CN105624285A - Mycoplasma pneumoniae fluorescent PCR detection reagent kit - Google Patents
Mycoplasma pneumoniae fluorescent PCR detection reagent kit Download PDFInfo
- Publication number
- CN105624285A CN105624285A CN201510887313.8A CN201510887313A CN105624285A CN 105624285 A CN105624285 A CN 105624285A CN 201510887313 A CN201510887313 A CN 201510887313A CN 105624285 A CN105624285 A CN 105624285A
- Authority
- CN
- China
- Prior art keywords
- mycoplasma pneumoniae
- probe
- pcr
- nucleic acid
- primer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- C12Q1/686—Polymerase chain reaction [PCR]
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a reagent kit for specific detection of existence of mycoplasma pneumoniae nucleic acid in a sample by adopting a real-time fluorescent PCR method, and in particular to a mycoplasma pneumoniae fluorescent PCR detection reagent kit. With the utilization of the mycoplasma pneumoniae fluorescent PCR detection reagent kit, whether mycoplasma pneumoniae nucleic acid exists in a sample or not can be quickly detected; besides, the detection limit of the reagent kit for mycoplasma pneumoniae nucleic acid is 10 copies/reaction system and the overall reaction can be completed within 2 hours, so that the reagent kit has an important application value in fields like disease surveillance and clinical diagnosis.
Description
Technical field
The present invention relates to and a kind of adopt the test kit of mycoplasma pneumoniae nucleic acid in real time fluorescent PCR method specific detection sample, belong to the in-vitro diagnosis field of mycoplasma pneumoniae.
Background technology
Mycoplasma pneumoniae (M.Pneumonia) is the pathogen causing mankind's mycoplasma pneumonia, and generally based on interstitial pneumonia, concurrent bronchopneumonia, is called primary atypical pneumonia sometimes. Mycoplasma pneumoniae is mainly through droplet infection, and general 2 ~ 3 weeks of incubation period, sickness rate is the highest with teenager; The performance of its clinical symptoms is relatively light, even at all asymptomatic, if having also is the general respiratory symptoms such as headache, pharyngalgia, heating, cough. In the detection, PCR method to substitute owing to its fast and convenient feature presents gradually based on the trend of antibacterial culturing and the traditional detection method of Serologic detection. And for PCR method, the specificity of primer is the basis of the specificity of its detection and sensitivity.
The present invention is directed to the special target sequence design primer of the conservative region of mycoplasma pneumoniae gene order and Taqman probe, the method utilizing real-timePCR, be used for quickly detecting the nucleic acid that whether there is mycoplasma pneumoniae in sample.
Summary of the invention
What present invention mainly solves technical problem is that to detect in sample whether there is mycoplasma pneumoniae rapidly and accurately, it is provided that the fluorescent PCR kit of a kind of specific detection mycoplasma pneumoniae.
The technical problem to be solved is achieved through the following technical solutions:
The test kit of mycoplasma pneumoniae nucleic acid in a kind of specific detection sample, its component includes PCR reactant liquor, enzyme mixation, negative quality-control product and positive quality control product, and wherein PCR reactant liquor mainly contains relevant primer and probe, reaction buffer, Mg2+With dNTP etc., enzyme mixation mainly contains hot start Taq polymerase, and negative quality-control product is without RNase and DNase water, and positive quality control product is detect the recombiant plasmid of target sequence containing mycoplasma pneumoniae.
Wherein in PCR reactant liquor, for the primer of nucleic acid amplification, to be P1 and P2, P1 and P2 be for the design of mycoplasma pneumoniae gene group specific and conserved sequence the specific primer that filters out through preliminary experiment; The oligonucleotide probe monitored for fluorescence signal in PCR reactant liquor is Probe1, Probe1 is the specific probe designing for mycoplasma pneumoniae gene group specific and conserved sequence and filtering out through preliminary experiment, wherein fluorescent reporter group X1For HEX, fluorescent quenching group Y1For Eclipse(in Table 1).
Table 1 detects primer and the probe sequence of mycoplasma pneumoniae
| Title | Sequence |
| P1 | 5`- TATGCTAGCAGGATACGCGAAT -3` |
| P2 | 5`- GGACCGCTCGAAATTCATAAAAG -3` |
| Probe1 | 5`-X1- TGAACGAGACCACGCCAGAACGGCT -Y1 -3` |
It is a further object to provide this fluorescence PCR detection reagent kit application process at detection mycoplasma pneumoniae, including:
The PCR reaction system that test kit is selected is 20 �� L, including 2 �� PCR buffer 10 �� L, 10mmol/LdNTP1.0 �� L, 25 ��m of each 0.5 �� L of ol/L primer, 10 ��m of ol/L probe 0.2 �� L, hot start Taq polymerase mixed liquor 0.4 �� L, sample DNA 2 �� L, add the extremely whole system 20 �� L of aquesterilisa.
The PCR reaction cycle parameter of test kit is 94 DEG C, 2min; Entering the cycle stage: 94 DEG C of degeneration 10s, 56 DEG C of annealing 50s, 72 DEG C extend 15s, coreaction 40 circulation.
Quality control: experiment should set up positive and negative to compare every time, and negative control is without Ct value (or Ct value is 0), and positive control Ct value��30, otherwise experimental result is false.
Result interpretation:
Positive: " S " type amplification curve, Ct value��35 occur; Suspicious: appearance " S " type amplification curve, but Ct value > 35; Negative: " S " type amplification curve does not occur, although or curve exceeded threshold value, but in " S " type; To suspect results, should repeating experiment, if repeating experiment or " S " type amplification curve occur, negative control does not pollute, and can determine whether as the positive.
Sample requires: clinical sample type includes patient's throat swab; Clinical sample should transport by band ice after gathering ,-20 DEG C of preservations, it is impossible to multigelation; DNA is extracted, it is proposed that adopting commercial kit stable and reliable for performance, concrete grammar is referring to corresponding commodity test kit description, and the DNA of extraction should detect immediately from clinical sample, otherwise, should by-80 DEG C��-20 DEG C preservations after DNA subpackage.
Test kit provided by the invention has good specificity, it is possible to detection mycoplasma pneumoniae, but can not detect the nucleic acid of non-mycoplasma pneumoniae; Monitoring lower-cut to mycoplasma pneumoniae nucleic acid is the 10 every reaction systems of copy; Have only to 2 hours to complete detection, experimental basis can be provided for the disease surveillance of mycoplasma pneumoniae and clinical diagnosis.
Accompanying drawing explanation
Fig. 1 is the amplification curve diagram of substance real-time PCR detection mycoplasma pneumoniae positive criteria product. Every suite line corresponding concentration from left to right is followed successively by 2 �� 106-2��102Copies/ �� L, the detection of mycoplasma pneumoniae is limit and is the 10 every reaction systems of copy by test kit. Abscissa is reaction cycle number, and vertical coordinate is the �� Rn value of different period fluorescent assay signal.
Fig. 2 is the amplification curve diagram of substance real-time PCR detection system 10 kinds of bacterial genomes DNA of detection. 10 kinds of antibacterials include: mycoplasma pneumoniae and 9 kinds of negative control microorganisms. There is S type amplification curve in mycoplasma pneumoniae, and S type amplification curve do not occur in other 9 kinds of pathogenic microorganisms.
Detailed description of the invention
The preferred embodiment of the present invention is described in detail below in conjunction with specific embodiment. It is pointed out that the embodiment listed here is merely exemplary descriptive purpose, and it should not be constructed as any limitation on the scope of the present invention. The reagent such as the test kit that wherein uses, buffer are only reagent specifically chosen in this specific embodiment, it should be appreciated that those skilled in the art can select the corresponding reagent of other companies to realize the purpose of the present invention as required.
The design of primer and TaqMan probe and synthesis
Utilize Blast instrument that the mycoplasma pneumoniae genome sequence in Genbank and domestic and foreign literature is analyzed, select its stable conservative region as detection target sequence respectively. Mycoplasma pneumoniae detection target sequence derives from card toxin gene; And for detection target sequence design and synthetic primer and probe (see table 1). Primer and probe are by the synthesis of TaKaRa Dalian treasured biotech firm of Japan, detection probe 5 ' the end labelling HEX fluorophor of mycoplasma pneumoniae; 3 ' end labelling Eclipse fluorescent quenching groups.
The preparation of detection strain
The mycoplasma pneumoniae used in the present embodiment and other negative control bacterial strain (corynebacterium ulcerrans, Corynebacterium pseudodi phtheriae, corynebacterium haemolyticum, staphylococcus aureus, streptococcus pneumoniae, escherichia coli, corynebacterium minutissimum, diphtheria corynebacterium, Bacillus anthracis) all buy in Nat'l Pharmaceutical & Biological Products Control Institute.
The extracting of bacterial strain DNA
Select Qiagen company QIAampDNAMiniKit(article No.: 51306) extract bacterial strain DNA. Concrete steps test kit is with reference to operating instruction.
The screening of primer and probe
Adopt the primer of design and the genomic DNA of the mycoplasma pneumoniae of probe in detecting extraction and negative control bacterial strain, through repeatedly testing, filter out the primed probe combination that sensitivity, specificity and repeatability are best. (see sequence table, mycoplasma pneumoniae (forward primer P1, reverse primer P2 and probe Probe1)
The structure of standard substance and preparation
Utilize P1 and P2 primer amplification mycoplasma pneumoniae genomic DNA, PCR primer is cloned into pMD-18T carrier, convert DH5 �� escherichia coli, utilize alkaline lysis method of extracting positive colony plasmid, utilize ultraviolet-uisible spectrophotometer to measure the absorptance at wavelength 260nm place, 280nm place DNA respectively, then calculate plasmid concentration and purity. Then 10 times of gradient dilutions copy every microlitre to 200, prepare the positive criteria product of mycoplasma pneumoniae.
Reaction condition optimization
The key elements such as primer, probe, enzyme are optimized one by one, the reaction system determined is: 2 �� PCR buffer 10 �� L, 10mmol/LdNTP1.0 �� L, 25 ��m of each 0.5 �� L of ol/L primer, 10 ��m of ol/L probe 0.2 �� L, Takara polymerase mixture 0.4 �� L, template 2 �� L, adds the extremely whole system 20 �� L of aquesterilisa.
Annealing temperature according to amplified fragments length, primer and probe and enzyme viability, mainly annealing temperature and extension time to reaction are optimized, and finally determine that loop parameter is: 94 DEG C, 2min; Entering the cycle stage: 94 DEG C of degeneration 10s, 56 DEG C of annealing 50s, 72 DEG C extend 15s, 40 circulations, and each annealing stage that circulates in gathers fluorescence signal.
By identical conditions analytical data after amplification terminates, it is determined that the Ct value of each sample.
The evaluation of detection limit
Judge the detection limit of valency test kit provided by the invention with above-mentioned positive criteria, positive criteria product concentration is: 2 �� 106copies/��L��2��105copies/��L��2��104copies/��L��2��103copies/��L��2��102Copies/ �� L, the Monitoring lower-cut of mycoplasma pneumoniae nucleic acid is the 10 every reaction systems of copy by test kit provided by the invention.
Detect specific evaluation
The specificity of this test kit is have rated with above-mentioned bacterial strains DNA for template. All visible clear and definite amplification curve when Mycoplasma pneumonia DNA is detected, during to above-mentioned 9 kinds of other pharyngeal encountered pathogenic microbial DNAs detection, do not produce positive amplification curve, illustrate to be absent from cross reaction between probe and primer and other bacterial strains that we select that we use.
Although above in a preferred manner; some embodiment of the present invention is illustrated by specific embodiment; but it will be understood by a person skilled in the art that; the present invention is not limited to embodiment disclosed above; but according to the knowledge of the technical field of the invention, it can be modified, made an amendment without departing from the scope of protection of present invention. Such as, fluorescent real time PCR used in the present invention can also adopt the mark substance beyond the fluorescent reporter group and fluorescent quenching group pointed out in embodiment listed in description as required, such as labels such as Tet, TAMRA, ROX, Cy3, TxRd, JOE; Or use other labelling systems outside Taqman technology, for instance the fluorescent probe labelling techniques such as MGB probe, molecular beacon MB probe, scorpion shape probe, fluorescence double cross probe; Or use the saturable dyes such as unsaturated dyestuff and LCGreen such as the chimeric method such as SYBRGreenI of dyestuff, as long as it using specific primer sequence of the present invention, get final product qualitative or detection by quantitative genes of interest existence, and then detect the existence of mycoplasma pneumoniae specifically. So, change and the replacement of customary means that these those skilled in the art are appreciated by also fall in protection scope of the present invention. Protection scope of the present invention should be limited by the accompanying claims.
SEQUENCELISTING
<110>Jiangsu and wound bio tech ltd
Fourth, little quiet
<120>mycoplasma pneumoniae fluorescence PCR detection reagent kit
<130>
<160>3
<170>PatentInversion3.3
<210>1
<211>22
<212>DNA
<213>artificial sequence
<220>
<221>primer_bind
<222>(1)..(22)
<223>primer sequence according to genome conserved regions design, for nucleic acid amplification and detection
<400>1
tatgctagcaggatacgcgaat22
<210>2
<211>23
<212>DNA
<213>artificial sequence
<220>
<221>primer_bind
<222>(1)..(23)
<223>primer sequence according to genome conserved regions design, for nucleic acid amplification and detection
<400>2
ggaccgctcgaaattcataaaag23
<210>3
<211>25
<212>DNA
<213>artificial sequence
<220>
<221>misc_binding
<222>(1)..(25)
<223>primer sequence according to genome conserved regions design, for nucleic acid amplification and detection
<400>3
tgaacgagaccacgccagaacggct25
Claims (3)
1., for a test kit for mycoplasma pneumoniae nucleic acid in specific detection sample, including PCR reactant liquor, enzyme mixation, negative quality-control product and positive quality control product, in PCR reactant liquor, the primer sequence for nucleic acid amplification reaction is as follows:
P1:5`-TATGCTAGCAGGATACGCGAAT-3`,
P2:5`-GGACCGCTCGAAATTCATAAAAG-3`,
The sequence of the oligonucleotide probe monitored for fluorescence signal in PCR reactant liquor is as follows:
Probe:5`-X1-TGAACGAGACCACGCCAGAACGGCT-Y1-3`,
Probe fluorescent reporter group X1For HEX, fluorescent quenching group Y1For Eclipse.
2. test kit as claimed in claim 1, it is further characterized in that PCR reaction system is 20 �� L, including 2 �� PCR buffer 10 �� L, 10mmol/LdNTP1.0 �� L, 25 ��m of each 0.5 �� L of ol/L primer, 10 ��m of ol/L probe 0.2 �� L, hot start Taq polymerase mixed liquor 0.4 �� L, sample DNA 2 �� L, add the extremely whole system 20 �� L of aquesterilisa.
3. test kit as claimed in claim 1, is further characterized in that PCR reaction cycle parameter is:
94 DEG C, 2min; Entering the cycle stage: 94 DEG C of degeneration 10s, 56 DEG C of annealing 50s, 72 DEG C extend 15s, coreaction 40 circulation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510887313.8A CN105624285A (en) | 2015-12-07 | 2015-12-07 | Mycoplasma pneumoniae fluorescent PCR detection reagent kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510887313.8A CN105624285A (en) | 2015-12-07 | 2015-12-07 | Mycoplasma pneumoniae fluorescent PCR detection reagent kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105624285A true CN105624285A (en) | 2016-06-01 |
Family
ID=56039627
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510887313.8A Pending CN105624285A (en) | 2015-12-07 | 2015-12-07 | Mycoplasma pneumoniae fluorescent PCR detection reagent kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105624285A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106191239A (en) * | 2016-07-11 | 2016-12-07 | 宁波基内生物技术有限公司 | A kind of real-time PCR detection mycoplasma pneumoniae nucleic acid and the primer of medicament-resistant mutation, probe, method and test kit |
| CN106916902A (en) * | 2017-05-09 | 2017-07-04 | 广州和实生物技术有限公司 | A kind of mycoplasma pneumoniae Constant Temperature Detection kit |
| CN108531556A (en) * | 2018-03-05 | 2018-09-14 | 南京岚煜生物科技有限公司 | The kit and its application method of multiple pathogens are detected based on micro-fluidic chip |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102230013A (en) * | 2011-06-21 | 2011-11-02 | 中国疾病预防控制中心传染病预防控制所 | Target sequence, primer, probe and kit for detecting Mycoplasma pneumonia |
| CN103060451A (en) * | 2013-01-10 | 2013-04-24 | 湖南圣湘生物科技有限公司 | Detection kit for mycoplasma pneumonia (MP) |
| CN104946769A (en) * | 2015-06-30 | 2015-09-30 | 中国疾病预防控制中心传染病预防控制所 | Kit for rapid detection and genotyping of mycoplasma pneumoniae |
-
2015
- 2015-12-07 CN CN201510887313.8A patent/CN105624285A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102230013A (en) * | 2011-06-21 | 2011-11-02 | 中国疾病预防控制中心传染病预防控制所 | Target sequence, primer, probe and kit for detecting Mycoplasma pneumonia |
| CN103060451A (en) * | 2013-01-10 | 2013-04-24 | 湖南圣湘生物科技有限公司 | Detection kit for mycoplasma pneumonia (MP) |
| CN104946769A (en) * | 2015-06-30 | 2015-09-30 | 中国疾病预防控制中心传染病预防控制所 | Kit for rapid detection and genotyping of mycoplasma pneumoniae |
Non-Patent Citations (1)
| Title |
|---|
| JONAS M. WINCHELL等: "Evaluation of Three Real-Time PCR Assays for Detection of Mycoplasma pneumonia in an Outbreak Investigation", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106191239A (en) * | 2016-07-11 | 2016-12-07 | 宁波基内生物技术有限公司 | A kind of real-time PCR detection mycoplasma pneumoniae nucleic acid and the primer of medicament-resistant mutation, probe, method and test kit |
| CN106916902A (en) * | 2017-05-09 | 2017-07-04 | 广州和实生物技术有限公司 | A kind of mycoplasma pneumoniae Constant Temperature Detection kit |
| CN108531556A (en) * | 2018-03-05 | 2018-09-14 | 南京岚煜生物科技有限公司 | The kit and its application method of multiple pathogens are detected based on micro-fluidic chip |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103642936B (en) | The specific detection of 55 type adenoviruss primed probe combination and test kit | |
| CN102660645B (en) | The specific detection of streptococcus pneumoniae and hemophilus influenza primed probe combination and test kit | |
| CN107746879A (en) | Detect RPA primers, probe, kit and the detection method of staphylococcus aureus | |
| Hu et al. | Detection of eight respiratory bacterial pathogens based on multiplex real‐time PCR with fluorescence melting curve analysis | |
| US20220098645A1 (en) | Fast and portable microfluidic detection system as an alternative to salmonella's classical culture method | |
| CN101113473A (en) | Method for detecting food-derived pathogenic vibrio bacteria by composite fluorescence PCR technique | |
| CN102634596B (en) | The specific detection of Bordetella pertussis and Bordetella parapertussis primed probe combination and test kit | |
| US8790899B2 (en) | Real-time PCR assays for rapid detection and differentiation of the Clostridium botulinum toxin genes A, B, E, and F | |
| CN105624285A (en) | Mycoplasma pneumoniae fluorescent PCR detection reagent kit | |
| Ogino et al. | Genotyping of Japanese field isolates of Mycoplasma synoviae and rapid molecular differentiation from the MS-H vaccine strain | |
| CN105567802A (en) | Fluorescence PCR (polymerase chain reaction) detection kit for Chlamydia pneumoniae | |
| CN102676664A (en) | Fluorescent quantitative polymerase chain reaction (PCR) primers and probes for detecting pathogenic bacteria of multiple aquatic products simultaneously and detection method | |
| CN104328175A (en) | Loop-mediated isothermal amplification (LAMP) primers, kit and method for detecting mouse Klebsiella pneumoniae | |
| CN105624284A (en) | Klebsiella pneumonia fluorescent PCR detection reagent kit | |
| CN105624286A (en) | Legionella pneumophila fluorescence PCR detection kit | |
| CN102643920B (en) | The specific detection of Neisseria meningitidis and discriminating primed probe combination and test kit | |
| CN104328174B (en) | A kind of detect the LAMP primer of Mus Klebsiella pneumonia, test kit and method | |
| CN1724686B (en) | Target sequence used for detecting mycoplasma pnoumoniae and reagent box | |
| CN109371150A (en) | Pseudomonas aeruginosa fluorescence PCR detection reagent kit | |
| EP3227459B1 (en) | Compositions and methods for detecting mecc containing methicillin-resistant staphylococcus aureus | |
| CN103993090A (en) | Specific nucleotides for providencia O31, O41, O42, O43 and O50 and application of specific nucleotides | |
| CN109439780A (en) | Moraxelle catarrhalis fluorescence PCR detection reagent kit | |
| CN105274199B (en) | Kit and its application method a kind of while that detect staphylococcus aureus and the rugged Cronobacter sakazakii of slope | |
| CN105567821A (en) | Bacillus-anthracis fluorescence PCR detection kit | |
| CN103642901B (en) | The specific detection of Neisseria meningitidis Z group primed probe combination and test kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160601 |
|
| RJ01 | Rejection of invention patent application after publication |