common phosphate buffer, and lysate is divided into A
With two kinds of components of B.Component A mainly contains enzyme mixation, including lysozyme, cellulase, glusulase etc. it is therein a kind of or
Several to combine, surfactant Buddhist Sha in addition also containing low concentration is graceful；B component mainly contains chemical cleavage agents, contains
Buddhist Sha is graceful, lauryl sodium sulfate, ethanol solution etc..
A kind of preferable formula is as follows：
Component A agent prescription is：Lysozyme 10mg/mL, cellulase 5mg/mL, surfactant Sha graceful concentration of ancient India are
0.002%, prepared with 1 × phosphate buffer or 1 × TE.
B component agent prescription is：Surfactant Sha graceful concentration 0.01% of ancient India, lauryl sodium sulfate 0.01%, is used
20% ethanol solution (V/V) is prepared.
Another preferable formula is as follows：
Component A agent prescription is：Lysozyme 5mg/mL, cellulase 5mg/mL, surfactant Sha graceful concentration of ancient India are
0.002%, prepared with 1 × phosphate buffer or 1 × TE.
B component agent prescription is：Surfactant Sha graceful concentration 0.008% of ancient India, lauryl sodium sulfate 0.006%, is used
20% ethanol solution (V/V) is prepared.
Another preferable formula is as follows：
Component A agent prescription is：Lysozyme 5mg/mL, cellulase 5mg/mL, glusulase 5mg/mL, surfactant Sha
Graceful concentration of ancient India is 0.002%, is prepared with 1 × phosphate buffer or 1 × TE.
B component agent prescription is：Surfactant Sha graceful concentration 0.005% of ancient India, lauryl sodium sulfate 0.005%, is used
20% ethanol solution (V/V) is prepared.
The use of 4 kits of embodiment
The component that this kit provides has 10 × phosphate buffer, lysate A, lysate B, PCR reaction solution, enzyme mixing
Liquid, negative Quality Control, positive quality control.
(1) Ye Shi lungs pityrosporion ovale nucleic acid extraction
Protozoon one kind was belonged in the past because the classification of Ye Shi lungs pityrosporion ovale is upper, fungi one kind was belonged to now, for this kind of
Biotinylated nucleic acid extraction is a very big obstacle, and cell cracks the whether abundant efficiency for being directly connected to nucleic acid extraction.Therefore,
The present invention specially develops a kind of lysate of high efficiency extraction nucleic acid for the bacterium.
First collect Patients with Lung washing liquid, collected after centrifugation precipitation (sputum sample with 1 × phosphate buffer dilution after from
The heart), add 500 1 × phosphate buffers of μ L cleaning precipitation once, then add lysate A50 μ L, after the precipitation that suspends, 37 DEG C
More than 30min is incubated, is then directly added into the μ L of lysate B 50, whirlpool concussion 5min again, then 95 DEG C of heating 5min, then
Stay supernatant stand-by after 12000rpm centrifugations 2min.
(2) detection of nucleic acids
It is following (50 μ L systems) per person-portion nucleic acid amplification reaction system, it is shown in Table 1：
The nucleic acid PCR amplification reaction system of table 1
Above-mentioned PCR amplification system enters performing PCR response procedures and is shown in Table 2：
The PCR response procedures of table 2
Note：* position is the fluorescent collecting stage.
As a result judge：(1) negative control should be without Ct values, and positive control has Ct values；(2) internal reference should be positive, but sample
If strong positive, negative findings can occur in internal reference.Wherein sample negative findings as shown in figure 1, positive findings such as Fig. 2 and
Shown in Fig. 3.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
God any modification, equivalent substitution and improvements made etc., should be included in the scope of the protection with principle.
Claims (8)
1. a kind of PCR detection kit of Ye Shi lungs pityrosporion ovale, it is characterised in that including nucleic acid extraction liquid, PCR reaction solutions, enzyme
Mixed liquor, positive control and negative control, the positive control be artificial synthesized Ye Shi lung pityrosporion ovales specific sequence, institute
It is aqua sterilisa to state negative control, archaeal dna polymerase containing Taq and UNG enzymes in the enzyme mixation；The PCR reaction solutions include being directed to
The pair for amplification primer and a specific amplification probe of the target sequence design of Ye Shi lung pityrosporion ovales, pair for amplification primer is by forward direction
Amplimer forms with direction amplimer, and the positive amplimer has the nucleotide sequence shown in SEQ ID NO.1, institute
Stating reverse amplimer has a nucleotide sequence shown in SEQ ID NO.2, the specific amplification probe by annulus sequence and
Two short handle sequence compositions, two short handle sequences are located at the both ends of the annulus sequence respectively, and the annulus sequence has SEQ
Nucleotide sequence shown in ID NO.4, short handle sequence are rearranged by 4~8 GC bases mixing, and the 5 ' of specific amplification probe
End is marked with fluorescent reporter group, and the end of specific amplification probe 3 ' is marked with fluorescent quenching group；
The sequence of the SEQ ID NO.1 is 5 '-GGAGTCGAGAGGGAAACAGC-3 '；
The sequence of the SEQ ID NO.2 is 5 '-GCTCCCTTCCTGAGATCATTACAC-3 '；
The sequence of the SEQ ID NO.4 is 5 '-GATGGCTGTTTCCAAGCCCACTTC-3 '.
2. the PCR detection kit of Ye Shi lungs pityrosporion ovale according to claim 1, it is characterised in that the amplifying specific
Property the short handle held of probe 5 ' there is nucleotide sequence described in SEQ ID NO.5, the short handle that the specific amplification probe 3 ' is held
With the nucleotide sequence described in SEQ ID NO.6；The sequence of the SEQ ID NO.5 is 5 '-GGCGGC-3 '；The SEQ
ID NO.6 sequence is 5 '-GCCGCC-3 '.
3. the PCR detection kit of Ye Shi lungs pityrosporion ovale according to claim 2, it is characterised in that the amplifying specific
Property probe there is nucleotide sequence shown in SEQ ID NO.3, the sequence of the SEQ ID NO.3 for 5 '-
GGCGGCGATGGCTGTTTCCAAGCCCACTTCGCCGCC-3′；5 ' ends of the specific amplification probe are marked with fluorescence report
Announcement group is FAM, and it is DABCYL that 3 ' ends, which are marked with fluorescent quenching group,.
4. the PCR detection kit of Ye Shi lungs pityrosporion ovale according to claim 1, it is characterised in that the nucleic acid extraction
Liquid is made up of cleaning fluid and lysate, and the lysate is made up of component A and B component, and the component A is mainly that enzyme is lived with surface
Property solution made of agent, the B component is that Sha is of ancient India graceful with the ethanol solution of lauryl sodium sulfate, the enzyme be selected from lysozyme,
Cellulase and the one or more in glusulase, the surfactant are that Buddhist Sha is graceful.
5. the PCR detection kit of Ye Shi lungs pityrosporion ovale according to claim 4, it is characterised in that molten in the component A
The concentration of bacterium enzyme is 1~15mg/mL, 1~15mg/mL of cellulase, 1~15mg/mL of glusulase, and Sha's graceful concentration of ancient India is
0.001wt%~0.02wt%；Sha's graceful concentration of ancient India is 0.002wt%~0.5wt% in the B component, lauryl sodium sulfate
0.002wt%~0.5wt%.
6. the PCR detection kit of Ye Shi lungs pityrosporion ovale according to claim 1 or 2, it is characterised in that the PCR is anti-
Answer the interior label primer for also having a pair of humanizeds in liquid and the internal standard specific probe of humanized, the interior label primer of a pair of humanizeds by
Positive interior label primer forms with reverse interior label primer, and positive interior label primer has the nucleotide sequence described in SEQ ID NO.7, instead
There is the nucleotide sequence described in SEQ ID NO.8 to interior label primer, the internal standard specific probe of humanized has SEQ ID
Nucleotide sequence described in NO.9,5 ' ends of the internal standard specific probe of humanized are marked with fluorescent reporter group, humanized's
The end of internal standard specific probe 3 ' is marked with fluorescent quenching group；
The sequence of the SEQ ID NO.7 is 5 '-CTGAGGAGAAGTCTGCCGTTAC-3 '；
The sequence of the SEQ ID NO.8 is 5 '-CACATGCCCAGTTTCTATT GG-3 '；
The sequence of the SEQ ID NO.9 is 5 '-GCAAGGTGAACGTGGATGAAG-3 '.
7. the PCR detection kit of Ye Shi lungs pityrosporion ovale according to claim 6, it is characterised in that the internal standard of humanized
5 ' ends of specific probe are marked with fluorescent reporter group and are selected from HEX, JOE, ROX, TET, Texas Red, CY3 or CY5, people
The end of internal standard specific probe 3 ' of source property is marked with fluorescent quenching group and is selected from Tamra, BHQ1 or BHQ2.
8. the PCR detection kit of Ye Shi lungs pityrosporion ovale according to claim 1, it is characterised in that the Ye Shi lungs spore
The target sequence of daughter bacteria is selected from conserved sequence, Ye Shi lung spore sclerotium bases on Ye Shi lung pityrosporion ovale mitochondria large subunit rRNA genes
Because of the conserved sequence of the upper ITS genes of group, the conserved sequence of HSP70 genes, the conserved sequence of DHPS genes or DHFR genes
Conserved sequence.
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