CN110129466A - For detecting the LAMP primer composition and application of Candida albicans in intraocular liquid - Google Patents
For detecting the LAMP primer composition and application of Candida albicans in intraocular liquid Download PDFInfo
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- 241001225321 Aspergillus fumigatus Species 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
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Abstract
The invention discloses a kind of for detecting the LAMP primer composition and application of Candida albicans in intraocular liquid.Primer combination provided by the invention, 6 single strand dnas shown in sequence 1 to 6 form.The present invention also protects application of the primer combination in detection Candida albicans.The present invention also protects the method whether sample to be tested has infected Candida albicans that detects.Using LAMP primer and method of the invention, Candida albicans can be fast and accurately detected.
Description
Technical field
The present invention relates to a kind of for detecting the LAMP primer composition and application of Candida albicans in intraocular liquid.
Background technique
Mycotic endophthalmitis is that a kind of pair of eyesight threatens greatly infectious eye disease, and blind rate is high, invasioning delitescence
It is long, the even several months usual several weeks.Early clinic symptom is unobvious, difficult diagnosis, and therefore, clinically, especially early stage is normal
Affected adversely treatment by mistaken diagnosis.Clinical antifungal drug curative effect is insufficient and adverse reaction is larger, and fungi is destructive to ocular tissue strong
Etc. reasons lead to the poor prognosis of mycotic endophthalmitis.In recent years, corticosteroid is largely abused with postoperative and chronic inflammation to swash
Element, antibiotic and immune formulation, cause human flora to lack of proper care, and immune function decline, mycotic endophthalmitis disease incidence rises year by year.
Mycotic endophthalmitis is divided into endogenous endophthalmitis and two kinds of Exogenous endophthalmitis.Endogenous mycotic endophthalmitis is passed through by fungi
Caused by hematogenous spread arrival eye causes intraocular infection, patient, which usually has, is used for a long time hormone, antibiotic or immunosuppressor, or
The medical histories such as diabetes, malignant tumour, immune deficiency.The most common pathomycete is Candida albicans and Aspergillus.It is exogenous true
Bacterium property entophthamia is secondary to after penetrating ocular injuries, intraocular surgery or fungal keratitis, secondary to the fungi after intraocular surgery
Property entophthamia is mostly caused by Aspergillus.
Candida albicans (Monilia albican or canidia Albicans) is also known as candida albicans bacterium, works as body
Immune function or the decline of general phylactic power defensive power or normal flora mutual restrictive function imbalance then this bacterium mass propagation and change growth shape
Formula (blastomycete silk phase) intrusion cell causes disease.Candida albicans intraocular inflammation is by being directly inoculated with sense when hematogenous spread or operation
Dye shows as blurred vision, floating blind spot and ophthalmodynia.
The primarily discrete culture of the clinical detection method for fungi at present, smear for microscopic examination and serodiagnosis.These inspections
Survey method is cumbersome, and detection time is long, and positive rate is low, and is easy to happen missing inspection and false retrieval, be unable to satisfy it is clinical quickly detect want
It asks.The molecular detection technology to grow up in recent years, especially round pcr, in answering for microorganism Rapid identification and context of detection
With, for fungi it is quick detect open new approach.But that there are detection times is long, easy to pollute, false positive rate is high by PCR
Disadvantage is restricted its application.Loop-mediated isothermal amplification technique (loop-mediated isothermal
Amplification, LAMP) it is a kind of sensitive, special, the simple, fast nucleic acid amplification technologies developed in recent years, it is former
Reason is the 4-6 primer in 6-8 region to be identified, in isothermal item under the action of a kind of archaeal dna polymerase with strand-displacement activity
Under part quickly, specifically amplifying target genes, can be applied to and fast and accurately detect fungi.
Summary of the invention
The object of the present invention is to provide a kind of for detecting the LAMP primer composition and application of Candida albicans in intraocular liquid.
The present invention provides primer combinations, by primers F 3, primer B3, primers F IP, primer BIP, primer LF and primer LB group
At；
The primers F 3 is following (a1) or (a2):
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) sequence 1 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 1
The DNA molecular of identical function；
The primer B3 is following (a3) or (a4):
(a3) single strand dna shown in the sequence 2 of sequence table；
(a4) sequence 2 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 2
The DNA molecular of identical function；
The primers F IP is following (a5) or (a6):
(a5) single strand dna shown in the sequence 3 of sequence table；
(a6) sequence 3 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 3
The DNA molecular of identical function；
The primer BIP is following (a7) or (a8):
(a7) single strand dna shown in the sequence 4 of sequence table；
(a8) sequence 4 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 4
The DNA molecular of identical function；
The primer LF is following (a9) or (a10):
(a9) single strand dna shown in the sequence 5 of sequence table；
(a10) sequence 5 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 5
The DNA molecular of identical function；
The primer LB is following (a11) or (a12):
(a11) single strand dna shown in the sequence 6 of sequence table；
(a12) sequence 6 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 6
The DNA molecular of identical function.
The purposes of the primer combination is following (b1) or (b2) or (b3) or (b4):
(b1) it identifies or assists to identify whether fungi to be measured is Candida albicans；
(b2) prepare for identify or assist to identify fungi to be measured whether be Candida albicans kit；
(b3) whether detection sample to be tested has infected Candida albicans；
(b4) whether preparation has infected the kit of Candida albicans for detecting sample to be tested.
The present invention also protects the application of the primer combination, following (b1) or (b2) or (b3) or (b4):
(b1) it identifies or assists to identify whether fungi to be measured is Candida albicans；
(b2) prepare for identify or assist to identify fungi to be measured whether be Candida albicans kit；
(b3) whether detection sample to be tested has infected Candida albicans；
(b4) whether preparation has infected the kit of Candida albicans for detecting sample to be tested.
The present invention also protects the kit containing the primer pair；The purposes of the kit is following (c1) or (c2):
(c1) it identifies or assists to identify whether fungi to be measured is Candida albicans；
(c2) whether detection sample to be tested has infected Candida albicans.
The present invention also protects the preparation method of the kit, includes the steps that individually packing each primer.
The present invention, which also protects, identifies whether fungi to be measured is Candida albicans method, includes the following steps: with fungi to be measured
Genomic DNA be template, using the primer combine carry out LAMP amplified reaction, if using the primer combination can be real
It is now Candida albicans by the positive amplification of template, fungi to be measured of the genomic DNA, if not using primer combination
Being able to achieve by the positive amplification of template, fungi to be measured of the genomic DNA is non-white candida albicans.
The present invention, which also protects, identifies whether fungi to be measured is Candida albicans method, includes the following steps: to detect to be measured true
With the presence or absence of the target sequence of primer combination in the genomic DNA of bacterium, if there are the primer sets in the genomic DNA
The target sequence of conjunction, fungi to be measured are Candida albicans, if there is no the target sequences of primer combination in the genomic DNA
Column, fungi to be measured are non-white candida albicans.
The present invention also protects whether detection sample to be tested has infected the method for Candida albicans, include the following steps: with to
The total DNA of test sample sheet is template, is combined using the primer and carries out LAMP amplified reaction, if using primer combination can be with
It realizes and has infected Candida albicans by the positive amplification of template, sample to be tested of the total DNA, if combined using the primer
It can not achieve and do not infect Candida albicans by the positive amplification of template, sample to be tested of the total DNA.
The present invention also protects whether detection sample to be tested has infected the method for Candida albicans, includes the following steps: to detect
With the presence or absence of the target sequence of primer combination in the total DNA of sample to be tested, if there are primer combinations in the total DNA
Target sequence, sample to be tested infected Candida albicans, if in the total DNA there is no the primer combination target sequence,
Sample to be tested does not infect Candida albicans.
It in any description above method, can specifically judge by the following method " positive amplification ": be detected with fluorescent PCR instrument glimmering
Optical signal is then positive amplification if there is positive amplification curve.The positive amplification curve concretely S type amplification curve.
Any description above is carried out in the reaction system of LAMP amplification using primer combination, each primer in primer combination
Concentration are as follows: 0.5 μM of F3,0.5 μM of B3,2 μM of FIP, 2 μM of BIP, 1 μM of LF, 1 μM of LB.
Any description above carries out the reaction system of LAMP amplification concretely using primer combination: 1 μ L 10 ×
ThermoPol Buffer, 1.6 μ L 5M glycine betaines, 0.1 μ L 50mg/ml BSA, 0.4 μ L 100mMMgSO4, 0.3 μ L 20 ×
EvaGreen, 0.15 μ L 100mM dNTPs, 0.4 μ L 8U/ml Bst archaeal dna polymerase large fragment, 1 μ L primer mixture (F3,
The mixture of B3, FIP, BIP, LF and LB), template DNA (50pg-50ng, concretely 2ng) uses ddH2O complements to 10 μ L.
Any description above LAMP amplification response procedures concretely: 65 DEG C of constant temperature 50min.
Any description above sample to be tested concretely eye clinical sample culture, ocular fluid body or aspirate.
Any description above fungi to be measured concretely Candida albicans, aspergillus fumigatus, Aspergillus flavus or Candida glabrata.
The present invention provides a kind of for detecting the LAMP primer composition and application of Candida albicans in intraocular liquid.The present invention
The LAMP primer composition includes outer primer F3 and B3, inner primer FIP and BIP and ring primer LF and LB, specific can be known
Six isolated areas on other target sequence are expanded, and have high specific.It, can be fast using LAMP primer and method of the invention
Speed, accurate detection go out Candida albicans.
Detailed description of the invention
Fig. 1 is the LAMP amplification curve diagram of embodiment 3.
Fig. 2 is the LAMP amplification curve diagram of embodiment 5.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
Candida albicans: ATCC 90028.
Aspergillus fumigatus: ATCC 10894.
Aspergillus flavus: ATCC 16870.
Candida glabrata: ATCC 2001.
Embodiment 1, design of primers and preparation
It carries out a large amount of sequence analyses, compare several primers obtained for identifying Candida albicans.By each primer into
Row preliminary experiment compares the performances such as sensitivity, specificity, finally obtains the LAMP primer group for identifying Candida albicans.
For identifying the primer sets of Candida albicans, including 2 outer primers (F3, B3), 2 inner primers (FIP, BIP) and 2
Ring primer (LF, LB), each primer sequence are (5 ' → 3 ') as follows:
F3 (sequence 1 of sequence table): CgATACgTAATATgAATTgCAgAT；
B3 (sequence 2 of sequence table): CCATTgTCAAAgCgATCC；
FIP (sequence 3 of sequence table): AggCATgCCCTCCggAATACTCgTgAATCATCgAATCTTTgAAC；
BIP (sequence 4 of sequence table): gAgCgTCgTTTCTCCCTCAAACCgCCTTACCACTACCgTCTT；
LF (sequence 5 of sequence table): AgAgggCgCAATgTgC；
LB (sequence 6 of sequence table): TgTTgAgCAATACgACTTgggTTTg.
Embodiment 2, detection method are established
1, it extracts the genomic DNA of fungi to be measured or extracts the total DNA of biological sample to be measured.
2, the DNA for taking step 1 to obtain carries out LAMP expansion using the LAMP primer group prepared in embodiment 1 as template
Increase.
The reaction system of LAMP amplification: 1 μ 10 × ThermoPol of L Buffer, 1.6 μ L 5M glycine betaines, 0.1 μ L
50mg/ml BSA, 0.4 μ L 100mM MgSO4, 0.3 μ L 20 × EvaGreen, 0.15 μ L 100mM dNTPs, 0.4 μ L 8U/
Ml Bst archaeal dna polymerase large fragment, 1 μ L primer mixture (mixture of F3, B3, FIP, BIP, LF and LB), 2ng template
DNA uses ddH2O complements to 10 μ L.The concentration of each primer is as follows in reaction system: 0.5 μM of F3,0.5 μM of B3,2 μM of FIP, 2 μ
M BIP、1μM LF、1μM LB。
The response procedures of LAMP amplification: 65 DEG C of constant temperature 50min.In reaction process, using fluorescent PCR instrument detection fluorescence letter
Number.
If using the available positive amplification curve of LAMP primer group, show fungi to be measured be Candida albicans or
Sample to be tested contains Candida albicans, if positive amplification curve cannot be obtained, shows that fungi to be measured is not Candida albicans
Or sample to be tested does not contain Candida albicans.
Embodiment 3, specificity
Bacterium to be measured: Candida albicans, aspergillus fumigatus, Aspergillus flavus and Candida glabrata.
The genomic DNA for extracting bacterium to be measured is detected using the detection method of embodiment 2.Setting is using water as mould
The negative control of plate DNA.
The result is shown in Figure 1.In Fig. 1, abscissa is recurring number, and ordinate is fluorescence signal intensity.The result shows that Candida albicans
Bacterium genomic DNA obtains S type amplification curve, and amplification is the positive, aspergillus fumigatus genomic DNA, Aspergillus flavus genomic DNA
S type amplification curve is not obtained with Candida glabrata genomic DNA, amplification is feminine gender.
The result shows that LAMP primer group prepared by embodiment 1 has very high specificity.
Embodiment 4, sensitivity
Sample to be tested: candida albicans gene group DNA.
1, with 10 times of gradient dilution samples to be tested of sterile water, each dilution is obtained.
2, each dilution for taking step 1 to obtain respectively is detected as template using the detection method of embodiment 2.
Since the dilution of use is different, following different reaction system is formed:
In reaction system 1, the initial content of genomic DNA are as follows: 10000 copies；
In reaction system 2, the initial content of genomic DNA are as follows: 1000 copies；
In reaction system 3, the initial content of genomic DNA are as follows: 500 copies；
In reaction system 4, the initial content of genomic DNA are as follows: 100 copies.
The result shows that candida albicans gene group DNA content obtains S type amplification curve when being 500-10000 copy, it is white
Color candida albicans genomic DNA concentration does not obtain S type amplification curve when being 100 copies.
The result shows that LAMP primer group sensitivity with higher prepared by embodiment 1.
Embodiment 5, clinical sample detection
Sample to be tested are as follows: by the eye aspirate sample (sample 1) and process that clinical identification is the Candida albicans positive
There is no the eye aspirate samples (sample 2) of the healthy person of Candida albicans for clinical identification.
The total DNA for extracting sample to be tested, is detected using the detection method of embodiment 2.Setting is using water as template
The negative control of DNA, positive control of the setting using the genomic DNA of Candida albicans as template.
As a result see Fig. 2.In Fig. 2, abscissa is recurring number, and ordinate is fluorescence signal intensity.The result shows that only sample
1 and positive control obtain S type amplification curve, sample 2 and negative control do not obtain S type amplification curve, with clinical identification result
It is consistent.
The result shows that the Candida albicans dientification of bacteria is carried out using LAMP primer group prepared by embodiment 1, for people's normal gene
Non-specific amplification is not present for group, as a result accurately and reliably.
<110>intelligence moral in Beijing is attained and Co., Ltd, medical test institute
<120>for detecting the LAMP primer composition and application of Candida albicans in intraocular liquid
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Claims (8)
1. primer combines, it is made of primers F 3, primer B3, primers F IP, primer BIP, primer LF and primer LB；
The primers F 3 is following (a1) or (a2):
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) have by sequence 1 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 1 identical
The DNA molecular of function；
The primer B3 is following (a3) or (a4):
(a3) single strand dna shown in the sequence 2 of sequence table；
(a4) have by sequence 2 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 2 identical
The DNA molecular of function；
The primers F IP is following (a5) or (a6):
(a5) single strand dna shown in the sequence 3 of sequence table；
(a6) have by sequence 3 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 3 identical
The DNA molecular of function；
The primer BIP is following (a7) or (a8):
(a7) single strand dna shown in the sequence 4 of sequence table；
(a8) have by sequence 4 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 4 identical
The DNA molecular of function；
The primer LF is following (a9) or (a10):
(a9) single strand dna shown in the sequence 5 of sequence table；
(a10) have by sequence 5 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 5 identical
The DNA molecular of function；
The primer LB is following (a11) or (a12):
(a11) single strand dna shown in the sequence 6 of sequence table；
(a12) have by sequence 6 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 6 identical
The DNA molecular of function.
2. the application of primer combination described in claim 1, for as follows (b1) or (b2) or (b3) or (b4):
(b1) it identifies or assists to identify whether fungi to be measured is Candida albicans；
(b2) prepare for identify or assist to identify fungi to be measured whether be Candida albicans kit；
(b3) whether detection sample to be tested has infected Candida albicans；
(b4) whether preparation has infected the kit of Candida albicans for detecting sample to be tested.
3. the kit containing primer pair described in claim 1；The purposes of the kit is following (c1) or (c2):
(c1) it identifies or assists to identify whether fungi to be measured is Candida albicans；
(c2) whether detection sample to be tested has infected Candida albicans.
4. the preparation method of kit described in claim 4 includes the steps that individually packing each primer.
5. identifying whether fungi to be measured is Candida albicans method, is included the following steps: using the genomic DNA of fungi to be measured as mould
Plate is combined using primer described in claim 1 and carries out LAMP amplified reaction, if use primer combination may be implemented with
The genomic DNA is the positive amplification of template, fungi to be measured is Candida albicans, if cannot be real using primer combination
It is now non-white candida albicans by the positive amplification of template, fungi to be measured of the genomic DNA.
6. identifying whether fungi to be measured is Candida albicans method, includes the following steps: to detect in the genomic DNA of fungi to be measured
With the presence or absence of the target sequence of primer described in claim 1 combination, if there are primer combinations in the genomic DNA
Target sequence, fungi to be measured be Candida albicans, if in the genomic DNA there is no the primer combination target sequence, to
Survey fungi is non-white candida albicans.
7. whether detection sample to be tested has infected the method for Candida albicans, include the following steps: be with the total DNA of sample to be tested
Template is combined using primer described in claim 1 and carries out LAMP amplified reaction, if may be implemented using primer combination
Candida albicans is infected by the positive amplification of template, sample to be tested of the total DNA, if cannot using primer combination
It realizes and does not infect Candida albicans by the positive amplification of template, sample to be tested of the total DNA.
8. the method whether detection sample to be tested has infected Candida albicans includes the following steps: the total DNA for detecting sample to be tested
In with the presence or absence of primer described in claim 1 combination target sequence, if in the total DNA there are the primer combination target
Sequence, sample to be tested have infected Candida albicans, if there is no the target sequences, to be measured of primer combination in the total DNA
Sample does not infect Candida albicans.
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| CN107541509A (en) * | 2016-06-29 | 2018-01-05 | 博奥生物集团有限公司 | For detecting the LAMP primer composition and its application of 6 kinds of infectious agents of mastitis for milk cows |
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