CN104450937A - Fluorescent quantitative PCR (polymerase chain reaction) primers, probe and kit for detecting pathogenic aspergilli - Google Patents
Fluorescent quantitative PCR (polymerase chain reaction) primers, probe and kit for detecting pathogenic aspergilli Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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Abstract
The invention discloses fluorescent quantitative PCR (polymerase chain reaction) primers, a probe and a kit for detecting pathogenic aspergilli. An upstream primer sequence is shown as sequence 1, a downstream primer sequence is shown as sequence 2, and a fluorescent probe sequence is shown as sequence 3. A quick, sensitive and specific kit for detecting pathogenic aspergilli is developed for overcoming the defects and limitations of traditional clinical pathogenic aspergillus detection, four kinds of common pathogenic aspergilli can be simultaneously detected in a reaction, and the kit has relatively high sensitivity and specificity.
Description
Technical field
The invention belongs to biological technical field, especially a kind of fluorescence quantification PCR primer, probe and test kit detecting pathogenic aspergillus tubigensis.
Background technology
Clinical common deep fungal infection is more common in candidiasis, aspergillus tubigensis, Cryptococcus neoformans etc., wherein candida infection is maximum, secondly be aspergillin infection, and aggressive aspergillin infection rate raises just year by year, case fatality rate is higher, and the survival of its infected patient depends on early diagnosis and early treatment.Traditional laboratory diagnostic method is consuming time oversize, probably misses best occasion for the treatment while wasting time, and susceptibility is lower simultaneously, therefore addresses this problem rather important technically.
At present, conventional clinically deep fungal diagnostic method has: culture-based method, imaging examination method, Serological testing.All there is certain defect in above method: culture-based method is the most direct method of diagnosis fungi infestation, and accuracy is high, but its length consuming time, easily pollute, shortcoming that susceptibility is low make it be unfavorable for clinical patients is diagnosed; Imaging examination taps into Mobile state by high-resolution machine to patient's focus position straight and observes, and has important prompting and is worth, but this method not only specific degree is low and only have early diagnosis to high risk patient and be worth; Serological method has certain susceptibility and specificity, but the false positive rate of diagnosis is higher.
At present relatively the most advanced, also the most promising is molecular biology method, i.e. PCR, polymerase chain reaction method.Though traditional round pcr has higher sensitivity, specific degree is low, because the problems such as external source pollution easily cause false positive results.Real-Time Fluorescent Quantitative PCR Technique can detect the fungal DNA of denier in tissue within the extremely short time, has higher sensitivity and specificity.
More than 95% is had to be caused by these four kinds of common causative aspergillin infections of Aspergillus fumigatus, flavus, terreus and aspergillus niger in the invasive aspergillosis of patient infection, in current bibliographical information useful many probe identification to kind, first for the diagnosis of invasive aspergillosis clinically without the need to identifying kind, only need identify Eurotium and corresponding methods for the treatment of just can be taked to carry out antifungal therapy; Secondly, many probes coexist in same quantitative fluorescent PCR system, and multiple fluorescence PCR condition is groped loaded down with trivial details and each other or have certain influence; In addition, TaqMan fluorescent probe price is higher, and test kit cost is increased greatly.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, a kind of fluorescence quantification PCR primer, probe and the test kit that detect pathogenic aspergillus tubigensis are provided, the present invention is by same primers and TaqMan probe, set up the fluorescent quantitative PCR detection method that one can detect 4 kinds of principal causative aspergillus tubigensis (Aspergillus fumigatus, flavus, terreus and aspergillus niger) simultaneously and have higher sensitivity and specificity, the early diagnosis and therapy for invasive aspergillosis is significant.
The technical scheme that the present invention realizes object is as follows:
Detect a fluorescence quantification PCR primer for pathogenic aspergillus tubigensis, sequence is as follows:
Upstream primer sequence is as shown in sequence 1, and downstream primer sequence is as shown in sequence 2.
Detect a quantitative fluorescent PCR probe for pathogenic aspergillus tubigensis, described fluorescent probe sequence is as shown in sequence 3.
Detect a PCR kit for fluorescence quantitative for pathogenic aspergillus tubigensis, comprise primer according to claim 1, comprise probe as claimed in claim 2.
And described test kit is provided with internal reference Quality Control, its primer and specificity detection probe sequence as follows:
Upstream primer sequence is as shown in sequence 4, and downstream primer sequence is as shown in sequence 5, and fluorescent probe sequence is as shown in sequence 6.
The advantage that the present invention obtains and beneficial effect are:
1, the present invention is in order to overcome the shortcomings and limitations of traditional clinical pathogenic aspergillus tubigensis detection, develop a kind of quick, sensitive, special detection to cause a disease the detection kit of aspergillus tubigensis, 4 kinds of common causative aspergillus tubigensis can be detected with a reaction simultaneously, and there is higher sensitivity and specificity.
2, this test kit adds internal reference Quality Control simultaneously, complete monitoring nucleic acid extraction and fluorescent PCR testing process, and avoid the generation that clinical detection pollutes (false positive) to greatest extent, Dual channel detection result precision is higher more reliable.
3, the present invention detects specific probe and the primer sequence of aspergillus tubigensis by autonomous design, Aspergillus fumigatus, flavus, terreus and the clinical common causative aspergillus tubigensis of aspergillus niger 4 kinds can be detected simultaneously, through experimental verification, there is higher sensitivity and specificity, whole testing process only needs 2-3 hour, compare traditional detection method not only efficiency greatly improve, and susceptibility and specificity have had great improvement.
4, test kit of the present invention uses uridylic-DNA-glycosylase and dUTP, helps avoid the pollution of amplified production.
Accompanying drawing explanation
Fig. 1 is the specific outcome figure of quantitative fluorescent PCR;
Fig. 2 is the sensitivity results figure of this kit fluorescence quantitative PCR detection;
Fig. 3 is fluorescence quantitative PCR detection sensitivity experiment internal reference passage result figure;
Fig. 4 display, to patient diagnosed's sample FAM Air conduct measurement curve, establishes negative control and positive control simultaneously;
Fig. 5 display, to patient diagnosed's sample internal reference Air conduct measurement curve, establishes negative control and positive control simultaneously.
Embodiment
Below by specific embodiment, the invention will be further described, and following examples are descriptive, is not determinate, can not limit protection scope of the present invention with this.
A kind of aspergillus tubigensis detection kit provided by the invention, is made up of PCR reaction solution pipe, Taq enzyme, uracil dna glycosylase, positive control QC, negative control QC, internal reference and box body.PCR reaction solution contains PCR reaction buffer, magnesium chloride, deoxyribonucleoside triphosphate (dNTPs) 0.1mM, each 200nM of Auele Specific Primer, fluorescently-labeled probe 100nM form; Negative control is the deionized water that nuclease free is polluted; Positive control is have object to detect the DNA of sequence and be inserted into the recombinant chou that pMD-T carrier forms; Internal reference is S. cervisiae thalline lyophilized powder.
The object that positive control inserts detects gene order:
GAAGGATCATTACCGAGTGCGGGTCCTTTGGGCCCAACCTCCCATCCGTGTCTATTGTACCCTGTTGCTTCGGCGGGCCCGCCGCTTGTCGGCCGCCGGGGGGGCGCCTCTGCCCCCCGGGCCCGTGCCCGCCGGAGACCCCAACACGAACACTGTCTGAAAGCGTGCAGTCTGAGTTGATTGAATGCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCG
Namely the core forming this test kit detects primer and the specificity fluorescent probe of conventional aspergillus tubigensis of causing a disease, and sequence is as follows respectively:
Upstream primer sequence: 5 '-ATGCCTGTCCGAGCGTCAT-3 '
Downstream primer sequence: 5 '-GTATCCCTACCTGATCCGAGGTC-3 '
Fluorescent probe sequence: 5 '-FAM-CCTCGAGCGTATGGGGCTTYGT-TAMRA-3 '
In addition, this test kit is provided with internal reference Quality Control, its primer and specificity detection probe sequence as follows:
Upstream primer sequence: 5 '-GCTGTCATCGGTTACTTAGATTTAG-3 '
Downstream primer sequence: 5 ’ – CCCTTCTTCTTTAGCAGCAATG-3 '
Fluorescent probe sequence: 5 '-VIC-CGATGTTGCTGTTTTGCCATTTTCCA-TAMRA-3 '
The using method of test kit of the present invention is:
(1) Fluorescence PCR system is as follows:
(2) fluorescent PCR amplified reaction program:
(3) sample extraction and detection
The liquid sample Trace bio-element test kit (P5002) using Tianjin Baorui Biological Technology Co., Ltd. to provide carries out sample nucleic extraction.
(4) result judges
Aspergillus (FAM) Ct value >35 or " Undet. " (ABI 7500) person " blank " (LightCycler480), and internal reference (VIC) Ct value <40 is negative; Aspergillus (FAM) Ct value≤31, and have good Increasing Curve of Logarithm to be the positive; Aspergillus (FAM) 31 < Ct sample < 33 is gray scale zone, and suggestion duplicate test confirms.
Detected result and accompanying drawing analysis
Fig. 1 is the specific outcome figure of quantitative fluorescent PCR.
Adopt LL cracking process to extract the genomic dna of aspergillus tubigensis and clinical all the other pathogenic bacterium common, increase according to quantitative fluorescent PCR reaction system and response procedures subsequently, to detect the specificity of this test kit primed probe.
Following clinical bacteria is specific to: Rhizopus oryzae, volume branch Mucor, Cryptococcus neoformans, Candida albicans, candida kefyr, monilia guilliermondii, Candida parapsilosis, candida krusei, candida tropicalis, Candida glabrata, candida sake, staphylococcus epidermidis, enterococcus faecalis, micrococcus luteus, intestinal bacteria, streptococcus dysgalactiae, streptococcus aureus, Pseudomonas aeruginosa through this test kit of experimental verification.
Experiment shows, this test kit has good specificity.
Fig. 2 is the sensitivity results figure of this kit fluorescence quantitative PCR detection.
Preparation aspergillus tubigensis spore suspension, and to dilute successively according to 10 times of gradient dilution methods be 1 × 106-1 × 101CFU/mL spore suspension.Double fluorescent quantitative PCR amplification and detection is carried out, the minimum aspergillus tubigensis to be measured detecting 1 × 101CFU/mL of result display after adopting LL cracking process to extract genomic dna.
Fig. 3 is fluorescence quantitative PCR detection sensitivity experiment internal reference passage result figure.
The monitoring result show nucleic acid of internal reference Quality Control extracts and pcr amplification, detection whole process all do not cause external source to pollute, and can determine that this detection does not exist false positive results.The result that this Dual channel detection obtains is more accurate, and confidence level is higher.
Concrete detection example
Embodiment 1: the fluorescent quantificationally PCR detecting kit of aspergillus tubigensis and use thereof
(1) collection of specimens, transport and preservation:
The patient that aseptic technique gathers doubtful aspergillin infection falls ill early stage blood specimen, and separation of serum is in suitable container, for subsequent use.
(2) detecting step and interpretation of result:
Sample nucleic acid extraction: liquid sample trace dna extracts test kit and carries out sample nucleic extraction.(P5002 provided by Tianjin Baorui Biological Technology Co., Ltd.) operation steps completes according to test kit specification sheets.
Real-time fluorescence PCR: nucleic acid product and 2 μ l positive control and the negative controls of getting 2 μ l said extracted respectively, add in real-time fluorescence PCR reaction tubes, add the PCR reaction solution of 23 μ l respectively, reaction cumulative volume is 25ul.
ABI7500 real-time fluorescence PCR instrument is used to carry out real-time fluorescence PCR reaction.
Fluorescence PCR program: 40 DEG C of 5min, 95 DEG C of 5min; 95 DEG C of 15s, 60 DEG C of 40s, 40 circulations.
Reaction terminates rear preservation and detects data file, and to data analysis.
Fig. 5 is the detected result of the internal reference passage of detected result, detects sample and is respectively 23.047,23.057,22.704 with Ct value that is negative, positive control, and show that extracting testing process does not have external source to pollute, result is effective;
Fig. 4 is the detected result of FAM passage, and the Ct value of its positive control, detection sample and negative control is respectively 24.304,28.946, Undet.By analysis, this detection sample is that aspergillus tubigensis is positive.
Claims (4)
1. detect a fluorescence quantification PCR primer for aspergillus tubigensis of causing a disease, it is characterized in that: sequence is as follows:
Upstream primer sequence is as shown in sequence 1, and downstream primer sequence is as shown in sequence 2.
2. detect a quantitative fluorescent PCR probe for aspergillus tubigensis of causing a disease, it is characterized in that: described fluorescent probe sequence is as shown in sequence 3.
3. detect a PCR kit for fluorescence quantitative for aspergillus tubigensis of causing a disease, it is characterized in that: comprise primer according to claim 1, comprise probe as claimed in claim 2.
4. detection according to claim 3 is caused a disease the PCR kit for fluorescence quantitative of aspergillus tubigensis, it is characterized in that: described test kit is provided with internal reference Quality Control, its primer and specificity detection probe sequence as follows:
Upstream primer sequence is as shown in sequence 4, and downstream primer sequence is as shown in sequence 5, and fluorescent probe sequence is as shown in sequence 6.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116064899A (en) * | 2022-09-01 | 2023-05-05 | 四川省三物科技有限公司 | A method and kit for rapid detection of deep infection Aspergillus |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030129600A1 (en) * | 1997-05-02 | 2003-07-10 | Dept. Of Health & Human Ser., C/O Ctr. For Disease Control & Prevention, Ofc. Of Technology Transfer | Nucleic acids for detecting Aspergillus species and other filamentous fungi |
| US6872523B1 (en) * | 2000-05-30 | 2005-03-29 | The Board Of Regents Of The University Of Nebraska | Materials and methods for molecular detection of clinically relevant pathogenic fungal species |
| WO2007101664A2 (en) * | 2006-03-08 | 2007-09-13 | Fungus Bioscience Gmbh | Improved aspergillosis test in clinical samples |
| CN101038254A (en) * | 2007-04-29 | 2007-09-19 | 南方医科大学 | PCR kit for fluorescence quantitative detecting aspergilli |
| CN102321738A (en) * | 2011-07-29 | 2012-01-18 | 广州呼吸疾病研究所 | Fluorescence quantitative PCR (polymerase chain reaction) universal premier for detecting pathogenic aspergillus, detection probe and kit |
-
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030129600A1 (en) * | 1997-05-02 | 2003-07-10 | Dept. Of Health & Human Ser., C/O Ctr. For Disease Control & Prevention, Ofc. Of Technology Transfer | Nucleic acids for detecting Aspergillus species and other filamentous fungi |
| US6872523B1 (en) * | 2000-05-30 | 2005-03-29 | The Board Of Regents Of The University Of Nebraska | Materials and methods for molecular detection of clinically relevant pathogenic fungal species |
| WO2007101664A2 (en) * | 2006-03-08 | 2007-09-13 | Fungus Bioscience Gmbh | Improved aspergillosis test in clinical samples |
| CN101038254A (en) * | 2007-04-29 | 2007-09-19 | 南方医科大学 | PCR kit for fluorescence quantitative detecting aspergilli |
| CN102321738A (en) * | 2011-07-29 | 2012-01-18 | 广州呼吸疾病研究所 | Fluorescence quantitative PCR (polymerase chain reaction) universal premier for detecting pathogenic aspergillus, detection probe and kit |
Non-Patent Citations (3)
| Title |
|---|
| 孙于谦等: "荧光实时定量PCR方法在异基因造血干细胞移植后侵袭性曲霉菌感染早期诊断中的价值", 《中华医院感染学杂志》 * |
| 张梦宇等: "实时荧光定量PCR检测血中曲霉菌基因的实验研究", 《临床检验杂志》 * |
| 张梦宇等: "荧光定量PCR检测侵袭性曲霉菌感染方法的建立", 《南方医科大学学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116064899A (en) * | 2022-09-01 | 2023-05-05 | 四川省三物科技有限公司 | A method and kit for rapid detection of deep infection Aspergillus |
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