CN106893782B - It is a kind of to detect and identify cryptococcal target gene, primer and probe and kit - Google Patents
It is a kind of to detect and identify cryptococcal target gene, primer and probe and kit Download PDFInfo
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Abstract
It the invention discloses a kind of detection and identifies cryptococcal target gene, primer and probe and kit, belongs to the field of Medical Mycology.According to cryptococcal target gene, the primer sequence of the detection cryptococcus gene is designed as shown in SEQ ID No.3 and SEQ ID No.4 and/or SEQ ID No.6 and SEQ ID No.7, wherein SEQ ID No.3 and SEQ ID No.4 corresponds to neogenesis cryptococcus, SEQ ID No.6 and SEQ ID No.7 corresponds to lattice spy cryptococcus, can be used for two kinds of cryptococcus of specific detection.Based on quantitative fluorescent PCR principle, the present invention also provides one group of detection neogenesis cryptococcus and/or the special cryptococcal probes of lattice, and the detection kit based on primer and probe, simultaneously or two kinds of pathogenic cryptococcus: neogenesis cryptococcus dative spy cryptococcus can be detected respectively and identified in same system.Have the advantages that sensibility height, high specificity, reproducible, detection time is short.
Description
Technical field
The invention belongs to the fields of Medical Mycology, and in particular to primer and spy for cryptococcus particular target genetic test
Needle and kit.
Background technique
The type of Cryptococcus is more at present, extensive in distributed in nature, with human infection it is most close mainly have new life
Cryptococcus and Ge Te cryptococcus, they can lead to human lung or cryptococcal meningitis, whole body can also be caused to send out
Sexuality dye.Lattice spy cryptococcus once caused Vancouver, CAN area and northwestern US area eruption and prevalence, in recent years Europe and
Also there is cases of infection report in China, and in China, lattice spy cryptococcus is mainly distributed on Shanghai, Jiangsu, Fujian, Zhejiang, Guangdong, sea
There are Sporadic cases in the provinces such as south, Hebei, neogenesis cryptococcus various regions.Neogenesis cryptococcus main infection immune deficient patients, Ge Te
Cryptococcus then mainly causes immune normal population illness.It is generally believed that lattice spy cryptococcus is pathogenic and drug resistance is above new life
Cryptococcus.
Due to the atypical clinical manifestations after Cryptococcus infections, being easy mistaken diagnosis is bacterial infection, and pathogeny detection becomes
The foundation of diagnosing and treating.Although cerebrospinal fluid ink dyeing can be in the form of preliminary observation be cryptococcal, sensibility is low；Cryptococcus
Antigenic agents can quickly detect cryptococcal antigen, but cannot distinguish between two kinds of main pathogenic cryptococcus；Cryptococcus culture mirror
Fixed not only to take longer, but also most automation assessing instruments are difficult to identify neogenesis cryptococcus and Ge Te cryptococcus, lead to laboratory
It can only report cryptococcus, or be possible to that lattice spy cryptococcus is mistakenly reported as neogenesis cryptococcus.Correctly detects and identify new life
Cryptococcus and Ge Te cryptococcus depend on new technology and methods.
Summary of the invention
The object of the present invention is to provide a kind of target genes for cryptococcus specific detection, can be quasi- as detection target
Really detects and identify two kinds of important pathogenic cryptococcus without being misdiagnosed as bacterium.
Another object of the present invention is the detection primer and probe designed based on the specific target gene position, accurately
Identify the two different pathogenic cryptococcus types of neogenesis cryptococcus dative spy cryptococcus.
It is yet another object of the invention to provide a kind of quick detection kits, can conveniently and efficiently detect and identify new life
Cryptococcus dative spy cryptococcus.
To achieve the goals above, technical solution of the present invention is as follows:
The present invention provides a kind of cryptococcal specific target gene of detection, target-gene sequence such as SEQ ID No.1 and/or SEQ
Shown in ID No.2, wherein SEQ ID No.1 corresponds to neogenesis cryptococcus, and SEQ ID No.2 corresponds to lattice spy cryptococcus, specific
Target-gene sequence is applied to preparation cryptococcus detection reagent.
For detecting the primer and probe of neogenesis cryptococcus, arranged with nucleotides sequence shown in SEQ ID No.1 as stencil design,
It is arranged with nucleotides sequence shown in SEQ ID No.3 and SEQ ID No.4 as primer, is with the column of nucleotides sequence shown in SEQ ID No.5
Probe, particular sequence are as follows:
Upstream primer CN2F (5 '-cttactcggcacaactccac-3 '): SEQ ID No.3；
Downstream primer CN2R (5 ' cggtgaatcaaatgacgtacctc-3 '): SEQ ID No.4；
Probe CN2P (HEX-ccaccgatcacacatccacgtgctc-BHQ1): SEQ ID No.5；
Wherein 5 ' end mark fluorescent reporter groups of probe sequence, 3 ' end label quenching groups.
For detecting the kit of neogenesis cryptococcus, including for detecting neogenesis cryptococcus primer CN2F, CN2R and spy
Needle CN2P further includes buffer, MgCl2, dNTPs, Taq archaeal dna polymerase etc..
For detecting the special cryptococcal primer and probe of lattice, arranged with nucleotides sequence shown in SEQ ID No.2 as stencil design,
It is arranged with nucleotides sequence shown in SEQ ID No.6 and SEQ ID No.7 as primer, is with the column of nucleotides sequence shown in SEQ ID No.8
Probe, particular sequence are as follows:
Upstream primer CG3F (5 '-atcaccttcacccaggagaa-3 '): SEQ ID No.6；
Downstream primer CG3R (5 '-ggaaatcaaacgacttacg-3 '): SEQ ID No.7；
Probe CG2P (FAM-aggagaaggagggtgctcccgttac-BHQ1): SEQ ID No.8；
Wherein 5 ' end mark fluorescent reporter groups of probe sequence, 3 ' end label quenching groups.
For detecting the special cryptococcal kit of lattice, including for detecting lattice special cryptococcal primer CG3F, CG3R and spy
Needle CG2P further includes buffer, MgCl2, dNTPs, Taq archaeal dna polymerase etc..
For detecting cryptococcal kit, neogenesis cryptococcus and Ge Te cryptococcus can be detected simultaneously, including for examining
Primer CN2F, the CN2R and probe CN2P for surveying neogenesis cryptococcus further include for detecting lattice special cryptococcal primer CG3F, CG3R
It further include buffer, MgCl with probe CG2P2, dNTPs, Taq archaeal dna polymerase etc..
Compared with prior art, the invention has the following advantages:
Key to the invention is that the selection of specific target gene position and the design of primer and probe.It is experimentally confirmed that using
Designed primer and probe detects and identifies that neogenesis cryptococcus and the cryptococcal specificity of Ge Te and sensibility are preferable.This hair
The bright sequence difference by comparing between neogenesis cryptococcus and Ge Te cryptococcus different genotype and its with other fungal genes, choosing
The sequence fragment at copper-zinc superoxide dismutase position is selected as target gene, primer and probe is designed, makes to neogenesis cryptococcus
With sensibility height, the high specificity during lattice spy cryptococcus progress detection and identification.Detection time is short, and the present invention will test newly
Required raw material is placed in same reaction system when raw cryptococcus and Ge Te cryptococcus, can be detected respectively and be identified neogenesis cryptococcus
With lattice spy cryptococcus, can also detect simultaneously, specify Species of Pathogens, make to carry out neogenesis cryptococcus and Ge Te cryptococcus detection and
The time of identification greatly shortens, and helps clinical diagnosis, wins treatment time.
Detailed description of the invention
Fig. 1 shows the neogenesis cryptococcus in the amplification curve of various concentration DNA (500ng, 50ng, 5ng, 0.5ng)；
Fig. 2 shows the lattice spy cryptococcus in the amplification curve of various concentration DNA (500ng, 50ng, 5ng, 0.5ng)；
Fig. 3 shows the specific amplification curve of the neogenesis cryptococcus, and parallel test detects escherichia coli, golden yellow grape
Coccus, streptococcus pneumonia, B race streptococcus, haemophilus influenzae, mycobacterium tuberculosis and Candida glabrata, S. cervisiae, Ah
Sa Xi trichosporon bacteria, result are negative (curve below baseline)；
Fig. 4 shows the special cryptococcal specific amplification curve of the lattice, and parallel test detects escherichia coli, golden yellow grape
Coccus, streptococcus pneumonia, B race streptococcus, haemophilus influenzae, mycobacterium tuberculosis and Candida glabrata, S. cervisiae, Ah
Sa Xi trichosporon bacteria, result are negative (curve below baseline)；
Fig. 5 shows that the neogenesis cryptococcus repeats the specific amplification curve of three reactions；
Fig. 6 shows that the lattice spy cryptococcus repeats the specific amplification curve of three reactions；
When Fig. 7 shows 60 DEG C of the annealing temperature, the amplification curve (Ct:25.32) of neogenesis cryptococcus；
When Fig. 8 shows 60 DEG C of the annealing temperature, the special cryptococcal amplification curve (Ct:24.51) of lattice；
When Fig. 9 shows 62 DEG C of the annealing temperature, the amplification curve (Ct:24.63) of neogenesis cryptococcus；
When Figure 10 shows 62 DEG C of the annealing temperature, the special cryptococcal amplification curve (Ct:24.56) of lattice；
When Figure 11 shows 58 DEG C of the annealing temperature, the amplification curve (Ct:25) of neogenesis cryptococcus；
When Figure 12 shows 58 DEG C of the annealing temperature, the special cryptococcal amplification curve (Ct:23.83) of lattice.
Specific embodiment
In order to enable those skilled in the art to better understand the solution of the present invention, below by specific embodiment to this hair
Primer, probe and the method that the neogenesis cryptococcus of bright offer and Ge Te cryptococcus quickly detect are described further.
Embodiment 1: target gene, primer, probe and its kit of neogenesis cryptococcus dative spy cryptococcus detection and identification
Step 1: the selection of target gene position
The present inventor by compare between neogenesis cryptococcus and Ge Te cryptococcus different genotype and its and other
The sequence difference of fungal gene selects the sequence fragment at copper-zinc superoxide dismutase position as target gene position, the gene
Segment is peculiar for cryptococcus, and has differences between neogenesis cryptococcus and Ge Te cryptococcus.A kind of obtained detection new life is hidden
The nucleotide sequence of coccus and Ge Te cryptococcus target gene is as shown in SEQ ID No.1 and/or SEQ ID No.2, wherein SEQ
ID No.1 corresponds to neogenesis cryptococcus, and SEQ ID No.2 corresponds to lattice spy cryptococcus.
Step 2: the design of primer and probe
Design of primers is carried out using primer design software for the target gene position of selection.Pass through comparative analysis repeatedly
And the preliminary experiment of early period.It is final to obtain following two pairs of primers and two probes.
For detecting the primer and probe of neogenesis cryptococcus, particular sequence is as follows:
Upstream primer CN2F:SEQ ID No.3；
Downstream primer CN2R:SEQ ID No.4；
Probe CN2P:SEQ ID No.5；
Wherein 5 ' end mark fluorescent reporter groups of probe sequence, 3 ' end label quenching groups.
For detecting the special cryptococcal primer and probe of lattice, particular sequence is as follows:
Upstream primer CG3F:SEQ ID No.6；
Downstream primer CG3R:SEQ ID No.7；
Probe CG2P:SEQ ID No.8；
Wherein 5 ' end mark fluorescent reporter groups of probe sequence, 3 ' end label quenching groups.
Step 3: fluorescence quantitative PCR detection
A. experimental material
A. experimental strain
Experimental strain uses the neogenesis cryptococcus for passing through gene sequencing confirmation and Ge Te cryptococcus clinical strains and warp
(including escherichia coli, staphylococcus aureus, streptococcus pneumonia, B race streptococcus, influenza are thermophilic for other bacteriums of general survey
Blood bacillus, mycobacterium tuberculosis) and fungi (including Candida glabrata, S. cervisiae, Trichosporon asahii).
B. reagent and instrument
DNA, which is extracted, uses Tiangeng company yeast genes extracts kit, with the EPOCH microplate of BioTek company
Reader detects its OD260 absorbance value, and obtains its concentration and purity；The synthesis of primer and probe is by the limited public affairs of bioengineering
Department completes；Archaeal dna polymerase and dNTPs use the Light cycler of Roche company purchased from Takara company fluorescence quantitative PCR instrument
480。
B. experimental method
A.DNA is extracted and concentration mensuration: extracting DNA according to Tiangeng company yeast genes extracts kit specification, uses
The EPOCH microplate reader of BioTek company detects its OD260 absorbance value, and obtains its concentration and purity.
B. primer is prepared: will be configured to 50 μM of preservation liquid after newly synthesized all primer and probes dissolution, then by CN2F,
CN2R, CN2P, CG3F, CG3R, CG2P are formulated as 8 μM of use liquid respectively.CN2P and CG2P will be kept in dark place.
C. reaction system: reaction system amounts to 25 μ L.It include: 10 × buffer, 2.5 μ L, 25mM MgCl23.5 μ L,
2 μ L of 2.5mM dNTPs, 8 μM of each 2 μ L of primer (CN2F, CN2R, CG3F, CG3R), 8 μM of probe (CN2P, CG2P) is respectively
1.25 μ L, DNA profiling 2 μ L, Taq archaeal dna polymerase (5u/ μ L) 0.25 μ L, complement to 25 μ L with water.Fluorescent quantitative PCR is anti-
The concentration for answering each primer (CN2F, CN2R, CG3F, CG3R) in system is 0.64 μM, and the concentration of probe (CN2P, CG2P) is 0.4 μ
M。
D. 95 DEG C, 45 circulations (including 95 DEG C of 10s, 60 DEG C of 60s) reaction condition: are entered after 10min.It returns after reaction
To room temperature.
Step 4: the judgement of result
In conjunction with specific amplification curve form (there is baseline period, exponential phase, linear phase and plateau) and reach luminescence threshold
Recurring number (CT value)≤26 interpretations needed for value are the positive.
Step 5: susceptibility and repeatability detection
A. sensibility is high: the neogenesis cryptococcus of extraction and the cryptococcal template DNA of Ge Te are diluted to following concentration:
500ng, 50ng, 5ng, 0.5ng, through fluorescence quantifying PCR method detect to obtain neogenesis cryptococcus and Ge Te it is cryptococcal it is minimum can
Detectable concentration is 5ng, as depicted in figs. 1 and 2.
B. high specificity: designed primer carries out specific amplification, institute to neogenesis cryptococcus and Ge Te cryptococcus respectively
The probe of design can further identify neogenesis cryptococcus and Ge Te cryptococcus.Designed by being used from the bacterial strain for being clinically separated out
Primer, probe and method detected, obtained lattice spy cryptococcus and neogenesis cryptococcus qualification result is analyzed through gene sequencing
Confirmed.Parallel test detection escherichia coli, staphylococcus aureus, streptococcus pneumonia, B race streptococcus, influenza are bloodthirsty
Bacillus, mycobacterium tuberculosis and Candida glabrata, S. cervisiae, Trichosporon asahii, result are feminine gender, without non-spy
Specific amplification curve, such as Fig. 3 and Fig. 4.
C. reproducible: neogenesis cryptococcus (100ng) He Gete cryptococcus (90ng) to be repeated to test respectively, respective three
The result of reaction is consistent, such as Fig. 5 and Fig. 6.
D. detection time is short: annealing and elongating temperature are set as mutually synthermal in amplified reaction, and annealing time is respectively set
It is 60 DEG C, 62 DEG C and 58 DEG C, optimizes by comparison, determines 60 DEG C for best annealing and elongating temperature, such as Fig. 7, Fig. 8, Fig. 9, figure
10, shown in Figure 11 and Figure 12.Since the more general amplified reaction of circulating temperature in reaction reduces one, amplified reaction is shortened
The required time.
Step 6: the kit of neogenesis cryptococcus dative spy cryptococcus detection and identification is prepared, include: 10 in kit ×
2.5 μ L, 25mM MgCl of buffer23.5 μ L, 2.5mM dNTPs, 2 μ L, 8 μM of primer (CN2F, CN2R, CG3F, CG3R) are each
2 μ L, 8 μM of each 1.25 μ L of probe (CN2P, CG2P), DNA profiling 2 μ L, Taq archaeal dna polymerase (5u/ μ L) 0.25 μ L are mended with water
Enough to 25 μ L.
Embodiment 2: the primer of neogenesis cryptococcus genetic test and identification, probe and kit
By the special cryptococcal primer of the detection neogenesis cryptococcus dative of step 3 in embodiment 1 (CN2F, CN2R, CG3F,
CG3R the primer CN2F and CN2R of detection neogenesis cryptococcus, the special cryptococcal probe of detection neogenesis cryptococcus dative) are changed to
(CN2P, CG2P) is changed to the probe CN2P of detection neogenesis cryptococcus, and completes experiment remaining step, and designed primer pair is newborn
Cryptococcus carries out specific amplification, and designed probe can further identify neogenesis cryptococcus.
Embodiment 3: primer, probe and the kit of lattice spy cryptococcus genetic test and identification
By the special cryptococcal primer of the detection neogenesis cryptococcus dative of step 3 in embodiment 1 (CN2F, CN2R, CG3F,
CG3R the special cryptococcal primer CG3F and CG3R of detection lattice, the special cryptococcal probe of detection neogenesis cryptococcus dative) are changed to
(CN2P, CG2P) is changed to the special cryptococcal probe CG2P of detection lattice, and completes experiment remaining step, and designed primer plaid matching is special
Cryptococcus carries out specific amplification, and designed probe can further identify lattice spy cryptococcus.
Detection of pathogens is carried out to clinical infection patient using primer and probe of the invention, clinical case is verified as down
Table:
Neogenesis cryptococcus dative spy Cryptococcus infections patient relevant clinical data and bacterial strain qualification result
Detailed Jie has been carried out to the cryptococcal target gene of detection provided by the present invention, primer and probe and kit above
It continues.Used herein a specific example illustrates the principle and implementation of the invention, and the explanation of above embodiments is only
It is to be used to help understand core of the invention thought.It should be pointed out that for those skilled in the art, not
, can be with several improvements and modifications are made to the present invention under the premise of being detached from the principle of the invention, these improvement and modification are also fallen into
In the protection scope of the claims in the present invention.
SEQUENCE LISTING
<110>Chinese People's Liberation Army General Hospital
<120>a kind of to detect and identify cryptococcal target gene, primer and probe and kit
<130> P20170026
<160> 8
<170> PatentIn version 3.3
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<211> 636
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tggaatggtc aaggtacgta cagtagccct cccggttggt ctggaatatc gctcaccgca 60
ggtccccttc cttcctctgt ctctcccacc tctcgggtct ccgcgctctc ccgcccatcc 120
tcctccccta tcttattgcg caatcatgaa accacgccgg tcttcttact cggcacaact 180
ccaccgatca cacatccacg tgctcgcctc atcgccccta cgatgtttca tccaaacaat 240
gcgaaataac cattataggc tgttgttgtc ctcaagggtg aatcctacgt ccacggcact 300
gtatgcttca cccaggagtc ggaaaatgct cccgtttgca tcactggtga ggtacgtcat 360
tggattcatc gtggagtaac atccggtcgc tgacatgata atcactgatc agattaagga 420
catggacgct gacgccaagc gaggcatgca cgtccacgag tttggagaca acaccaacgg 480
ctgtacctct gctggccccc attacaaccc ctttaaaaag caccacggtg ctcccaccga 540
ctccgagagg cacgttggtg acctcggtat gtgcccattg tcatttcagt attgattgcg 600
tttgccgact gtatacatag gtaatatcca gacgaa 636
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ccacgtgctc gcgtcatcac cgctctatcc ctcatccaaa caaccactgt aataatccca 60
cttatacagg ctgttgctgt cctcaagggt gactcccctg tcaccggtgt tatcaccttc 120
acccaggaga aggagggtgc tcccgttacc gtttccggtg acgtaagtcg tttgatttcc 180
aatagggtaa ccatttgctg acttggtaat cactgattag atcaagaacc tcgacgccaa 240
cgccgagcga ggcttccacg tccacgagtt tggagacaac accaacggct gtacctctgc 300
cggtccccac ttcaaccccc acggcaagaa ccacggtgct ccctctgact ctgagaggca 360
cgttggtgac ctcggtatgt gccccttgcc tcatggcgtt gcttttgctg actgtatata 420
cataggtaat gtcaagactg acggcaacgg tgttgcttcc gtcaacattt ccggtaagtg 480
tttacgtcct atatatttta tactatacaa tgctaaagcc aaacctctac agacaagagt 540
ctctccctct ttggccctta ctccatcatt ggtcgaacca tcgtcgtcca cgccggtact 600
gacgatttcg gaaagggcgg caacgccgag tccctcaaga ctggtaacgc cggtgcccgt 660
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ccaccgatca cacatccacg tgctc 25
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aggagaagga gggtgctccc gttac 25
Claims (8)
1. the primer and probe for detecting neogenesis cryptococcus, which is characterized in that with target-gene sequence as shown in SEQ ID No.1
Nucleotides sequence is classified as stencil design, is arranged with nucleotides sequence shown in SEQ ID No.3 and SEQ ID No.4 as primer, with SEQ ID
Nucleotides sequence shown in No.5 is classified as probe, and particular sequence is as follows:
Upstream primer CN2F (5 '-cttactcggcacaactccac-3 '): SEQ ID No.3；
Downstream primer CN2R (5 ' cggtgaatcaaatgacgtacctc-3 '): SEQ ID No.4；
Probe CN2P (HEX-ccaccgatcacacatccacgtgctc-BHQ1): SEQ ID No.5；
Wherein 5 ' end mark fluorescent reporter groups of probe sequence, 3 ' end label quenching groups.
2. the kit for detecting neogenesis cryptococcus, which is characterized in that including described in claim 1 newborn hidden for detecting
The primer and probe of coccus.
3. according to claim 2 for detecting the kit of neogenesis cryptococcus, which is characterized in that further include buffer,
MgCl2, dNTPs, Taq DNA polymerase.
4. for detecting the special cryptococcal primer and probe of lattice, which is characterized in that with target-gene sequence as shown in SEQ ID No.2
Nucleotides sequence is classified as stencil design, is arranged with nucleotides sequence shown in SEQ ID No.6 and SEQ ID No.7 as primer, with SEQ ID
Nucleotides sequence shown in No.8 is classified as probe, and particular sequence is as follows:
Upstream primer CG3F (5 '-atcaccttcacccaggagaa-3 '): SEQ ID No.6；
Downstream primer CG3R (5 '-ggaaatcaaacgacttacg-3 '): SEQ ID No.7；
Probe CG2P (FAM-aggagaaggagggtgctcccgttac-BHQ1): SEQ ID No.8；
Wherein 5 ' end mark fluorescent reporter groups of probe sequence, 3 ' end label quenching groups.
5. for detecting the special cryptococcal kit of lattice, which is characterized in that including as claimed in claim 4 hidden for detecting lattice spy
The primer and probe of coccus.
6. according to claim 5 for detecting the special cryptococcal kit of lattice, which is characterized in that further include buffer,
MgCl2, dNTPs, Taq DNA polymerase.
7. neogenesis cryptococcus and Ge Te cryptococcus can be detected simultaneously for detecting cryptococcal kit, which is characterized in that packet
Include the primer and probe of claim 2 and 5.
8. according to claim 7 for detecting cryptococcal kit, which is characterized in that further include buffer, MgCl2、
DNTPs, Taq DNA polymerase.
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| CN108060263B (en) * | 2018-02-10 | 2020-03-06 | 杭州缔蓝生物技术有限公司 | Primer probe combination and fluorescent quantitative PCR kit for simultaneously detecting three cryptococci |
| CN111763756A (en) * | 2020-03-10 | 2020-10-13 | 首都医科大学附属北京世纪坛医院 | Method for rapidly detecting cryptococcus gatherensis |
| CN113881789B (en) * | 2021-09-30 | 2024-01-19 | 北京大学第一医院 | Probe and primer pair composition for detecting cryptococcus and detection method and application |
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| CN117265162B (en) * | 2023-10-11 | 2024-09-13 | 首都医科大学附属北京世纪坛医院 | Primer probe combination, method and kit for detecting cryptococcus garvieae |
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