CN102071247B - Detection method for distinguishing non-pneumophilia legionella and legionella pneumophilia and kit - Google Patents
Detection method for distinguishing non-pneumophilia legionella and legionella pneumophilia and kit Download PDFInfo
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Abstract
The invention discloses a detection method for distinguishing non-pneumophilia legionella and legionella pneumophilia and a kit. The method detects legionella and legionella pneumophilia through single tube amplification of multi-wavelength fluorescent polymerase chain reaction (PCR) technology of a TaqMan probe by adopting a legionella target sequence SEQ ID No.1 design-based primer probe, and distinguishes the non-pneumophilia legionella and the legionella pneumophilia according to the result; and meanwhile, an artificially constructed internal reference system for avoiding false negative detection results is also adopted in the kit. The kit comprises nucleic acid extract, PCR buffer solution, a fluorescent probe, Taq enzyme, an internal reference, and negative and positive contrasts; and two steps comprising sample treatment and amplification detection are used in the kit. The kit is simple and convenient to operate, has high sensitivity, can avoid false positive generated by nucleic acid pollution of PCR products, can solve the problem of false negative caused by a PCR inhibitor in the sample, and can be widely applied to disease control and quick detection of the legionella and the legionella pneumophilia in clinic.
Description
Technical field
The invention belongs to a kind of fluorescent PCR that utilizes and distinguish the non-detection method addicted to lung and legionella pneumophilia and test kit.
Background technology
Legionella (Legionella) is a class Gram-negative bacteria, has found that there is 34 kinds, 53 serotypes at present.Legionella is often concealed in air-conditioning water cooler (tower), hot water pipeline, the first-class place of shower spraying, pass through airborne transmission, sucking human body through respiratory tract in the form of an aerosol causes the mankind to cause légionaires' disease, patient there will be high fever, shivers, coughs, the symptom being similar to upper respiratory disease such as uncomfortable in chest, cause death time serious, its mortality ratio of now general report is 5 ~ 30%.Extensively exist in place due to legionella human lives, this disease is once outbreak of epidemic, and it is sizable for playing harm; It is reported, it is higher that the legionella inside cooling tower detects positive rate, from 15% to 30% not etc.2006, the Ministry of Health also found in the sampling observation of national public place central air conditioning, and legionella recall rate is up to 24.5%.In addition, detect result from legionella patient and ambient water, legionella pneumophilia (Legionella pneumophila) recall rate is the highest, and the harm caused clinically is also more serious than other legionella bacterium.
It is isolated culture that traditional legionella detects Main Means, but the method is time-consuming, troublesome poeration.In addition also have adopt observations of Dieterle silver impregnation method smear, immunochromatography, directly fluorescence antibody mensuration, PCR electrophoresis or probe hybridization detection method carry out diagnosing.But there is the defects such as sensitivity deficiency in these methods, easily causes undetected.In recent years, along with molecular biological progress, real-time fluorescence PCR technology is used widely in pathogen detection, and normal PCR electrophoretic detection compares and has many advantages: (1) its stopped pipe adopted detects the amplified production pollution problem that disappears, and improves accuracy in detection; (2) add fluorescent probe in PCR system, improve detection specificity and sensitivity; (3) rapidly, without the need to the post-processing detection step of normal PCR, level of automation is high in operation.If introduce internal reference and double wave length fluorescent probe in reaction system, the detected result false negative because PCR inhibitor possible in sample causes can also be monitored.
At present, have in clinical labororatory and utilized fluorescent PCR to detect legionella and distinguish the report of legionella pneumophilia, such as (the J Clin Microbiol such as Templeton, 2003 (41): 4016-4021) utilize molecular beacon (Molecular Beacon) multicolor fluorescence round pcr to detect legionella 16S rRNA, mip gene and external standard reference gene PhHV, legionella pneumophilia is distinguished mutually with other legionella.(the Appl Environ Microbiol such as Stolhaug, 2006 (72): 6394-6398) utilize FRET fluorescence probe round pcr to detect legionella 16S rRNA gene, and by the melting curve analysis of amplified production, legionella pneumophilia is distinguished mutually with other legionella; But can analyze, due to different serotypes strain gene sequence variation, legionella pneumophilia melting curve may Yin Tezheng not obvious and cause result to judge.
The invention provides and a kind ofly distinguish the non-detection method addicted to lung and legionella pneumophilia and test kit, utilize TaqMan probe multicolor fluorescence round pcr, only needing without the need to detecting multiple characterizing gene to detect legionella 16S rRNA gene order, only just legionella pneumophilia can be distinguished mutually with non-legionella pneumophilia with the fluorescently-labeled probe in detecting of difference without the need to the melting curve analysis after fluorescent PCR; Present invention further introduces artificial constructed internal reference system, for avoiding the false negative of detected result simultaneously.Compare with the public technology of legionella pneumophilia addicted to lung with existing differentiation detection is non-, detected result of the present invention is reliable and stable, and test kit is easy to use, is suitable for the rapid detection of disease control and clinical middle legionella and legionella pneumophilia.
Summary of the invention
The object of this invention is to provide and a kind ofly distinguish the non-detection method addicted to lung and legionella pneumophilia and test kit, it adopts the primed probe designed based on legionella target sequence SEQ ID No.1, detect legionella and legionella pneumophilia by TaqMan probe multi-wavelength Fluorescence PCR assay Single tube amplification, and distinguish that differentiation is non-addicted to lung and legionella pneumophilia according to this result; Test kit also uses artificial constructed internal reference system for avoiding detected result false negative simultaneously.Reagent constituents comprises nucleic acid extraction liquid, PCR damping fluid, fluorescent probe, Taq enzyme, internal reference, feminine gender and positive control; Its use comprises sample process and augmentation detection two step.Test kit is easy and simple to handle, highly sensitive, not only can avoid the false positive that self PCR primer pollution of nucleic acid produces, and the Problem of False Negative because PCR inhibitor in sample causes can be solved, the rapid detection of disease control and clinical middle legionella and legionella pneumophilia can be widely used in.
1. the Design and synthesis of real-time fluorescence PCR primer probe
By inquiring about legionella 16S rRNA gene order in GenBank, find after various sources legionella, legionella pneumophilia genes sequence being compared with molecular biology professional software, wherein one section of sequence SEQ ID No.1 is comparatively conservative in all legionella bacteriums, and has legionella pneumophilia specific sequence.Therefore, according to principle and the SEQ ID No.1 sequence of the design of TaqMan fluorescent PCR, devise a pair legionella Auele Specific Primer, legionella bacterial fluorescence probe, a legionella pneumophilia fluorescent probe and detect in real time for legionella and legionella pneumophilia.Primer and probe base sequence as follows:
Upstream primer is 5 '-TgTgAAATTCCTgggCTTAAC-3 ', as shown in SEQ ID No.2;
Downstream primer is 5 '-TTCgTgCCTCAgTgTCAg-3 ', as shown in SEQ ID No.3;
Legionella fluorescent probe is 5 '-CAggTAgCCgCCTTCgCC-3 ', 5 ' end flag F AM (6-Fluoresceincarboxylic acid), 3 ' end mark BHQ (Black Hole Quencher)), as shown in SEQ ID No.4.
Legionella pneumophilia fluorescent probe be 5 '-TgggACggTCAgATAATACTggTT-3 ' (5 ' end mark HEX (6-carboxyl chlordene fluorescein) or JOE (6-carboxyl-4 ', 5 '-two chloro-2 ', 7 '-dimethoxyfluorescein), VIC fluorophor, 3 ' end mark BHQ), as shown in SEQ ID No.5.
It also can be that above-mentioned sequence extends the sequence of 10 ~ 20 Nucleotide forward or backward according to target sequence SEQ ID No.1 that legionella and legionella pneumophilia detect primer probe sequence, or is greater than more than 85% with above-mentioned sequence homology.
Another aspect of the present invention have employed artificial constructed internal reference system, for monitoring PCR inhibition possible in sample amplification reaction, avoid detected result false negative, internal reference system comprises internal reference probe and internal reference DNA, and wherein internal reference probe adopts SEQ ID No.6 sequence:
5 '-TTCAgCgAgCgTgggACATCAgg-3 ' (5 ' end mark Cy5 or ROX (carboxy-X-rhodamine), TAMRA (6-carboxyl tetramethylrhodamine) fluorophor, 3 ' end mark BHQ), internal reference probe also can be the sequence being greater than more than 85% with above-mentioned sequence homology.
Internal reference DNA in internal reference system is artificial constructed DNA profiling, it contains a primer sequence in nucleic acid to be checked (legionella DNA) amplimer and another Primers complementary sequences and internal reference probe sequence or its complementary sequence, and a primer sequence and another Primers complementary sequences lay respectively at the upstream and downstream of internal reference probe sequence or complementary sequence.
By above-mentioned design, the pair of primers sequence of acquisition by legionella bacterium special, two fluorescent probes are respectively legionella bacterium and legionella pneumophilia institute is special; And internal reference system comprises internal reference probe and internal reference DNA, it is 25 μMs that above-mentioned primed probe is dissolved to concentration with aseptic double-distilled water after being synthesized by business sequent synthesis company (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd etc.) ,-20 DEG C of preservations.
2. the preparation of test kit
Legionella kit for detecting nucleic acid (PCR-fluorescence probe method) (32 person-portion) containing above-mentioned primer and probe, its main component is as follows:
(1) nucleic acid extraction liquid 1.5ml, main component is 10 ~ 50mM NaOH, 5 ~ 10mM EDTA, 1% ~ 10%Triton X-100,1% ~ 10%NP-40.
(2) PCR damping fluid 0.7ml, main component has Tris-HCl (pH8.3), KCl, gelatin, dATP, dGTP, dCTP, dUTP, MgCl
2, primer.
(3) fluorescent probe 0.1ml, containing legionella, legionella pneumophilia fluorescent probe.
(4) Taq enzyme 70 μ l, containing Taq archaeal dna polymerase, uracil-DNA glycosylase (UNG enzyme).
(5) internal reference 16 μ l, containing internal reference probe and internal reference DNA.
(6) negative control 200 μ l is culture of Escherichia coli.
(7) positive control 200 μ l is legionella pneumophilia culture.
The above-mentioned test kit be assembled into is-20 DEG C of preservations.
The 30 μ l reaction system each main component concentration prepared by (2) ~ (5) and template is as follows:
Tris-HCl (pH8.3) 10mM, KCl 50mM, each 0.2 ~ 0.4mM of gelatin 0.1mg/ml, dATP, dGTP, dCTP, dUTP, MgCl
2each 0.1 ~ 0.5 μM of each 0.2 ~ 0.5 μM of 2.5 ~ 5mM, primer, legionella, legionella pneumophilia fluorescent probe and internal reference probe, Taq archaeal dna polymerase 1 ~ 5U, UNG enzyme 0.1 ~ 1U.
In another preference of the present invention, the 30 μ l reaction system each main component concentration prepared by (2) ~ (5) and template is as follows:
Tris-HCl (pH8.3) 10mM, KCl 50mM, gelatin 0.1mg/ml, dATP, dGTP, dCTP, dUTP each 0.3mM, MgCl
2each 0.2 μM of each 0.35 μM of 3.5mM, primer, legionella fluorescent probe and legionella pneumophilia fluorescent probe, internal reference probe 0.15 μM, Taq archaeal dna polymerase 2U, UNG enzyme 0.2U.
3. test kit using method
One of test kit of the present invention use step in sample legionella and legionella pneumophilia testing process is:
(1) sample preparation
Get the sample of collection, if water sample to be measured or air, need first to be placed in centrifuge tube through filter membrane (0.22 ~ 0.45 μm, aperture) filtration, wash-out; If clinical throat swab scavenging solution, bronchoalveolar lavage fluid, sputum (need first liquefy) sample are then directly placed in centrifuge tube, sample liquid 13, centrifugal 6 minutes of 000rpm, carefully inhales with pipettor and abandons supernatant (for preventing siphoning away precipitation, can retain 5 μ l raffinates).In precipitation, add 50 μ l nucleic acid extraction liquid, latter 100 DEG C of vortex vibration mixing keeps 10 minutes, and 13,000rpm gets supernatant in centrifugal 6 minutes and carries out fluorescent PCR augmentation detection.Test kit is negative identical with sample liquid with positive control treatment process.
(2) augmentation detection
When test kit breaks a seal first and uses, all join in PCR damping fluid after internal reference being looked after the low-speed centrifugal several seconds, on vortex vibrator, vibration mixing uses.According to sample number n+2 to be detected, get PCR damping fluid 21 × (n+2), fluorescent probe 3 × (n+2), Taq enzyme 2 × (n+2) component, each amplification pipe packing 26 μ l after mixing, adds respectively according to the test kit negative control of sample processing method process, positive control and each 4 μ l of the above-mentioned n handled well a sample form.Each reaction amplification pipe volume is 30 μ l, is put into by above-mentioned amplification pipe on hyperchannel real-time fluorescence PCR instrument and runs according to the circulate program of 40 times of 94 DEG C of 10sec after 50 DEG C of 2min, 94 DEG C of 2min, 60 DEG C of 45sec.After end of run, result judges according to following table.
Note: if during Ct value=40, some fluorescent PCR instrument software may be shown as > 40, without value or UNDET etc.
4. beneficial effect
The present invention is according to the primed probe of above-mentioned design and technical scheme, and be developed into legionella kit for detecting nucleic acid (PCR-fluorescence probe method) by optimizing reaction system and amplification condition, its principal feature is as follows:
(1) adopt specific target sequence and design primed probe accordingly and increased by fluorescent PCR, detect same target sequence in real time with the fluorescently-labeled probe of difference and detect legionella, and distinguishing non-addicted to lung and legionella pneumophilia, result is reliable and stable.
(2) the internal reference system introduced, the internal reference signal of detection can monitored results whether false negative, can avoid the mistaken diagnosis caused thus;
(3) test kit employs sequence-specific and different fluorescently-labeled three kinds of fluorescent probes, and determined wavelength is different and can not mutually disturb between fluorescent signal, ensure that detection specificity;
(4) test kit stopped pipe detects, fast easy and simple to handle;
(5) the dUTP-UNG enzyme system in amplification system further avoids the result false positive that gene-amplification product pollution causes.
The These characteristics of test kit, is and adopts the target sequence of screening and directly cause in conjunction with the application of multi-wavelength Fluorescence PCR assay system in combination.From the principle, test kit of the present invention passes through the same target sequence of real-time fluorescent PCR amplification and the analysis to two sense channel signals in légionaires' disease substance clinical sample detects, judge the existence of legionella and legionella pneumophilia nucleic acid specific fragment, and in reaction system, whether there is the false negative because inhibition causes.As the means of a kind of legionella and legionella pneumophilia nucleic acid rapid detection, Technical Design of the present invention is reasonable, can be widely used in the Rapid identification of disease control and clinical middle legionella and legionella pneumophilia.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can do various technical change or amendment by technology general knowledge to the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
Main agents involved in the present invention illustrates:
Taq archaeal dna polymerase, uracil-DNA glycosylase (UNG enzyme), dNTPs (dATP, dUTP, dCTP, dGTP) are Shanghai Hong Yi company and produce, and primed probe and other chemical reagent are purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Embodiment 1: the preparation of legionella kit for detecting nucleic acid (PCR-fluorescence probe method)
(1) primed probe synthesis
Design primed probe according to target sequence SEQ ID No.1, and entrust business sequent synthesis company (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) to synthesize:
Upstream primer is 5 '-TgTgAAATTCCTgggCTTAAC-3 ', as shown in SEQ ID No.2;
Downstream primer is 5 '-TTCgTgCCTCAgTgTCAg-3 ', as shown in SEQ ID No.3;
Legionella fluorescent probe is 5 '-CAggTAgCCgCCTTCgCC-3 ', 5 ' end flag F AM (6-Fluoresceincarboxylic acid), 3 ' end mark BHQ (Black Hole Quencher)), as shown in SEQ ID No.4.
Legionella pneumophilia fluorescent probe be 5 '-TgggACggTCAgATAATACTggTT-3 ' (5 ' end mark HEX (6-carboxyl chlordene fluorescein) or JOE (6-carboxyl-4 ', 5 '-two chloro-2 ', 7 '-dimethoxyfluorescein), VIC fluorophor, 3 ' end mark BHQ), as shown in SEQ ID No.5.
Being dissolved to concentration with aseptic double-distilled water is respectively 25 μMs ,-20 DEG C of preservations.
(2) PCR damping fluid is prepared
17.2ml 5*PCR Buffer is added successively, dATP, dGTP, dCTP, dUTP (the equal 100mM of concentration) each 258 μ l, upstream and downstream primer (25 μMs) each 1.2ml, 100mM MgCl in 36ml aseptic deionized water
23ml, vibration mixing, cumulative volume use water constant volume is to 60ml.According to 700 μ l/ pipe packing.
(3) fluorescent probe is prepared
In 8.4ml aseptic deionized water, add 25 μMs of legionella fluorescent probes and each 0.8ml of legionella pneumophilia fluorescent probe successively, vibration mixing, according to 100 μ l/ pipe packing.
(4) Taq enzyme is prepared
In 5.6ml Taq enzyme diluent, add 1.6ml Taq enzyme (5U/ μ l) and 0.8ml UNG enzyme (1U/ μ l), put upside down and repeatedly mix.According to 70 μ l/ pipe packing.
(5) nucleic acid extraction liquid is prepared
In 800ml water, add 2.0 grams of NaOH, 10ml Triton X-100,10ml NP-40,18.6 grams of EDTA, 10ml 1M Tris-HCl (pH8.0), magnetic stirring apparatus stirs and within 1 hour, ensures to dissolve and fully mixing; 1L is settled to, autoclaving with water.According to the packing of 1.5ml/ pipe.
(6) internal reference is prepared
In 8ml aseptic deionized water, containing 9.5 μMs of internal reference probes and 10
5copy/ml internal reference DNA.According to 16 μ l/ pipe packing.
(7) test kit feminine gender, positive reference substance is prepared
Cultivate intestinal bacteria and legionella reference culture respectively, and measure nutrient solution concentration by viable plate count method, be diluted to 1 × 10
4individual bacterium/ml.According to 200 μ l/ pipe packing.
(8) kind of the component of 6 after above-mentioned packing is placed in same container, is assembled into test kit, encapsulation.
Preparation technology is summarized as follows:
(1) primed probe synthesized quantitatively is prepared, Concentration Testing, sampling quality inspection.
(2) PCR damping fluid, Taq enzyme, nucleic acid extraction liquid, internal reference is aseptic subpackaged, sampling quality inspection.
(3) feminine gender, positive reference substance are carried out concentration determination, packing, sampling quality inspection.
(4) assemble finished product test kit, be stored in-20 DEG C.
Embodiment 2: the application of legionella kit for detecting nucleic acid (PCR-fluorescence probe method)
(1) sample preparation
Get the water sample of collection, be first placed in centrifuge tube through filter membrane (0.22 ~ 0.45 μm, aperture) filtration, wash-out, centrifugal 6 minutes of 13,000rpm, careful suction abandons supernatant (for preventing siphoning away precipitation, can retain 5 μ l raffinates).In precipitation, add 50 μ l nucleic acid extraction liquid, latter 100 DEG C of vortex vibration mixing keeps 10 minutes, and 13,000rpm gets supernatant in centrifugal 6 minutes and carries out fluorescent PCR augmentation detection.Test kit is negative identical with sample eluent with positive control treatment process.
(2) augmentation detection
According to the method in summary of the invention, internal reference wherein, by each component freeze thawing vibration mixing, is added PCR damping fluid and mixing of vibrating by unpacking test kit subsequently.Then according to sample number n+2 to be detected, get PCR damping fluid 21 × (n+2), fluorescent probe 3 × (n+2), Taq enzyme 2 × (n+2) component, each amplification pipe packing 26 μ l after mixing, adds each 4 μ l of above-mentioned test kit negative control, positive control and the n sample form handled well respectively.Each reaction amplification pipe volume is 30 μ l, above-mentioned amplification pipe is put into the program operation of 40 times that ABI7500 real-time fluorescence PCR instrument (Applied biosystems) circulates according to 94 DEG C of 10sec after 50 DEG C of 2min, 94 DEG C of 2min, 60 DEG C of 45sec.
(3) detected result
After instrument end of run, according to the result determination methods in summary of the invention, negative control is negative, and positive control is positive, as FAM passage amplification curve is obvious S shape and Ct < 40 after sample amplification, HEX channel C t=40 then judges that non-legionella pneumophilia is positive, as FAM and HEX passage amplification curve, Ct < 40 is judged as that legionella pneumophilia is positive all in obvious S shape and simultaneously, if FAM, HEX channel C t is 40 and Cy5 channel C t < 40 judges that legionella is negative, if FAM, HEX and Cy5 channel C t value is 40, then sample extraction thing aseptic deionized water can be diluted, or resampling remove PCR inhibition possible in sample after recheck.
Embodiment 3: the application of legionella kit for detecting nucleic acid (PCR-fluorescence probe method)
(1) sample preparation
Get the throat swab of clinical acquisitions, sputum, bronchoalveolar lavage fluid (throat swab first first liquefies with the mixing of 4%NaOH equal-volume with the rinsing of 1ml physiological saline, sputum), throat swab rinsing liquid, liquefaction sputum and bronchoalveolar lavage fluid are placed in centrifuge tube respectively, sample liquid 13, centrifugal 6 minutes of 000rpm, supernatant (for preventing siphoning away precipitation, can retain 5 ~ 10 μ l raffinates) is abandoned in careful suction.In precipitation, add 50 μ l nucleic acid extraction liquid, latter 100 DEG C of vortex vibration mixing keeps 10 minutes, and 13,000rpm gets supernatant in centrifugal 6 minutes and carries out fluorescent PCR augmentation detection.Test kit is negative identical with sample liquid with positive control treatment process.
(2) augmentation detection
According to the method in summary of the invention, internal reference wherein, by each component freeze thawing vibration mixing, is added PCR damping fluid and mixing of vibrating by unpacking test kit subsequently.Then according to sample number n+2 to be detected, get PCR damping fluid 21 × (n+2), fluorescent probe 3 × (n+2), Taq enzyme 2 × (n+2) component, each amplification pipe packing 26 μ l after mixing, adds each 4 μ l of above-mentioned test kit negative control, positive control and the n sample form handled well respectively.Each reaction amplification pipe volume is 30 μ l, above-mentioned amplification pipe is put into the program operation of 40 times that LC480 real-time fluorescence PCR instrument (Roche company) circulates according to 94 DEG C of 10sec after 50 DEG C of 2min, 94 DEG C of 2min, 60 DEG C of 45sec.
(3) detected result
After instrument end of run, according to the result determination methods in summary of the invention, negative control is negative, and positive control is positive, as FAM passage amplification curve is obvious S shape and Ct < 40 after sample amplification, HEX channel C t=40 then judges that non-legionella pneumophilia is positive, as FAM and HEX passage amplification curve, Ct < 40 is judged as that legionella pneumophilia is positive all in obvious S shape and simultaneously, if FAM, HEX channel C t is 40 and Cy5 channel C t < 40 judges that legionella is negative, if FAM, HEX and Cy5 channel C t value is 40, then sample extraction thing aseptic deionized water can be diluted, or resampling remove PCR inhibition possible in sample after recheck.
Nucleotides sequence list
SEQUENCE LISTING
<110> Fuxing Medical Science-Technology Development Co., Ltd., Shanghai
Shanghai Fosun Pharmaceutical (Group) Co., Ltd.
Wu great Zhi, Li Zhiyuan, Xia Yi
<120> mono-kind distinguishes the non-detection method addicted to lung and legionella pneumophilia and test kit
<130>LegP
<140>CN
<141>2009-11-22
<160>6
<170>PatentIn version 3.3
<210>1
<211>510
<212>DNA
<213>Legionella sp.
<220>
<221>SEQ ID No.1
<222>(1)..(500)
<400>1
atgggggcaa ccctgatcca gcaatgccgc gtgtgtgaag aaggcctgag ggttgtaaag 60
cactttcagt ggggaggagg gttgataggt taagagctaa ttaactggac gttacccaca 120
gaagaagcac cggctaactc cgtgccagca gccgcggtaa tacggagggt gcaagcgtta 180
atcggaatta ctgggcgtaa agggtgcgta ggtggttgat taagttatct gtgaaattcc 240
tgggcttaac ctgggacggt cagataatac tggttgactc gagtatggga gagggtagtg 300
gaatttccgg tgtagcggtg aaatgcgtag agatcggaag gaacaccagt ggcgaaggcg 360
gctacctggc ctaatactga cactgaggca cgaaagcgtg gggagcaaac aggattagat 420
accctggtag tccacgctgt aaacgatgtc aactagctgt tggttatatg aaaataatta 480
gtggcgcagc aaacgcgata agttgaccgc 510
<210>2
<211>21
<212>DNA
<213>Legionella sp.
<220>
<221>SEQ ID NO.2
<222>(1)..(21)
<400>2
tgtgaaattc ctgggcttaa c 21
<210>3
<211>18
<212>DNA
<213>Legionella sp.
<220>
<221>SEQ ID No.3
<222>(1)..(18)
<400>3
ttcgtgcctc agtgtcag 18
<210>4
<211>18
<212>DNA
<213>Legionella sp.
<220>
<221>SEQ ID No.4
<222>(1)..(18)
<400>4
caggtagccg ccttcgcc 18
<210>5
<211>24
<212>DNA
<213>Legionella pneumophila
<220>
<221>SEQ ID No.5
<222>(1)..(24)
<400>5
tgggacggtc agataatact ggtt 24
<210>6
<211>23
<212>DNA
<213>Inter-control
<220>
<221>SEQ ID No.6
<222>(1)..(23)
<400>6
ttcagcgagc gtgggacatc agg 23
Claims (8)
1. distinguish the non-detection kit addicted to lung and legionella pneumophilia for one kind, it is characterized in that, adopt the primed probe designed based on legionella target sequence SEQID No.1, the legionella pneumophilia fluorescent probe wherein distinguished addicted to lung and non-legionella pneumophilia is SEQID No.5 sequence, detect legionella and legionella pneumophilia by TaqMan probe multi-wavelength Fluorescence PCR assay Single tube amplification, and distinguish that differentiation is non-addicted to lung and legionella pneumophilia according to this result.
2. test kit as claimed in claim 1, it is characterized in that, target sequence SEQ ID No.1 used can be used for distinguishing the non-conserved sequence addicted to lung and legionella pneumophilia in legionella 16S rRNA gene, according to the primer of SEQ ID No.1 sequences Design can increase its continuous 70 ~ 300 Nucleotide, design two TaqMan fluorescent probes can detect legionella and legionella pneumophilia respectively, described primer length is 15 ~ 35 Nucleotide.
3. test kit as claimed in claim 1, is characterized in that, test kit primer sequence used is the sequence of SEQ ID No.2 and SEQ IDNo.3; Legionella fluorescent probe is SEQ IDNo.4 sequence, its 5 ' end flag F AM fluorophor; 5 ' end mark HEX or JOE, the VIC fluorophor of legionella pneumophilia fluorescent probe sequence SEQ ID No.5; Article two, mark BHQ quenching group held by probe 3 '.
4. test kit as claimed in claim 1, is characterized in that, introduce an artificial constructed internal reference system for avoiding detected result false negative, internal reference system comprises internal reference probe and internal reference DNA.
5. test kit as claimed in claim 4, is characterized in that, internal reference probe adopts SEQ ID No.6 sequence, and probe 5 ' holds mark Cy5 or ROX, TAMRA fluorophor, 3 ' end mark BHQ quenching group.
6. test kit as claimed in claim 4, it is characterized in that, internal reference DNA used is artificial constructed DNA profiling, it contains a primer sequence in legionella DNA cloning primer and another Primers complementary sequences and internal reference probe sequence or its complementary sequence, and a primer sequence and another Primers complementary sequences lay respectively at the upstream and downstream position of internal reference probe sequence or complementary sequence.
7. test kit as claimed in claim 1, comprises nucleic acid extraction liquid, PCR damping fluid, fluorescent probe, Taq enzyme, internal reference, feminine gender and positive control.
8. test kit as claimed in claim 1, is characterized in that the reaction system be mixed with according to following concentration carries out amplified reaction: Tris-HCl 10mM, the KCl 50mM of pH8.3, each 0.2 ~ 0.4mM of gelatin 0.1mg/ml, dATP, dGTP, dCTP, dUTP, MgCl
2each 0.2 ~ 0.5 μM of 2.5 ~ 5mM, primer, legionella fluorescent probe and legionella pneumophilia fluorescent probe and each 0.1 ~ 0.5 μM of internal reference fluorescent probe, Taq archaeal dna polymerase 1 ~ 5U, UNG enzyme 0.1 ~ 1U.
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| CN103184283A (en) * | 2013-03-07 | 2013-07-03 | 广州金域医学检验中心有限公司 | PCR enzyme digestion type kit for detecting legionella bacteria and application of kit |
| CN104313169B (en) * | 2014-11-03 | 2016-04-13 | 无锡市疾病预防控制中心 | A kind of quick differentiation is non-addicted to lung, addicted to lung and the triple PCR detection method addicted to lung I type legionella |
| CN105624286A (en) * | 2015-12-07 | 2016-06-01 | 江苏和创生物科技有限公司 | Legionella pneumophila fluorescence PCR detection kit |
| US20220145367A1 (en) * | 2019-01-31 | 2022-05-12 | Quest Diagnostics Investments Llc | Methods for detecting legionella |
| CN114410847A (en) * | 2022-03-03 | 2022-04-29 | 广东省人民医院 | Legionella pneumophila and dengue fever nucleic acid quantitative combined detection reagent |
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2009
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Non-Patent Citations (3)
| Title |
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| Taqman-MGB双重探针PCR技术检测军团菌;朱水荣等;《中国人兽共患病学报》;20090228;第25卷(第2期);174-178 * |
| Utility of Real-Time PCR for Diagnosis of Legionnaires’ Disease in Routine Clinical Practice;Diederen bmw et al;《J Clin Microbiol》;20071219;第46卷(第2期);671-677 * |
| 嗜肺军团菌荧光定量PCR检测;朱水荣等;《中国公共卫生》;20080930;第24卷(第9期);1111-1113 * |
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| CN102071247A (en) | 2011-05-25 |
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Address after: 200233 Yishan Road, Shanghai, No. 1289 Applicant after: Shanghai Fosun Pharmaceutical (Group) Corporation Applicant after: Shanghai Xingyao Medical Technology Development Co., Ltd. Address before: 200233 Yishan Road, Shanghai, No. 1289 Applicant before: Shanghai Fosun Pharmaceutical (Group) Corporation Applicant before: Shanghai Fosun Pharmaceutical (Group) Limited by Share Ltd. |
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Correction item: Patentee|Address|Patentee Correct: Shanghai Xingyao Medical Technology Development Co., Ltd.|201315 Shanghai city Pudong New Area Kangshi Road No. 17|Shanghai Fuxing Pharmaceutical (Group) Co., Ltd. False: Shanghai Fosun Pharmaceutical (Group) Corporation|200233 Yishan Road, Shanghai, No. 1289|Shanghai Xingyao Medical Technology Development Co., Ltd. Number: 09 Volume: 31 |
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Correction item: Patentee|Address|Patentee Correct: Shanghai Xingyao Medical Technology Development Co., Ltd.|201315 Shanghai city Pudong New Area Kangshi Road No. 17|Shanghai Fuxing Pharmaceutical (Group) Co., Ltd. False: Shanghai Fosun Pharmaceutical (Group) Corporation|200233 Yishan Road, Shanghai, No. 1289|Shanghai Xingyao Medical Technology Development Co., Ltd. Number: 09 Page: The title page Volume: 31 |