CN103205502B - Fluorescence quantification PCR (Polymerase Chain Reaction) primer, probe and kit for detecting dog leptospira nucleic acid - Google Patents

Abstract

The invention relates to a fluorescence quantification PCR (Polymerase Chain Reaction) primer, a fluorescence quantification PCR probe and a fluorescence quantification PCR kit for detecting dog leptospira nucleic acid. The primer and the probe are shown as SEQ ID NO. (Sequence Identification Number) 1-4. The kit comprises 22 microliters of 5xFRET (Fluorescence Resonance Energy Transfer)-qPCRStock and 22 microliters of 5xOligo mixture, wherein the Oligo mixture comprises the primer and the probe. With the adoption of the primer and the probe, the pathogenic dog leptospira can be detected quickly, specifically and sensitively.

Description

A kind of fluorescence quantification PCR primer, probe and test kit that detects dog Leptospira nucleic acid

Technical field

The present invention relates to fluorescence quantitative PCR detection primer, probe and the test kit of pathogenic dog Leptospira diagnosis.This invention is responsive, special, pathogenic Leptospira (the Leptospira interrogans Canicola Canicola of dog is infected in amplification fast, L.interrogans Icterohemorrhagiae Icterohemorrhagiae, L.interrogans Pomona Pomona, with L.kerschneri Grippotyphosa Grippotyphosa), and the nucleic acid of the Leptospira (L.biflexa) of the non-virulent that do not increase.

Background technology

Leptospira can cause the important zoonosis of many animals and people's morbidity.The main existing laboratory diagnosis technology of leptospiral infection comprises: 1) face disease diagnosis.Leptospira causes, and to face disease symptom not special, limited in the value of clinical diagnosis; 2) pathogen isolation: the time of these needs is long, and need to be through the professional of special training; 3) Serologic test detects anti-leptospiral antibody.The limitation of this technology is to carry out early diagnosis to Leptospira, and can not effectively distinguish diagnosis and distinguish leptospiral the infected, metainfective rehabilitation clients and accepted vaccine recipient; 3) round pcr detects leptospiral nucleic acid.Existing 16 kinds of Leptospira, approximately 38 serologic group, surpass 250 serotypes, infect multiple host.In addition, the mobility of leptospiral gene is very large, and the gene order between homophyletic does not differ greatly.Therefore, be difficult to the Leptospira that a single PCR method is accurate, responsive, detect fast all strains.Existing PCR method can not play molecular diagnosis fast and effectively to the leptospiral infection of dog.The particularly important is, the leptospiral quantity in leptospiral patient's blood, urine is low, and the present insufficient sensitivity of the diagnostic techniques on market.

Summary of the invention

The object of the present invention is to provide a kind of fast, the pathogenic leptospiral quantitative PCR detection technique of the infection dog of high specificity, highly sensitive, the detection that is applicable to clinical use, comprise and detect primer, probe and test kit.

Principle of the present invention and most crucial key problem in technology are scientifically to design amplification and detect pathogenic leptospiral special, the efficient primer of dog and probe.Guaranteeing the pathogenic dog Leptospira of the efficient amplification of primer of design, the leptospiral while of the pathogenic dog of probe specific detection of design, guaranteeing that this primer and probe do not increase and detect the Leptospira of non-virulent.From GenBank(www.ncbi.nlm.nih.gov) obtain the leptospiral LigA gene order of following pathogenic dog, and by the method for Clustal Multiple Alignment Algorithm, all sequences is alignd.Relevant information is in Table-1:

Table-1. pathogenic dog Leptospira.

Figure-1 shows that primer and the probe of the present invention's design is as follows:

Upstream primer: 5 '-GCTACWGGGATCTACTCTGACAACTC-3 ' (SEQ ID NO.1);

Downstream primer: 5 '-GGACTACTTACYTTTCCGAATGTGGCT-3 ' (SEQ ID NO.2)

6-FAM probe: 5 '-ATTTCAAACGCCMAAAAAAATCAAGGAAACKCTTA-(6-FAM)-3 ' (SEQ ID NO.3);

LCRed640 probe: 5 ' (LCRed640)-GGAGCAGCTACAGGARCAACGGATATT-(phosphoric acid is built)-3 ' (SEQ ID NO.4).

The invention also discloses the PCR kit for fluorescence quantitative that detects dog Leptospira nucleic acid, this test kit comprises 5x FRET-qPCR Stock22 microlitre, and the Oligo mixture of the 5x of 22 microlitres (being upstream primer, 5 μ M downstream primers, the 6-FAM probe of 1 μ M, the LCRed640 probe of 1 μ M of 5 μ M containing concentration).

Technical scheme of the present invention is to draw by following experiment:

(1) the leptospiral quantitative PCR of pathogenic dog is specific determines:

I) primer of design, for the blast search of GenBank, is confirmed the leptospiral nucleic acid of the primer specific ground pathogenic dog of amplification of the present invention's design, and do not increase other pathogenic agent and the leptospiral nucleic acid of non-virulent.With the PCR product of 176-bp, carry out the result demonstration of BLAST, pathogenic leptospiral sequence and PCR product have coincideing of 98-100%.In addition, the highest goodness of fit be and leptospiral 6% similarity of non-virulent;

Ii) by Integrated DNA Technology company, synthesized the sequence of pathogenic dog Leptospira amplification region type strain, that contain PCR.By the PCR system (upstream primer, downstream primer, 6-FAM probe, LCRed640 probe) of design, synthetic Leptospira DNA is carried out to pcr amplification;

Iii) positive (Leptospira interrogans Pomona) is carried out to pcr amplification;

Iv) the nucleic acid of negative control and other similar pathogenic agent is carried out to pcr amplification;

V) observe the variation of above amplification object fluorescence intensity (640nm) in PCR process, and pcr amplification product is in the size of the band of agarose gel electrophoresis.Fluorescence occurs or strengthens at 640nm wavelength, shows positive; The leptospiral pcr amplification product of pathogenic dog shows the band of the 176-bp of expection at agarose gel electrophoresis;

Iv) the PCR product of amplification is checked order, the result of order-checking and the sequence of GenBank compare.Result shows, the leptospiral nucleic acid of the primer specific ground pathogenic dog of amplification of design, and the Leptospira of do not increase other similar pathogenic agent or non-virulent.

(2) determining of pathogenic dog Leptospira quantitative PCR sensitivity: by the sequence of the synthetic leptospiral LigA of pathogenic dog of Intergrated DNA Technology.According to the DNA quantitative technique of molecular weight and absolute weight and the PicoGreen of synthetics, calculate the absolute number of the gene copy of the contained LigA of synthetics.Subsequently, synthetics is diluted, prepare the dilution reagent of every 10 μ l syntheticss containing the LigA of 10000 copies, 1000 copies, 100 copies, 10 copies.Pathogenic dog Leptospira with above-mentioned PCR system amplification containing different concns LigA, determines that the present invention detects the leptospiral sensitivity of pathogenic dog according to this.Result shows, this invention can amplified reaction system in the leptospiral LigA gene of pathogenic dog of 1 copy.

Compared with prior art, the invention has the advantages that and adopt primer of the present invention and probe can detect quick, special, delicately pathogenic dog Leptospira.The main existing laboratory diagnosis technology of leptospiral infection comprises: 1) face disease diagnosis.Leptospira causes, and to face disease symptom not special, limited in the value of clinical diagnosis; 2) pathogen isolation: the time of these needs is long, and need to be through the professional of special training; 3) Serologic test detects anti-leptospiral antibody.The limitation of this technology is to carry out early diagnosis to Leptospira, and can not effectively distinguish diagnosis and distinguish leptospiral the infected, metainfective rehabilitation clients and accepted vaccine recipient; 3) round pcr detects leptospiral nucleic acid.Existing 16 kinds of Leptospira, approximately 38 serologic group, surpass 250 serotypes, infect multiple host.In addition, the mobility of leptospiral gene is very large, and the gene order between homophyletic does not differ greatly.Therefore, be difficult to the Leptospira that single PCR method of design is accurate, responsive, detect fast all strains.The sensitivity of this technology is from 1 milliliter of blood or approximately 20 leptospiral genomes of urine detection, than at least 50 times of the conventional LipL32SYBR method susceptibility height of present North America market.The whole process that the present invention judges from nucleic acid purification to PCR result approximately 150 minutes.

Accompanying drawing explanation

Fig. 1: the leptospiral probe of pathogenic dog and primer.

Embodiment

The procedure detecting with test kit of the present invention is as follows:

1. the standard quantitative reagent (standards) that preparation PCR uses.By the synthetic leptospiral nucleotide sequence of pathogenic dog of Integrated DNA Technology, this sequence contains the pcr amplification region of this research.According to the DNA quantitative technique of molecular weight and absolute weight and the PicoGreen of synthetics, calculate the gene copy number of the contained LigA of synthetics.Subsequently, synthetics is diluted, prepare the dilution reagent of every 10 μ l syntheticss containing the rRNA of 10000 copies, 1000 copies, 100 copies, 10 copies, the standard quantitative reagent of using as PCR.

2. prepare the DNA profiling of sample to be checked.Collect whole blood and the urine sample of dog, for DNA purifying; The DNA of purifying is eluted in 100 μ l T ₁₀e _0.1(containing 10mM Tris-HCl, 0.1mM EDTA, pH8.5) is as the amplification template of PCR.

3.PCR amplification system.The commercialization Taq enzyme of the sample DNA template that the amplification system of 20 μ l comprises 10 μ l or quantitative criterion reagent (standard), 1xPCR damping fluid, 1.0 μ M upstream primers, 1.0 μ M downstream primers, the 6-FAM probe of 0.2 μ M, the LCRed640 probe of 0.2 μ M, 2 units, 200 μ M dNTP.

4.PCR amplification cycles parameter.Pcr amplification comprises that the rigorous circulation of the height of 18 lapses of temperature (high-stringency step-down) and 25 owe rigorous fluorescence and obtain circulation (relaxed-stringency fluorescence acquisition).The rigorous circulation of height of 18 lapses of temperature: 95 ° of C of 6x15sec@, 74 ° of C of 60sec@; 95 ° of C of 9x15sec@, 72 ° of C of 60sec@; 95 ° of C of 3x15sec@, 70 ° of C of 10sec@, C.25 72 ° of 15sec@owe rigorous fluorescence and obtain circulation: 95 ° of C of 25x15sec@, 58 ° of C of 8sec@, 65 ° of C of 10sec@, 72 ° of C of and15sec@.

The judgement of 5.PCR result and quantitative analysis.The preparation process of DNA profiling is equipped with feminine gender and the positive control of DNA extraction, and pcr amplification object subsequently comprises the DNA profiling of testing sample, the feminine gender of DNA extraction and positive control, (every 10 μ l standard are containing 10 for quantitative standard reagent ⁴, 10 ³, 10 ², 10 ¹the leptospiral LigA gene of pathogenic dog of copy).Pcr amplification efficiency of the present invention is high, can effectively increase containing the leptospiral LigA gene of pathogenic dog of single copy.

Embodiment-1: the pcr amplification of pathogenic dog Leptospira type strain (L.interragans Pomona Pomona)

The L.interragans Pomona Pomona type strain (being presented by veterinary college Ashutosh doctor Verma of Ross university) of cultivating, carries out the calculating of the molecular weight number of nucleic acid purification and L.interragans Pomona Pomona.FRET-PCR method of the present invention can detect the LigA gene of a copy of single reaction system.

Embodiment-2: relatively the present invention and in North America susceptibility and the specificity of conventional leptospiral quantitative PCR

In leptospiral diagnosis, there is a Lip32SYBR technology (Levett PN who is widely used, Morey RE, Galloway RL equals within 2005, to be published in J Med Microbiol, and 54:45-49. title of article is: Detection of pathogenic leptospires by real-time quantitative PCR.).Our system compared the technology of the present invention and Lip32SYBR technology at the susceptibility detecting on Leptospira. use the Leptospira type strain of cultivating, calculating for nucleic acid purification and molecule number, then 10-diluent is doubly for the nucleic acid amplification of 2 cover PCR systems. relatively find, the present invention has the repeatability of height, can detect the leptospiral nucleic acid of single copy, the recessiveness contrast of free nucleic acid is always negative. and Lip32SYBR technology can detect 154 copies/reaction effectively, and also one there is false-positive result; The present invention is than at least high 50 times in the sensitivity of the widely used LipL32SYBR of North America market.

Embodiment-3. detect and infect the trouble dog that hook end breechblock infects

A trouble dog that has typical case to face disease symptom, gathers blood and urine and detects for leptospiral PCR.Lip32SYBR technology detects Leptospira in suffering from the urine of dog and blood, and FRET-PCR of the present invention detects the Leptospira of low copy in urine.According to the diagnostic result of FRET-PCR, to suffering from dog, carried out effective treatment.

Claims (2)

1. detect primer and the probe of the quantitative fluorescent PCR of dog Leptospira nucleic acid, it is characterized in that described primer and probe are as follows:

Primer:

Upstream primer: 5 '-GCTACWGGGATCTACTCTGACAACTC-3 ';

Downstream primer: 5 '-GGACTACTTACYTTTCCGAATGTGGCT-3 ';

Probe:

6-FAM probe: 5 '-ATTTCAAACGCCMAAAAAAATCAAGGAAACKCTTA-(6-FAM)-3 ';

LCRed640 probe: 5 ' (LCRed640)-GGAGCAGCTACAGGARCAACGGATATT-(phosphorylation)-3 '.

2. a PCR kit for fluorescence quantitative that detects dog Leptospira nucleic acid, it is characterized in that this test kit comprises 5x FRET-qPCR Stock22 microlitre, and the Oligo mixture of the 5x of 22 microlitres, in described Oligo mixture, containing concentration, be upstream primer, 5 μ M downstream primers, the 6-FAM probe of 1 μ M, the LCRed640 probe of 1 μ M of 5 μ M; Wherein institute's primer and probe are as follows:

Upstream primer: 5 '-GCTACWGGGATCTACTCTGACAACTC-3 ';

Downstream primer: 5 '-GGACTACTTACYTTTCCGAATGTGGCT-3 ';

6-FAM probe: 5 '-ATTTCAAACGCCMAAAAAAATCAAGGAAACKCTTA-(6-FAM)-3 ';

LCRed640 probe: 5 ' (LCRed640)-GGAGCAGCTACAGGARCAACGGATATT-(phosphorylation)-3 '.