CN109536634A - Universal primer, kit and the detection method that fungal contamination detects in cell product - Google Patents
Universal primer, kit and the detection method that fungal contamination detects in cell product Download PDFInfo
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- CN109536634A CN109536634A CN201910073878.0A CN201910073878A CN109536634A CN 109536634 A CN109536634 A CN 109536634A CN 201910073878 A CN201910073878 A CN 201910073878A CN 109536634 A CN109536634 A CN 109536634A
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Abstract
The invention discloses universal primer, kit and detection methods that fungal contamination in a kind of cell product detects, wherein universal primer includes the upstream primer and downstream primer to expand fungi rDNA, the upstream primer and downstream primer sequence are complementary with the conservative region of fungi 26S rDNA sequence, and nucleotide sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.It is demonstrated experimentally that carrying out fluorescence quantitative PCR detection using primer pair sample to be tested provided by the present invention, the versatility of fungal detection is greatly improved；The homology that also can avoid the human genome sequence contained in testing goal segment and cell product simultaneously, provides fast and accurately method for the detection of cell products early stage.Detection method of the invention is easy to operate, without complicated nucleic acid extraction process, only the fungi that polluted in cell product need to being subjected to quantitative PCR detection by can be used as template with lysate cracking after of short duration centrifugal enrichment, the detection of Common fungi in cell product can be completed in 3 hours.
Description
Technical field
The invention belongs to technical field of microbial detection, and in particular to fungal contamination detection is general in a kind of cell product
Primer, kit and detection method.
Background technique
In recent years, with the rapid development of cell therapy research, external cell culture is had become in cell therapy procedures
An important link.In cell cultivation process, the seriously polluted of bacterium, fungi and mycoplasma etc. affects cell life
It is long.According to 2015 version " Chinese Pharmacopoeia " Sterility Test, the sterility test of test sample needs in defined culture medium and suitable
It is cultivated 14 days at a temperature of suitable, needs to carry out every experiment contrast during the experiment, while requirement of experiment is more stringent, and
Whether observed and recorded daily in the plate of culture during the cultivation process has fungi growth.Detection method in pharmacopeia is when detecting
Between the upper desired time it is longer, until can also observe to come already in cell cultivation process when cultivation detection is drawn a conclusion.
Therefore, it is necessary to a kind of modes of rapid sensitive to detect in cell cultivation process with the presence or absence of fungal contamination.Real time fluorescent quantitative
Round pcr as a kind of emerging detection means, have in the detection process high sensitivity, reproducible, detection time is short etc.
Advantage is gradually applied in detection of pathogens.
The gene of encoding ribosomal RNA has 4 kinds in fungal gene group, 26S rDNA, 5S rDNA, 18S rDNA and 5.8S
rDNA.26S is divided into conserved region and hypervariable region in structure, and the affiliation between biological species, hypervariable region reflection are reflected in conserved region
Difference between species.The 26S rDNA of fungi has certain conservative, and conserved region sequence is somebody's turn to do common to most of fungi
To avoid with the gene of bacterium, people cross reaction can occur for detection of the Duan Xulie for fungi, have very strong specificity.Fungi
26S rDNA molecule in large subunit, can be divided into the multiple regions such as D1, D2 ... D12, wherein D1/D2 regional sequence moderate length,
It is currently a popular fungal molecule identification and Phylogenetic analysis label, while is equally applicable to the method for quantitative PCR to fungi
It is detected.
Summary of the invention
For technological gap in the prior art, the object of the present invention is to provide fungal contaminations in a kind of cell product to detect
Universal primer, kit and detection method, this method to Common fungi detect it is versatile, be based on fluorescent quantitative PCR technique,
Homology of the sequence of amplification on cell product itself nucleic acid is avoided in design of primers, is solved in quantitative PCR detection
False positive issue, improve the reliability of testing result.
The present invention is achieved by the following technical solutions:
The first purpose of this invention is to propose a kind of universal primer group detected for fungal contamination in cell product,
Including the upstream primer and downstream primer to expand fungi rDNA, the upstream primer and downstream primer sequence and fungi 26S
The conservative region of rDNA sequence is complementary, and nucleotide sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
Further, the molar ratio for forming two single strand dnas of the primer sets is 1:1.
Second object of the present invention is to propose the kit for detecting fungal contamination in cell product, the reagent
Box includes universal primer group described in any of the above embodiments and PCR premixed liquid.
Further, the kit further includes standard items, and the standard items sequence is as shown in SEQ ID NO.3.
Third object of the present invention is to propose universal primer described in any of the above embodiments or described in any item reagents
Application of the box in cell culture fluid, the detection of cell culture medium fungal contamination.
Fourth object of the present invention is to propose the rapid detection method of fungal contamination in a kind of above-mentioned cell product, wrap
It includes
(1) quantitative fluorescent PCR is carried out by template of the plasmid with standard sequence, establishes standard curve, the standard sequence
Column are as shown in SEQ ID NO.3, the upstream and downstream primer nucleotide sequence of the quantitative fluorescent PCR such as SEQ ID NO.1 and SEQ
Shown in ID NO.2；
(2) test sample is handled, and obtains quantitative fluorescent PCR test sample template；
(3) augmentation detection: being that amplification template carries out quantitative fluorescent PCR, the fluorescent quantitation with the template in step (2)
The upstream and downstream primer nucleotide sequence of PCR is as shown in SEQ ID NO.1 and SEQ ID NO.2, the overall reaction system are as follows: template
Each 0.25 μ l of 1 μ l, 10 μM of upstream and downstream primer, 5 μ l of PCR premixed liquid, 3.5 μ l of aseptic deionized water；
(4) fluorescent quantitation interpretation of result: judge to supply according to the target amplification Ct value in the sample genomic dna of test sample
It whether there is fungal contamination in test product.
Further, test article treating method in the step (2) are as follows: after taking cell product to carry out of short duration centrifugal enrichment,
It reuses lysate cracking and directly obtains test sample template.
Further, the quantitative fluorescent PCR reaction condition is 95 DEG C of 3min initial denaturations；With 95 DEG C of 10s, 58 DEG C of 10s, 72
DEG C 30s is a circulation, totally 40 circulations.
Further, positive control and negative control are set in the method, and the positive control is that lysate cracks ferment
The solution of imperial mother, the negative control are aseptic deionized water.
Further, positive interference product are set in the method, and the positive interference product are to add in isometric test sample
Yeast liquid is added to be formed；The positive interference product processing method are as follows: after taking positive interference product to carry out of short duration centrifugal enrichment, then make
Positive interference product template is directly obtained with lysate cracking.
Compared with prior art, the advantages of the present invention are as follows:
(1) technical solution of the present invention is realized with Real-Time Fluorescent Quantitative PCR Technique to normal by design universal primer
The purpose for seeing the general detection of fungi efficiently solves the problems, such as that previous PCR method is only used for detecting single fungi, while
The homology for avoiding the human genome sequence contained in testing goal segment and cell product, greatly improves fungal detection
Versatility provides fast and accurately method for the detection of cell products early stage.
(2) detection method of the invention is easy to operate, only need to will be dirty in cell product without complicated nucleic acid extraction process
The fungi of dye carries out quantitative PCR detection by can be used as template after being cracked after of short duration centrifugal enrichment with lysate, can be small 3
When it is interior complete cell product in Common fungi detection.
Detailed description of the invention
Fig. 1 is 1 middle and upper reaches primer of embodiment and reference sequences comparison result schematic diagram；
Fig. 2 is 1 middle and lower reaches primer of embodiment and reference sequences comparison result schematic diagram；
Fig. 3 is recombinant clone plasmid encoding luciferase quantitative pcr amplification curve in embodiment 2；
Fig. 4 is recombinant clone plasmid encoding luciferase quantitative PCR solubility curve in embodiment 2；
Fig. 5 is quantitative fluorescent PCR standard curve in embodiment 2；
Fig. 6 is the fluorescence quantitative PCR detection result schematic diagram of sample to be tested in embodiment 3.
Specific embodiment
Applicant is in conjunction with specific embodiments described in further details technical solution of the present invention below, so that this field
Technical staff may be better understood the present invention and can be practiced, but range is claimed not as the present invention in illustrated embodiment
Restriction.
Embodiment 1: for quickly detecting the acquisition of the fluorescence PCR primer of fungal contamination in cell product
One, the design and synthesis of primer pair
In cell product fungi rapid detection method of the invention, upstream and downstream PCR primer sequence is from fungi 26S rDNA
The universal primer screened in the conservative region of sequence, in NCBI (https: //www.ncbi.nlm.nih.gov/
Nuccore after the 26S rDNA sequence for acquiring fungi on), sequence is analyzed with DNAMAN software, is found different true
Conservative fragments between strain belongs to are screened universal primer in these fungi conservative regions, are utilized simultaneously according to design of primers principle
Blast (https: //blast.ncbi.nlm.nih.gov/Blast.cgi) analyzes primer whether can expand in human genome
Increasing sequence similar in size ensures the reliability of primer, therefrom best a pair of of the combination of selection.Design of primers reference sequences are such as
Shown in table 1.
1 design of primers reference sequences of table
Fig. 1, Fig. 2 be design primer and with reference to species sequence alignment analysis as a result, the primer that designs as the result is shown with
Reference sequences have higher homology at 3 ' ends, show that primer can be used for a variety of fungal detection analyses.The primer sequence of acquisition
Primer is carried out by Beijing Qing Kexin industry Bioisystech Co., Ltd to synthesize.The upstream and downstream PCR primer sequence of design is as follows:
Upstream primer sequence: SEQ ID NO.1 5'-AACAGGACGYCATAGAGGGTGAGA-3'
Downstream primer sequence: SEQ ID NO.2 5'-CATATAACCATTAYGCCAGCATCC-3'
Embodiment 2: for detecting the specific detection of the fluorescence PCR primer of fungal contamination and sensitivity in cell product
It determines
1 pair of primer that the present embodiment obtains embodiment 1 using reference culture saccharomycete carries out specific detection and minimum
Detect the determination of limit.Standard items sequence is as shown in SEQ ID NO.3.
With the primer amplification reference culture saccharomycete of embodiment 1, gel extraction PCR product after purification connects target fragment
Be connected on pTOPO cloning vector, with positive colony of the vector primer M13F to recombination carry out sequence verification it is errorless after, extracting recombination
Cloned plasmids.Measurement concentration is simultaneously converted into (copy number/volume), is then carried out gradient dilution, is diluted to 1.0977 × 10-
1.0977×108Copy/μ l.It takes 1 μ l to carry out quantitative PCR detection experiment as template under each concentration, is done under each concentration
Three repetitions.
Augmentation detection: carrying out on fluorescence quantitative PCR instrument, and total reaction volume is 10 μ l, wherein it is dilute that gradient is added as required
The template 1 μ l, each 0.25 μ l of 10 μM of upstream and downstream primer, 5 μ l of quantitative fluorescent PCR premixed liquid (purchase is in TaKaRa company) released,
3.5 μ l of aseptic deionized water is supplied to 10 μ l.
Reaction condition: 95 DEG C of 3min initial denaturations；With 95 DEG C of 10s, 58 DEG C of 10s, 72 DEG C of 30s are a circulation, carry out 40 altogether
A circulation.The production of solubility curve is carried out after the completion of 40 circulations again.
Testing result:
Fluorescent quantitative PCR curve of the corresponding quantitative fluorescent PCR Ct value of plasmid copy number from Fig. 3 in table 2
(curve of mark P1-P8 is respectively that recombinant clone plasmid is diluted to 1.0977 × 108Copy/μ l-1.0977 × 10 copies/μ l
Sample), Fig. 4 be the experiment solubility curve.From the point of view of amplification curve and solubility curve, experimental repeatability is good, high specificity.
2 plasmid copy number of table is compareed with fluorescence quantitative PCR detection result
Standard curve: y=-4.6511x+46.797R2=0.9998
X: it indicates Lg (plasmid copy number) y: indicating fluorescent quantitation Ct value
From the point of view of the relationship corresponding with objective gene sequence copy number in every microlitre of fluorescent quantitation Ct value in table 2, this method
In the sensitivity minimization that is able to detect that be 110 copies/microlitre.Fig. 5 is standard sequence difference copy number and corresponding Ct value
Data obtain the standard curve of the linear relationship of Lg (plasmid copy number) and quantitative fluorescent PCR Ct value after processing.Standard is bent
The R of line2> 0.98, show that data and lienarized equation degree of agreement are high, it can be with quantitative detection test sample to be checked using the standard curve
The concentration of target gene in product, minimum detection limit about 110 copies/microlitre.
Embodiment 3: by real-time fluorescence quantitative PCR to the fungal detection of cell products
1, the processing of sample to be tested
It takes in cell culture fluid 3ml to 15ml centrifuge tube, 10000rpm is centrifuged 2min, is added 20 μ l's after removing supernatant
Lysis Buffer for Microorganism (purchase is in TaKaRa company) lysate, is placed in 80 DEG C of reactions in PCR instrument
20min takes 1 μ l as test sample template after the reaction was completed.
2, positive control
A small amount of yeast thallus is dipped with toothpick, in the Lysis Buffer for Microorganism lysate of 20 μ l
Several lower rear taking-ups are stirred, 80 DEG C of reaction 20min in PCR instrument is subsequently placed in, takes 1 μ l as positive control template after the reaction was completed.
3, negative control
1 μ l aseptic deionized water is taken to do negative control template.
4, the processing of positive interference product
Cell culture fluid mixing takes 3ml supernatant into 15ml centrifuge tube, while be added 2 μ l yeast liquids, 10000rpm from
Heart 2min goes after supernatant to add the Lysis Buffer for Microorganism lysate of 20 μ l, be placed in 80 in PCR instrument
DEG C reaction 20min, take 1 μ l as test sample template.
5, real-time fluorescence quantitative PCR detects
Carry out real-time fluorescence quantitative PCR detection on fluorescence quantitative PCR instrument to above-mentioned sample respectively, total reaction volume is
10 μ l, wherein 1 μ l of template, 10 μM of upstream and downstream primer each 5 μ l of 0.25 μ l, PCR premixed liquid, 3.5 μ l of aseptic deionized water；Reaction
Condition are as follows: 95 DEG C of 3min initial denaturations；With 95 DEG C of 10s, 58 DEG C of 10s, 72 DEG C of 30s are a circulation, totally 40 circulations.
6, testing result
Qualitative criteria:
(1) sample of the quantitative fluorescent PCR Ct value equal to 40.0 is feminine gender；
(2) sample of value≤32 CT, testing result are the positive；
(3) Ct value is suspicious region between 32~40, and sample is placed on 30 DEG C of shaking tables after shake culture one day and is reformed,
Being greater than 32 values after reforming is feminine gender.
The quantitative pcr amplification curve of each sample is shown in Fig. 6, and the corresponding Ct value of each sample is shown in Table 3.According to testing result,
Positive control can be expanded normally, and negative control and test sample do not read Ct value, show test sample without fungal contamination.Simultaneously
By sample to be tested 30 DEG C shake culture 7 days in cell culture medium, culture medium is still limpid, shows that testing result of the invention is quasi-
It is really reliable.
3 pattern detection of table corresponds to Ct value
In conclusion carrying out real-time fluorescence in the primer pair cell product provided by the present invention obtained using embodiment 1
Quantitative PCR detection is greatly improved the accuracy, specificity and versatility of fungal detection, solves the vacation in quantitative PCR detection
Positive problem improves the reliability of testing result.Detection method of the invention simultaneously is easy to operate, mentions without complicated nucleic acid
Take process, only need to by the fungi polluted in cell product pass through after of short duration centrifugal enrichment with lysate crack can be used as template into
Row quantitative PCR detection can complete the detection of Common fungi in cell product in 3 hours.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Sequence table
<110>Wuhan Rui Jian Pharmaceutical Technology Co., Ltd
<120>fungal contamination detects in cell product universal primer, kit and detection method
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
aacaggacgy catagagggt gaga 24
<210> 2
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
catataacca ttaygccagc atcc 24
<210> 3
<211> 435
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
aacaggacgt catagagggt gagaatcccg tgtggcgagg agtgcggttc tttgtaaagt 60
gccttcgaag agtcgagttg tttgggaatg cagctctaag tgggtggtaa attccatcta 120
aagctaaata ttggcgagag accgatagcg aacaagtaca gtgatggaaa gatgaaaaga 180
actttgaaaa gagagtgaaa aagtacgtga aattgttgaa agggaagggc atttgatcag 240
acatggtgtt ttgtgccctc tgctccttgt gggtagggga atctcgcatt tcactgggcc 300
agcatcagtt ttggtggcag gataaatcca taggaatgta gcttgcctcg gtaagtatta 360
tagcctgtgg gaatactgcc agctgggact gaggactgcg acgtaagtca aggatgctgg 420
cataatggtt atatg 435
Claims (10)
1. a kind of universal primer group detected for fungal contamination in cell product, which is characterized in that including to expand fungi
The conserved region of the upstream primer and downstream primer of rDNA, the upstream primer and downstream primer sequence and fungi 26S rDNA sequence
Domain is complementary, and nucleotide sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
2. a kind of universal primer group detected for fungal contamination in cell product according to claim 1, feature exist
In the molar ratio for forming two single strand dnas of the primer sets is 1:1.
3. the kit for detecting fungal contamination in cell product, which is characterized in that the kit includes claim
The described in any item universal primer groups of 1-2 and PCR premixed liquid.
4. according to claim 3 a kind of for detecting the kit of fungal contamination in cell product, which is characterized in that institute
Stating kit further includes standard items, and the standard items sequence is as shown in SEQ ID NO.3.
5. the described in any item universal primers of claim 1-2 or the described in any item kits of claim 3-4 are trained in cell
Application in nutrient solution, the detection of cell culture medium fungal contamination.
6. the rapid detection method of fungal contamination in a kind of cell product, which is characterized in that including
(1) quantitative fluorescent PCR is carried out by template of the plasmid with standard sequence, establishes standard curve, the standard sequence is such as
Shown in SEQ ID NO.3, the upstream and downstream primer nucleotide sequence of the quantitative fluorescent PCR such as SEQ ID NO.1 and SEQ ID
Shown in NO.2；
(2) test sample is handled, and obtains quantitative fluorescent PCR test sample template；
(3) augmentation detection: being that amplification template carries out quantitative fluorescent PCR with the template in step (2), the quantitative fluorescent PCR
Upstream and downstream primer nucleotide sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2, the overall reaction system are as follows: 1 μ l of template,
Each 0.25 μ l of 10 μM of upstream and downstream primer, 5 μ l of PCR premixed liquid, 3.5 μ l of aseptic deionized water；
(4) test sample fluorescent quantitation interpretation of result: is judged according to the target amplification Ct value in the sample genomic dna of test sample
In whether there is fungal contamination.
7. the rapid detection method of fungal contamination in a kind of cell product according to claim 6, which is characterized in that described
Test article treating method in step (2) are as follows: after taking cell product to carry out of short duration centrifugal enrichment, reuse lysate cracking and directly obtain
Obtain test sample template.
8. the rapid detection method of fungal contamination in a kind of cell product according to claim 6, which is characterized in that described
Quantitative fluorescent PCR reaction condition are as follows: 95 DEG C of 3min initial denaturations；With 95 DEG C of 10s, 58 DEG C of 10s, 72 DEG C of 30s are a circulation, totally 40
A circulation.
9. the rapid detection method of fungal contamination in a kind of cell product according to claim 6, which is characterized in that described
Positive control and negative control are set in method, and the positive control is that lysate cracks the solution after yeast, and the feminine gender is right
According to for aseptic deionized water.
10. the rapid detection method of fungal contamination in a kind of cell product according to claim 6, which is characterized in that institute
It states and positive interference product is set in method, the positive interference product are that addition yeast liquid is formed in isometric test sample；Institute
State positive interference product processing method are as follows: after taking positive interference product to carry out of short duration centrifugal enrichment, reuse lysate cracking and directly obtain
Obtain positive interference product template.
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| CN201911260273.9A CN110735003A (en) | 2019-01-25 | 2019-12-10 | Universal primer, kit and detection method for detecting fungal contamination in cell product |
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| CN114134239A (en) * | 2021-11-25 | 2022-03-04 | 广州烨善生物科技有限公司 | Kit for rapidly evaluating mammalian cell quality by PCR (polymerase chain reaction) method and detection method thereof |
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| CN114480596A (en) * | 2022-02-11 | 2022-05-13 | 宁波市中心血站 | Micro-fluidic blood and blood product bacterial contamination detection method |
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| US5763169A (en) * | 1995-01-13 | 1998-06-09 | Chiron Diagnostics Corporation | Nucleic acid probes for the detection and identification of fungi |
| JP2004201641A (en) * | 2002-12-26 | 2004-07-22 | Mitsubishi Kagaku Bio-Clinical Laboratories Inc | Detection of eumycetes |
| US20080228406A1 (en) * | 2007-03-12 | 2008-09-18 | Myconostica Ltd. | System and method for fungal identification |
| CN103509851A (en) * | 2012-06-19 | 2014-01-15 | 上海交通大学医学院附属上海儿童医学中心 | Microbial species analysis method |
| CN106701747B (en) * | 2015-08-28 | 2019-12-13 | 光明乳业股份有限公司 | Universal PCR primer pair and application thereof |
| CN105567800A (en) * | 2015-11-19 | 2016-05-11 | 英格尔检测技术服务（上海）有限公司 | Fungus species PCR identification method |
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| CN114134239A (en) * | 2021-11-25 | 2022-03-04 | 广州烨善生物科技有限公司 | Kit for rapidly evaluating mammalian cell quality by PCR (polymerase chain reaction) method and detection method thereof |
| CN114134239B (en) * | 2021-11-25 | 2023-09-15 | 广州烨善生物科技有限公司 | Kit for rapidly evaluating quality of mammalian cells by PCR method and detection method thereof |
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