CN101314792A

CN101314792A - Tripartite genome urinalysis method for kidney transplantation immunological rejection, reagent kit and diagnosis agent thereof

Info

Publication number: CN101314792A
Application number: CNA200710099861XA
Authority: CN
Inventors: 翟原
Original assignee: Tarcine BioMed Inc
Current assignee: Wuxi Tarcine BioMed Inc.
Priority date: 2007-05-31
Filing date: 2007-05-31
Publication date: 2008-12-03
Anticipated expiration: 2027-05-31
Also published as: CN101314792B

Abstract

The invention discloses a method for quantitatively measuring the expression levels of two transplanted-kidney rejection target genes (that are an IP-10 ad a granzyme-B) and a reference gene (HPRT) at the same time, and also provides a kit that is represented as UC-QPCR-TARCINE<TM>. Three detection-gene quantitative standard markers and three detection-gene PCR primers are arranged in the kit. The method adopts a triple gene urinalysis method of a multi-channel real-time fluorescent quantitative gene detection technology, has the advantages of shortcut, sensitivity, efficacy, specificity, non-invasive performance, etc. when diagnosing the kidney transplantation immunological rejection, and can reflect the allograft rejection on the transplanted-kidney caused by the immune system of a patient systematically.

Description

Tripartite genome urinalysis method for kidney transplantation immunological rejection and test kit and diagnostic reagent

Technical field

The present invention relates to molecular biology and medical field.Can be applicable to immunological rejection after the renal transplantation is carried out non-invasive monitoring.Particularly, the present invention relates to design and optimize the PCR primer that is applicable to three kinds of genes of while quantitative amplification in micro-urine cast-off cells, and clone's preparation is used for three kinds of gene standard substance segments of quantitative PCR.

Background technology

Organ transplantation is one of significant subject of modern medicine.Existing in the world at present more than 70 ten thousand uremic patients have been accepted organ transplantation and have been obtained new life since the first renal transplantation success of nineteen fifty-nine, and this numeral also increases in the speed with every year nearly 50,000.By 2003, China totaled renal transplantation 50,000 many cases, finished every year now to surpass 5000 examples, occupied the second place of the world next in number only to the U.S..China has more than 100 ten thousand uremic patients approximately, also has nearly 120,000 newly-increased patient every year.To these patients, renal transplantation is present most important treatment means.Yet transplanting the long-term surviving of kidney, is the subject matter (reference 1,2) of renal transplantation success or not all the time.Along with the clinical application of development of joining the type technology and neotype immunosuppressant, early stage acute cellular rejection incidence obviously descends, and transplanted kidney short-term survival rate significantly improves.Transplanted kidney function goes down and final transplanted kidney forfeiture most important reason (3) but the acute and chronic rejection of retardance remains.If long-term effect with half life (transplanting after the 1st year the time of 50% graft loss of function) prediction renal transplantation, over nearly 20 years, the long-term surviving rate of renal transplantation is not significantly improved, 10% to 15% transplanted kidney loss of function is arranged in 1 year approximately, the transplanted kidney survival rate is about 67% in 5 years, and survival rate is less than 38% in 10 years.The major obstacle of long-term survival in renal transplantation is chronic rejection or chronic allograft deterioration (4).In China, to transplant and use the organ multi-source in corpse, small part provides the live body kidney by relatives.After the renal transplantation, needs of patients is taken immunosuppressor (as Ciclosporin A and FK506) all the life and is used for anti-rejection treatment.But the treatment window of this type of medicine is very narrow, if the quantity not sufficient of using, the transplanted kidney that just can not effectively prevent preciousness is ostracised, if dosage is excessive, then can produce serious dosage correlation Toxicity of Kidney effect and make transplanted kidney lose merit, thereby and make the incidence of serious infection complication obviously increase the life that threatens the patient.So monitoring timely, accurate, special after the renal transplantation has important role to the early discovery of transplanted kidney immunological rejection and adjustment patient's immunosuppressor consumption.

Yet detect clinically at present method that transplanted kidney repels also only limit what according to clinical manifestation (as fervescence, dysarteriotony, stomachache, even hematuria is arranged, or the like) and serum creatinine (serum creatinine, serum creatinine SCr) rising judge.But these methods can not the early diagnosis rejection, in case above-mentioned symptom occurs often, transplanted kidney repels and taken place and entered the serious stage, is difficult to treatment clinically; Next is that these diagnosis indexs lack specificity, because when infecting appears in other position of human body, also can bring out above-mentioned clinical disease and serum creatinine (SCr) raises, can therefore judge by accident, blindly increase the immunosuppression pharmaceutical quantities, not only increase patient's economical load in rain, also can cause toxic side effect even jeopardize patient's life.The transplanted kidney aspiration biopsy is present unique clinically authoritative diagnostic method.But what the disadvantage of this method was it to transplant organ is traumatic, might cause complication such as kidney is hemorrhage, perirenal hematoma, arterio venous fistula, the survival (5,6) of harm transplanted kidney.Shou Shu enforcement simultaneously must be passed through a series of processes such as anesthesia, aseptic sterilization, preoperative and postoperative nursing, expense costliness.Just traumatic because of it, process is complicated and expensive, just can not popularize the means of making a definite diagnosis that are used as routine, momentarily use in all hospitals.At present in China so that the international boundary of transplanting, the aspiration biopsy operation generally behind renal transplantation 1 month, 3 months, 6 months and 1 year carry out, with such frequency, be difficult to accomplish to find early and handle.Bioptic another the serious defective of kidney puncture is, the tissue size that biopsy is got be tens0000 of kidney/, only be the minimum part of whole transplanted kidney, its situation may not necessarily reflect whether other position has pathology and cause mistaken diagnosis (7).

So be badly in need of clinically at present setting up a kind of quick, responsive, special and noninvasive immunologic detection method to renal transplantation after immunological rejection monitor.This is for early discovery and treatment rejection and instruct the clinician rationally to adjust the immunosuppressor consumption, make it to reduce or drug withdrawal (will alleviate patient's economical load greatly) in good time, improve patient's life quality, reduce transplant patient's mortality ratio, the survival time that prolongs transplanted kidney is all significant, will promote the development of organ transplantation cause effectively.

Organ transplant rejection prediction, diagnosis, monitoring field belong to a still undeveloped field in the world, the research institution of many countries drops into lot of manpower and material resources and is stepping up to research and develop, using the transplanting renal tissue of having made a definite diagnosis the generation immunological rejection comes examination and identifies the protein marker (8,9) that causes transplant rejection.Calendar year 2001, the scientists of the U.S. took the lead in being reported in the nephridial tissue of immunological rejection, and cytotoxic protein pore-forming protein (cytotoxic proteins perforin) and the horizontal abnormality of Granzyme B (granzyme B) increase (10).Then the research group of the professor Knechtle of Univ Wisconsin-Madison USA is by the observation to 99 routine renal transplantation postoperative patients, find IP-10 (IFN-gamma-inducible protein 10, (11) are obviously increased in being expressed in interferon inducible protein matter 10) when the transplanted kidney immunological rejection takes place, they prove that also IP-10 not only appears in the organ-tissue of being ostracised, also can in patient's urine cell, detect (11,12).Experiment showed, that IP-10 is a member of chemokine CXCR3 aglucon family, function is to attract activated T cells to arrive immune response (inflammation) zone, is one of initial committed step of the immunological rejection of transplant organ.Cytotoxic T cell is one of of paramount importance lymphocyte in the transplant organ immunological rejection.Its main molecules mechanism of killing the transplant organ cell is to discharge Granzyme B and perforin, and Perforin is combined in transplant cell membrane surface formation hole enters in the cell Granzyme B in a large number, causes the disintegration death of cell.Preliminary clinical experiment shows, the immunological rejection of organ not only can be accurately diagnosed in the increase of IP-10 and Granzyme B in renal transplantation live body or the urine sample cell, and immunological rejection or infection that can the differential diagnosis organ wait other inflammatory reactions, has diagnostics and be worth (13-15).A lot of for a long time medical research units are all utilizing these achievement in research research and development to detect the method that transplant organ repels, but because (for example there is the phase mutual interference in many technical difficult problems between the different genes primer, the quantitative problem of PCR product, the specificity of PCR reaction in the complex system, may cause mistake amplification of genomic dna or the like), there is not practical diagnostic kit to release so far as yet.

Summary of the invention

At above-mentioned this area urgent problem, in recent years, with the scientists of sunrise biotechnology (Beijing) company on above-mentioned every cutting edge technology and theoretical basis, design and optimized the PCR primer of three kinds of genes of quantitative amplification simultaneously, adopt hyperchannel real time fluorescent quantitative technique of gene detection to carry out the triple PCR reaction, successfully set up a kind of technological method that two transplanted kidney repel the expression level of a target gene (IP-10 and granzyme B) and a reference gene (HPRT), called after " tripartite genome urinalysis method for kidney transplantation immunological rejection " in the urine cell of trace, measured simultaneously.Simultaneously, we clone and have prepared three kinds of gene standard substance segments that are used for quantitative PCR, make our this three symbasis because of uroscopy an absolute reference standard arranged quantitatively.So different patients', the sample of same patient's different times can compare.

In more detail, the invention provides following:

1. test kit that is used to detect immunological rejection after the renal transplantation, described test kit comprise be used for detecting the cell transplanted kidney repel protein marker IP-10 expression one couple of PCR primers and be used for detecting second pair of PCR primer that the cell transplanted kidney repels the expression of protein marker granzyme B, forward in the described one couple of PCR primers and reverse primer are respectively the oligonucleotide sequences shown in sequence table SEQ ID NO:4 and the SEQ ID NO:5, forward in described second pair of PCR primer and reverse primer are respectively the oligonucleotide sequences shown in sequence table SEQ ID NO:6 and the SEQ ID NO:7, and preferred described cell is the urine cell.In one embodiment of the invention, described PCR primer may be mixed together or separates in different vessels, and can randomly be in the suitable medium well known to those skilled in the art.In addition, test kit of the present invention can also comprise working instructions, is used to instruct the user to use the detection applications of this test kit and this test kit of explanation.For example, a kind of test kit name of the present invention is called UC-QPCR-TARCINE ^TM

2. according to above 1 test kit, wherein said test kit also comprises the 3rd pair of PCR primer that is used for detecting cell reference gene HPRT, and forward in described the 3rd pair of PCR primer and reverse primer are respectively the oligonucleotide sequences shown in sequence table SEQ ID NO:8 and the sequence table SEQ ID NO:9.

3. according to above 1 or 2 test kit, wherein said test kit also is included in the fluorescence dye that is used for the quantitative PCR product in the PCR reaction process, preferred SYBR G I fluorescence dye.

4. according to above 1 or 2 test kit, wherein said test kit also is included in the TaqMan probe that is used for the quantitative PCR product in the PCR reaction process, preferred pin is the probe that comprises the oligonucleotide sequence shown in the sequence table SEQ ID NO:10 to the TaqMan probe of IP-10, TaqMan probe at granzyme B is the probe that comprises the oligonucleotide sequence shown in the sequence table SEQ ID NO:11, is the probe that comprises the oligonucleotide sequence shown in the sequence table SEQ ID NO:12 at the TaqMan probe of HPRT.

5. diagnostic reagent that is used to detect immunological rejection after the renal transplantation, described diagnostic reagent comprise be used for detecting the cell transplanted kidney repel protein marker IP-10 expression one couple of PCR primers and be used for detecting second pair of PCR primer that the cell transplanted kidney repels the expression of protein marker granzyme B, forward in the described one couple of PCR primers and reverse primer are respectively the oligonucleotide sequences shown in sequence table SEQ IDNO:4 and the SEQ ID NO:5, forward in described second pair of PCR primer and reverse primer are respectively the oligonucleotide sequences shown in sequence table SEQ ID NO:6 and the SEQ ID NO:7, and preferred described cell is the urine cell.

6. according to above 5 diagnostic reagent, wherein said diagnostic reagent also comprises the 3rd pair of PCR primer that is used for detecting cell reference gene HPRT, and forward in described the 3rd pair of PCR primer and reverse primer are respectively the oligonucleotide sequences shown in sequence table SEQ ID NO:8 and the SEQ ID NO:9.

7. according to above 5 or 6 diagnostic reagent, wherein said diagnostic reagent also is included in the fluorescence dye that is used for the quantitative PCR product in the PCR reaction process, preferred SYBR G I fluorescence dye.

8. according to above 5 or 6 diagnostic reagent, wherein said diagnostic reagent also is included in the TaqMan probe that is used for the quantitative PCR product in the PCR reaction process, preferred pin is the probe that comprises the oligonucleotide sequence shown in the sequence table SEQ ID NO:10 to the TaqMan probe of IP-10, TaqMan probe at granzyme B is the probe that comprises the oligonucleotide sequence shown in the sequence table SEQ ID NO:11, is the probe that comprises the oligonucleotide sequence shown in the sequence table SEQ ID NO:12 at the TaqMan probe of HPRT.

9. the method for the expression of IP-10 and granzyme B in the vitro detection urine cell, this method comprise use according in above 1 to 4 any one test kit and according to any one diagnostic reagent in above 5 to 8, the expression that comes quantitative IP-10 and granzyme B by PCR method.

10. according to above 9 method, wherein said urine cell is isolating from renal transplantation patient's urine.

The present invention has overcome many technical difficult problems in the prior art (for example have the phase mutual interference between the different genes primer, the quantitative problem of PCR product, the specificity of PCR reaction in the complex system may cause mistake amplification of genomic dna or the like).

With respect to using single target gene, the three symbasis advantage following that the present invention uses because of detection method has:

1. three symbasis just can detect three expression of gene that cause immunological rejection simultaneously because of detection method only needs the urine sample of trace.This point is very important for clinical practice, because only contain trace of albumin in the urine sample, the quantity of a urine sample is not enough uses single target gene method that three related genes are detected one by one.

2. three symbasis can detect three expression of gene that cause immunological rejection simultaneously because of detection method in a urine sample, so its detection sensitivity and specificity just significantly are better than using single target gene.

3. three symbasis have improved transplanted kidney immunological rejection detection efficiency greatly because of detection method.

Three symbasis can accurately, dynamically reflect the function status of transplanted kidney because of the detection index of detection method, therefore can dynamically monitor the result of treatment of immunosuppressor, instruct the doctor to regulate the consumption of immunosuppressor at any time according to the function of transplanted kidney.

Detailed Description Of The Invention

The real-time fluorescence quantitative PCR technology is the once leap of DNA quantitative technique.Exponential amplification can be carried out to the specific nucleotide segment in polymerase chain reaction (PCR).After amplified reaction finished, we can analyze amplified production qualitatively by the method for gel electrophoresis, also can carry out quantitative analysis by the densitometric scan that radionuclide mixes behind the mark.No matter qualitative still quantitative analysis, analysis all be the PCR end product.But in many cases, we interested be starting template amount before amplifying without the PCR signal.For example we wonder genetically modified copy number of a certain genetically modified animals and plants or the expression amount of a certain specific gene in particular organization.Fluorescent quantitative PCR technique arises at the historic moment under this demand.Thereby so-called real-time fluorescence quantitative PCR is exactly to realize starting template quantitatively and is qualitatively analyzed by the real-time detection to each circulation products fluorescent signal in the pcr amplification reaction.In the real-time fluorescence quantitative PCR reaction, introduced a kind of fluorescence chemical material, along with the carrying out of PCR reaction, the PCR reaction product constantly adds up, and fluorescence signal intensity also equal proportion increases.Every through a circulation, collect a fluorescence intensity signals, we just can pass through the variation of fluorescence intensity variation monitoring product amount like this, thereby obtain an amplified fluorescence graphic representation.

Generally speaking, amplified fluorescence curve amplification curve can be divided into three phases: fluorescence background signal phase, fluorescent signal index amplification stage and plateau.In the fluorescence background signal phase, the fluorescent signal of amplification is covered by the fluorescence background signal, and we can't judge the variation of product amount.And in plateau, amplified production no longer is exponential increase.There is not linear relationship between the end product amount of PCR and the starting template amount, so can not calculate the initiate dna copy number according to final PCR product amount.Only in the fluorescent signal index amplification stage, have linear relationship between the logarithmic value of PCR product amount and the starting template amount, we can be chosen in this stage and carry out quantitative analysis.For quantitative and convenience relatively, two important concept in the real-time fluorescence quantitative PCR technology, have been introduced: fluorescence threshold and CT value.Fluorescence threshold is an artificial value of setting on the amplified fluorescence curve, and it can be set on the fluorescent signal index amplification stage optional position, but generally we are 10 times of standard deviation of 3-15 round-robin fluorescent signal with the default setting of fluorescence thresholding.The cycle number that fluorescent signal in each reaction tubes is experienced when arriving the thresholding of setting is called as CT value (thresholdvalue).

The principles of chemistry of real-time fluorescence quantitative PCR comprise two kinds of probe class and non-probe classes, the probe class is to utilize the increase of indicating amplified production with the probe of target sequence specific hybridization, and non-probe class then is to utilize the primer of fluorescence dye or particular design to indicate the increase of amplification.The former is owing to increased the identification step of probe, and specificity is higher, but the latter is then simple and easy to do.The present invention can use any real-time fluorescence quantitative PCR.

SYBR Green I is a kind of double-stranded DNA combination dye that is incorporated in the ditch.After double-stranded DNA combined, its fluorescence strengthened greatly.This character makes its detection that is used for amplified production very desirable.The maximum absorption wavelength of SYBR Green I is about 497nm, and the emission wavelength maximum is about 520nm.In the PCR reaction system, add excessive SYBR fluorescence dye, after the SYBR fluorescence dye mixes the dna double chain specifically, the emitting fluorescence signal, and the SYBR dye molecule that does not mix in the chain can not launched any fluorescent signal, thereby the increase of the increase of assurance fluorescent signal and PCR product is synchronous fully.

The TaqMan probe is the patented technology that many people have.The TaqMan probe is a kind of oligonucleotide probe, and its fluorescence is relevant with the amplification of aim sequence.It is designed to and target sequence upstream primer and downstream primer between sequence pairing.Fluorophor is connected 5 ' end of probe, and quencher is then at 3 ' end.When complete probe and target sequence pairing, the fluorophor emitted fluorescence is because of approaching by cancellation with the quencher of 3 ' end.But when carrying out extension, 5 ' 5 prime excision enzyme activity of polysaccharase carries out enzyme with probe to be cut, and makes fluorophor separate with quencher.。Along with the increase of amplification cycles number, the fluorophor that discharges constantly accumulates.So proportional relation of quantity of fluorescence intensity and amplified production.

The present invention is directed to organ transplantation medical science urgent problem, that is, lack immunologic detection method, utilize modern transplantation medicine latest developments, selected two kinds of gene and a kind of reference genes that immunological rejection is relevant the transplant organ rejection.Utilize round pcr the most responsive among the molecular biology method, set up a kind of in the urine cell of trace the method for these three kinds of gene expression doses of quantitative assay simultaneously.

Technical scheme of the present invention comprises: 1) design of primer and probe and optimization; 2) target gene clone, the foundation of typical curve; With 3) foundation of triple PCR system.

Description of drawings

Fig. 1 .H (HPRT) gene single amplification result, typical curve and amplification curve;

Fig. 2 .I (IP10) gene single amplification result, typical curve and amplification curve;

Fig. 3 .G (Granzyme B) gene single amplification result, typical curve and amplification curve;

Fig. 4. triple system melt curve analysis are analyzed;

Fig. 5. multiple reaction PCR product electrophoresis result;

Fig. 6. spleen cDNA substance and multiplex amplification melt curve analysis are as a result analyzed;

Fig. 7. spleen rna substance and triple amplified production melt curve analysis are analyzed;

Fig. 8. (a) H gene amplification curve; (b) I gene amplification curve; (c) G gene amplification curve;

Fig. 9. (a) spleen cDNA and urine cell cDNA H gene amplification result; (b) spleen cDNA and urine cell cDNA I gene amplification result; (c) spleen cDNA and urine cell cDNA G gene amplification result; With

Figure 10. triple amplifications of spleen cDNA and single amplification product electrophoresis result.

Embodiment

Hereinafter will describe the present invention in detail by embodiment with reference to the accompanying drawings, described embodiment only is intended as illustrative explanation the present invention, rather than intention limits the scope of the invention.Scope of the present invention is specifically limited by accompanying Claim.

Embodiment 1. triple fluorescent real-time quantitative PCRs (Triplex Real-time PCR) primer probe design

HPRT (Hypoxantine phosphoribosyltransferase, hypoxanthine phosphoribosyltransferase) is housekeeping gene in a kind of common cell, its expression amount in the different sorts cell and similar cell under different situations all constant relatively (16,17), therefore be often used as reference gene, be used for the research of target gene differential expression.

IP10 (GenBank Accession No.NM_001565 according to the NCBI announcement, sequence table SEQ ID NO:1), Granzyme B (GenBank Accession No.NM_004131, SEQID NO:2) and hprt gene (GenBank Accession No.NM_000194, SEQ IDNO:3) cDNA sequence, use primer-design softwares such as Primer express 2.0 (Applied Biosystems) and Primer Premier 5 (PREMIER Biosoft International) `, and with reference to relevant document, we select shown in the following table 1 three groups of primers and TaqMan probe to be used for triple quantitative pcr amplifications.

Design multiple quantitative PCR primer and probe should be based on following principles:

Designed primer or probe have at least one to be positioned at two exon joints, so just can avoid the influence of genomic dna amplification for amplification.

The primer sequence of designing is compared mutually, when the same amplification system, do not influence amplification each other to greatest extent, and avoid wrong amplification to guarantee it.

Should guarantee that in single amplification system and triple amplification system, primer is for the amplification efficiency unanimity of single target.

Table 1

HEX in the table 1, BHQ1, ROX, BHQ2 and FAM are the fluorescence molecule and the quenching groups of this area taqman probe commonly used, and its full name is as follows:

6-FAM 6-carboxy-fluorescein

HEX 5-hexachloro-fluorescein

ROX 6-carboxy-x-rhodamine

BHQ1 Black?hole?quencher?1

BHQ2 Black?hole?quencher?2

Except as otherwise noted, all primers used herein are all synthetic by Shanghai biotechnology company limited.

The foundation of embodiment 2. target genes clone and typical curve

For accurate quantification is unified in genetic expression in the clinical sample, we have prepared the quantitative absolute standard product of target gene.We will contain the pulsating target gene segment of quantitative pcr amplification and be cloned on the carrier, extract plasmid purification and cut carrying out biochemistry after its linearization quantitative by restriction endonuclease, plasmid by adopting different concns has so just obtained being used for the typical curve of absolute quantitation genetic expression as the substrate of target gene PCR reaction.The concrete operations step is as follows:

1. gene clone primer design.

According to the sequence that the NIH human gene bank announces, we have used primer Premier 5 software designs clone's primer of three target genes, its primer sequence such as following table 2:

Table 2

2. the total RNA of people's spleen tissue extracts and reverse transcription

Extract the total RNA of people's spleen with the RNeasy Mini Kit of QIAGEN company.

With the synthetic cDNA of the Sensiscript RT Kits of QIAGEN company reverse transcription.

3. target gene pcr amplification

With the reverse transcription product is template, respectively target gene is increased with above three pairs of primers, and amplification system is as follows:

2×Hotstar?Mastermix(QIAGEN)：10μl

Forward?Primer(4μM)： 1μl

Reverse?Primer(4μM)： 1μl

DD?water： 7μl

cDNA?template： 1μl

Total： 20μl

The pcr amplification condition is as follows: 95 ℃ of pre-sex change 15min; 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 60s, 30 circulations of increasing; 72 ℃ are extended 10min.(PCR instrument producer, MJResearch PTC-200)

4. target gene is cloned on the T-easy Vector

The PCR product is carried out gel separation, and reclaim specific PCR product from gel, and be connected on the pGEM-T Easy Vectors of Promega company, institute carries out in steps to specifications.With the DH5a bacterium (TIANGEN Biotech (Beijing) Co., Ltd.) that connects the product transformed competence colibacillus, and carry out blue hickie screening.Positive bacterium colony at first carries out PCR and detects, and PCR detection positive is sent to the living worker in Shanghai and carries out the plasmid DNA order-checking.Sequencing result proof has obtained having the plasmid of the gene of target really.

5. plasmid DNA is extracted purifying and linearization for enzyme restriction

Culturing bacterium is extracted plasmid DNA with a large amount of plasmid extraction kits of TIANGEN company, and institute carries out in steps to specifications.The plasmid that extracts uses restriction enzyme (TAKARA) SacI (G plasmid and I plasmid) and Spe I (H plasmid) to carry out linearizing respectively.

Reclaim the linearizing plasmid of purifying with reclaiming test kit.TE solution dissolving plasmid with pH 8.0 carries out the concentration accurate quantification with spectrophotometer to it.According to measuring concentration it is diluted to 1 * 10 ⁸The concentration storage liquid.

Embodiment 3. triple fluorescent real-time quantitative PCRs (Triplex Real-time PCR) system is set up

1. before synthetic TAQMAN probe, we carry out the substance pcr amplification of each gene earlier with SYBR G I fluorescence dye (being purchased the biology from Beijing ancient cooking vessel state), to determine that each is to primer The amplification order The validity of mark gene. in addition, in triple amplification bodies be for detecting Between the different genes primer whether The phase mutual interference, we add three pairs of primers in the reaction system simultaneously, by electrophoresis and solubility curve analysis, judge whether to produce unusual product.

1.1 with the plasmid is template, and primer validity is verified

1.1.1 amplification system:

2×Quantitech?Multiplex?PCR

NoRox?Master?Mix(Qiagen)： 12.5μl

SYBRGI (10X) (Beijing ancient cooking vessel state biology) 1 μ l

Primer Mix (each 2 μ M): 2.5 μ l

Plasmid DNA: 2.5 μ l

dd?Water： 6.5μl

Total： 25μl

The final concentration of single primer is 0.2 μ M in the substance system, and in multiple system, the final concentration of each primer is 0.2 μ M.

1 95 ℃ of 15min of 1.1.2 amplification condition: Step

Step?2 94℃ 1min

Step?3 60℃ 1min

Step?4 Plate?read

Go?to?step?2?for?40?cycles

Melting?curve?from?60℃?to?94℃，read?every?0.2℃，hold?0.1s

End

(the quantitative fluorescent PCR instrument is: MJ Research Chromo 4)

1.1.3 plasmid DNA concentration

Sample is the standard substance plasmid DNA of 5 times of dilutions, and concentration is respectively 100000 molecules/μ l, 20000 molecules/μ l, 4000 molecules/μ l and 800 molecules/μ l.

1.1.4 result:

As Fig. 1, Fig. 2 and (analysis software shown in Figure 3, Opticon Monitor Version 2.03, MJ Research), containing separately with H, I and G primer amplification respectively, the standard substance plasmid DNA of different concns is a template, amplified product, the result shows that amplification system can distinguish the plasmid of different concns, and linear.The melt curve analysis analysis revealed has amplified single product.

Fig. 4 and Fig. 5.With in same test tube, increase the simultaneously mixture of three kinds of gene plasmids of H, I and G primer.Melt curve analysis is analyzed (Fig. 4) and is demonstrated 3 spikes, its position and the single amplified production of each gene to melt spike consistent.Electrophoresis result (Fig. 5) also demonstrates 3 bands corresponding to the expectation product that length is different.

1.2 when being template amplification with the plasmid, amplification system is simple.For illustrate this primer whether can be in complex system the specific target product that amplifies, the cDNA that we obtain with the total RNA reverse transcription of people's spleen is that template is carried out pcr amplification.

Extract spleen rna with Qiagen RNeasy Mini Kit, carry out reverse transcription with Qiagen ReverseTranscriptase Kit, institute carries out in steps to specifications.

Amplification system is as follows:

2×Quantitech?Multiplex?PCR

NoRox?Master?Mix(Qiagen)： 12.5μl

SYBR G I (10X) (ancient cooking vessel state biology) 1 μ l

Primer Mix (each 2 μ M): 2.5 μ l

Spleen?cDNA： 2.5μl

dd?Water： 6.5μl

Total： 25μl

Simultaneously, we are template with the RNA sample of not reverse transcription, detect the amplification whether above primer can cause genomic dna.We show the sub-interface place outside with design of primers when design, can avoid the influence of genomic dna amplification for experimental result like this.

1 95 ℃ of 15min of amplification condition: Step

Step?2 94℃ 1min

Step?3 60℃ 1min

Step?4 Plate?read

Goto?step?2?for?40?cycles

Melting?curve?from?60℃to?94℃，read?every?0.2℃，hold?0.1s

End

The result as shown in Figure 6, in the single amplification system, each gene has melt curve analysis peak (being respectively the green among the figure, blue and yellow peak) separately respectively, shows that product is single.Triple amplification system amplifications (red curve) have three peaks, and this position, three peaks respectively with single amplification in overlap, amplified each target gene segment in the triple amplification systems of this presentation of results really.

RNA template amplification product melt curve analysis (Fig. 7) analysis revealed, without any specific peak produce, illustrate when being template there is not wrong amplification initiation with RNA.

2.Taqman probe method amplification (18)

SYB G I experimental results show that designed primer, can amplify target product in triple systems respectively, so we have synthesized the Taqman probe of three genes respectively, is used to the target product that increases.

2.1 SYB G I experimental result has proved the validity of triple systems amplification targets, so we directly carry out triple quantitative pcr amplifications with the Taqman probe method, is template with the plasmid DNA.

2×Quantitech?Multiplex?PCR

NoRox?Master?Mix(5μM)： 12.5μl

H-primer?Mix(5μM)： 1μl

H-PROBE(5μM)： 1μl

G-primer?Mix(5μM)： 1μl

G-PROBE(5μM)： 1μl

I-primerMix(5μM)： 1μl

I-PROBE(5μM)： 1μl

dd?water(5μM)： 3.5μl

H?plasmid?DNA： 1μl

G?plasmid?DNA： 1μl

I?plasmid?DNA： 1μl

Total： 25μl

Amplification program is as follows:

Step?1 95℃ 15min

Step?2 94℃ 1min

Step?3 60℃ 90s

Step?4 Plate?read

Goto?step?2?for?40?cycles

End

Standard substance plasmid with 5 times of dilutions is a template, and concentration is 10000 molecules/μ l, 2000 molecule/μ l, 400 molecule/μ l, 80 molecule/μ l, 16 molecule/μ l, 3.2 molecule/μ l, 0.64 molecule/seven concentration of μ l.

2.2 result

Shown in Fig. 8 (a) and (b), (c): with 5 times of dilution plasmids is template, and in triple Taqman amplification systems, success amplifies target molecule respectively.Linearly dependent coefficient (the R of the amplification curve of three genes ²) difference 0.997,0.993 and 0.993.Presentation of results, each plasmid molecule in the quantitative system that this Taqman probe system can be accurate and effective.

2.3 the Taqman probe amplification of spleen cDNA and urine cell cDNA

Extract people's spleen rna and people and urinate cell RNA (Qiagen RNeasy Mini Kit) (10), be used for triple quantitative pcr amplifications after the reverse transcription (Qiagen Reverse Transcriptase Kit).Design the RNA contrast of not reverse transcription simultaneously.

The urine cell harvesting:

1) provides one of urine cup to patient, collect mud-stream urine 100ml.

2) the urine branch is installed to 2 aseptic centrifuge tubes of 50ml

3) 2 50ml centrifuge tubes that urine sample will be housed are at room temperature 2000g (3300rpm) centrifugal 30 minutes

4) abandon supernatant.

5) with the resuspended urine cell of the cold aseptic PBS of 1ml and shift in 1.5ml nut Eppendorf tube.

6) under the room temperature 16, centrifugal 10 minutes of 000g (13000rpm).

7) abandon clean supernatant and flick centrifuge tube.

8) add 150 μ l RNAlater (Qiagen, Cat#76104) also thorough mixing.

9) simple centrifugal to collect dispersion liquid.

10 and preserve cell samples in-70 ℃.

11) transport sample with dry ice

Reverse transcription:

Use the Sensiscript RT Kits of QIAGEN company, carry out reverse transcription according to following system

10X?Buffer?RT 2.0μl

dNTP?Mix(5mM?each?dNTP) 2.0μl

primers： 1μM

RNase?inhibitor(10units/μl) 1.0μl

Sensiscript?Reverse?Transcripase 1.0μl

RNase-free?water

Program:

60min 37℃

5min 93℃

Amplification system is as follows:

2×Quantitech?Multiplex?PCR

NoRox?Master?Mix： 12.5μl

Primers-probes?Mix： 5μl

(final concentration is 0.2 μ M separately)

cDNA：or(RNA)： 2.5μl

Add water to 25 μ l, 25 μ l

Amplification program is as follows:

Step?1 95℃ 15min

Step?2 94℃ 1min

Step?3 60℃ 90s

Step?4 Plate?read

Go?to?step?2?for?40?cycles

End

2.4 result:

Shown in Fig. 9 (a) and (b), (c): with spleen cDNA and urine cell cDNA is template, has amplified target molecule respectively, and when amplification each sample three parallel control institute amplification basically identicals.In the sample of RNA for contrast, there is not amplified signal, designed primer be described, can specific amplification target gene segment, and can not cause wrong the amplification from genome.For proving once more whether institute's amplifier molecule is the target gene segment, our product during with spleen cDNA amplified production and single amplification is electrophoresis together, the result as shown in figure 10, the product size during triple amplification is in full accord with the target product size of amplification.

Industrial usability

Innovation point of the present invention is:

(1) applying biological target gene expression is monitored the early stage repulsion of transplanted kidney organ.

(2) adopt " three symbasis are because of the uroscopy " of multichannel real time fluorescent quantitative technique of gene detection right In diagnosis kidney transplant immunological rejection have fast, responsive, effective, special and do not have an advantage such as wound.

(3) organ is not had any damage, to tested patient without any risk.

(4) can system reflect that patient's immune system is to the rejection of transplanted kidney.

Purposes of the present invention is:

The patient measures the target that causes transplanted kidney to repel regularly with its urine after accepting renal transplantation The mark gene expression dose is used for the prediction of clinical renal transplantation rejection, diagnosis and monitoring. This is for early Phase is found and treats rejection and instruct the clinician rationally to adjust the immunodepressant consumption, make it Reduce or drug withdrawal (will greatly alleviate patient's financial burden) in good time, improve quality of life of patients, reduce Transplant patient's the death rate, the time-to-live that prolongs transplanted kidney is all significant, will promote effectively The development of organ transplant cause.

Reference

1.Hariharan，S.，C.P.Johnson，B.A.Bresnahan，S.E.Taranto，M.J.Mclntosh，and?D.Stablein.2000.Improved?graft?survuival?after?renal?transplantation?in?the?United?States，1988to?1996.N?Engl?J?Med?342：605.

2.Gulanikar，A.C.，A.S.MacDonald，U.Sungurtekin，and?P.Belitsky.1992.The?incidence?andimpact?of?early?rejection?episodes?on?graft?outcome?in?recipients?of?first?cadaver?kidneytransplants.Transplantation?53：323.

3.Almond，P.S.，A.Matas，K.Gillingham，D.L.Dunn，W.D.Payne，P.Gores，R.Gruessner，and?J.S.Najarian.1993.Risk?factors?for?chronic?rejection?in?renal?allograft?recipients.Transplantation?55：752.

4.Cecka，J.M.1999.The?UNOS?Scientific?Renal?Transplant?Registry.Clin?Transpl：1.

5.Huraib，S.，H.Goldberg，A.Katz，C.J.Cardella，G.A.deVeber，G.T.Cook，and?P.R.Uldall.1989.Percutaneous?needle?biopsy?of?the?transplanted?kidney：technique?and?complications.Am?J?Kidney?Dis?14：13.

6.Beckingham，I.J.，M.L.Nicholson，and?P.R.Bell.1994.Analysis?of?factors?associated?withcomplications?following?renal?transplant?needle?core?biopsy.Br?J?Urol?73：73.

7.Colvin，R.B.，A.H.Cohen，C.Saiontz，S.Bonsib，M.Buick，B.Burke，S.Carter，T.Cavallo，M.Haas，A.Lindblad，J.C.Manivel，C.C.Nast，D.Salomon，C.Weaver，and?M.Weiss.1997.Evaluation?of?pathologic?criteria?for?acute?renal?allograft?rejection：reproducibility，sensitivity，and?clinical?correlation.J?Am?Soc?Nephrol?8：1930.

8.Henger，A.，H.Schmid，and?M.Kretzler.2004.Gene?expression?analysis?of?human?renalbiopsies：recent?developments?towards?molecular?diagnosis?of?kidney?disease.Curr?OpinNephrol?Hypertens?13：313.

9.Striker，L.J.，and?G.E.Striker.2003.Windows?on?renal?biopsy?interpretation：does?mRNAanalysis?represent?a?new?gold?standard？J?Am?Soc?Nephrol?14：1096.

10.Li，B.，C.Hartono，R.Ding，V.K.Sharma，R.Ramaswamy，B.Qian，D.Serur，J.Mouradian，J.E.Schwartz，and?M.Suthanthiran.2001.Noninvasive?diagnosis?of?renal-allograft?rejectionby?measurement?of?messenger?RNA?for?perforin?and?granzyme?B?in?urine.N?Engl?J?Med344：947.

11.Hu，H.，B.D.Aizenstein，A.Puchalski，J.A.Burmania，M.M.Hamawy，and?S.J.Knechtle.2004.Elevation?of?CXCR3-binding?chemokines?in?urine?indicates?acute?renal-allograftdysfunction.Am?J?Transplant?4：432.

12.Tatapudi，R.R.，T.Muthukumar，D.Dadhania，R.Ding，B.Li，V.K.Sharma，E.Lozada-Pastorio，N.Seetharamu，C.Hartono，D.Serur，S.V.Seshan，S.Kapur，W.W.Hancock，and?M.Suthanthiran.2004.Noninvasive?detection?of?renal?allograft?inflammationby?measurements?of?mRNA?for?IP-10?and?CXCR3?in?urine.Kidney?Int?65：2390.

13.Farber，J.M.1997.Mig?and?IP-10：CXC?chemokines?that?target?Iymphocytes.J?Leukoc?Biol61：246.

14.Hancock，W.W.，W.Gao，V.Csizmadia，K.L.Faia，N.Shemmeri，and?A.D.Luster.2001.Donor-derived?IP-10?initiates?development?of?acute?allograft?rejection.J?Exp?Med?193：975.

15.Le?Moine，A.，M.Goldman，and?D.Abramowicz.2002.Multiple?pathways?to?allograft?rejection.Transplantation?73：1373.

16.Hamalainen?HK，Tubman?JC，Vikman?S，Kyrola?T，Ylikoski?E，Warrington?JA，Lahesmaa?R.Identification?and?validation?of?endogenous?reference?genes?for?expression?profiling?of?Thelper?cell?differentiation?by?quantitative?real-time?RT-PCR.Anal?Biochem.2001?Dec1；299(1)：63-70.

17.Foss，D.L.，Baarsch，M.J.，Murtaugh，M.P.，1998.Regulation?of?hypoxanthinephosphoribosyltransferase，glyceraldehyde-3-phosphate?dehydrogenase?and?beta-actinmRNA?expression?in?porcine?immune?cells?and?tissues.Anim.Biotechnol.9，67-78.

18.Giuiietti?A，Overbergh?L，Valckx?D，Decallonne?B，Bouillon?R，Mathieu?C.An?overview?ofreal-time?quantitative?PCR：applications?to?quantify?cytokine?gene?expression.Methods.2001(4)：386-401.

Sequence table

＜110〉with sunrise biotechnology (Beijing) company limited

＜120〉tripartite genome urinalysis method for kidney transplantation immunological rejection and test kit and diagnostic reagent

<130>IB073849

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<170>PatentIn?version?3.1

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aaagatcaaa?aggttaataa?agtaattata?act 1173

<210>2

<211>923

<212>DNA

<213>2

<400>2

ccaagagcta?aaagagagca?aggaggaaac?aacagcagct?ccaaccaggg?cagccttcct 60

gagaagatgc?aaccaatcct?gcttctgctg?gccttcctcc?tgctgcccag?ggcagatgca 120

ggggagatca?tcgggggaca?tgaggccaag?ccccactccc?gcccctacat?ggcttatctt 180

atgatctggg?atcagaagtc?tctgaagagg?tgcggtggct?tcctgatacg?agacgacttc 240

gtgctgacag?ctgctcactg?ttggggaagc?tccataaatg?tcaccttggg?ggcccacaat 300

atcaaagaac?aggagccgac?ccagcagttt?atccctgtga?aaagacccat?cccccatcca 360

gcctataatc?ctaagaactt?ctccaacgac?atcatgctac?tgcagctgga?gagaaaggcc 420

aagcggacca?gagctgtgca?gcccctcagg?ctacctagca?acaaggccca?ggtgaagcca 480

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acactacaag?aggtgaagat?gacagtgcag?gaagatcgaa?agtgcgaatc?tgacttacgc 600

cattattacg?acagtaccat?tgagttgtgc?gtgggggacc?cagagattaa?aaagacttcc 660

tttaaggggg?actctggagg?ccctcttgtg?tgtaacaagg?tggcccaggg?cattgtctcc 720

tatggacgaa?acaatggcat?gcctccacga?gcctgcacca?aagtctcaag?ctttgtacac 780

tggataaaga?aaaccatgaa?acgctactaa?ctacaggaag?caaactaagc?ccccgctgta 840

atgaaacacc?ttctctggag?ccaagtccag?atttacactg?ggagaggtgc?cagcaactga 900

ataaatacct?cttagctgag?tgg 923

<210>3

<211>1005

<212>DNA

<213>3

<400>3

tcttgctgcg?cctccgcctc?ctcctctgct?ccgccaccgg?cttcctcctc?ctgagcagtc 60

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tgatgaacca?ggttatgacc?ttgatttatt?ttgcatacct?aatcattatg?ctgaggattt 180

ggaaagggtg?tttattcctc?atggactaat?tatggacagg?actgaacgtc?ttgctcgaga 240

tgtgatgaag?gagatgggag?gccatcacat?tgtagccctc?tgtgtgctca?aggggggcta 300

taaattcttt?gctgacctgc?tggattacat?caaagcactg?aatagaaata?gtgatagatc 360

cattcctatg?actgtagatt?ttatcagact?gaagagctat?tgtaatgacc?agtcaacagg 420

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ccttgactat?aatgaatact?tcagggattt?gaatcatgtt?tgtgtcatta?gtgaaactgg 720

aaaagcaaaa?tacaaagcct?aagatgagag?ttcaagttga?gtttggaaac?atctggagtc 780

ctattgacat?cgccagtaaa?attatcaatg?ttctagttct?gtggccatct?gcttagtaga 840

gctttttgca?tgtatcttct?aagaatttta?tctgttttgt?actttagaaa?tgtcagttgc 900

tgcattccta?aactgtttat?ttgcactatg?agcctataga?ctatcagttc?cctttgggcg 960

gattgttgtt?taacttgtaa?atgaaaaaat?tctcttaaac?cacag 1005

<210>4

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<212>DNA

<213>4

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<210>5

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<212>DNA

<213>5

<400>5

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<210>6

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<213>6

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<210>7

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<212>DNA

<213>7

<400>7

gcctttctct?ccagctgcag?t 21

<210>8

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<213>8

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<210>9

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<212>DNA

<213>9

<400>9

ggtggagatg?atctctcaac?tttaa 25

<210>10

<211>31

<212>DNA

<213>10

<400>10

acaatgaaaa?agaagggtga?gaagagatgt?c 31

<210>11

<211>26

<212>DNA

<213>11

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cccatccccc?atccagccta?taatcc 26

<210>12

<211>32

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<213>12

<400>12

ccatgttcaa?ttatatcttc?cacaatcaag?ac 32

Claims

1. test kit that is used to detect immunological rejection after the renal transplantation, described test kit comprise be used for detecting the cell transplanted kidney repel protein marker IP-10 expression one couple of PCR primers and be used for detecting second pair of PCR primer that the cell transplanted kidney repels the expression of protein marker granzyme B, forward in the described one couple of PCR primers and reverse primer are respectively the oligonucleotide sequences shown in SEQ ID NO:4 and the SEQ IDNO:5, forward in described second pair of PCR primer and reverse primer are respectively the oligonucleotide sequences shown in SEQ ID NO:6 and the SEQ ID NO:7, and preferred described cell is the urine cell.

2. according to the test kit of claim 1, wherein said test kit also comprises the 3rd pair of PCR primer that is used for detecting cell reference gene HPRT, and forward in described the 3rd pair of PCR primer and reverse primer are respectively the oligonucleotide sequences shown in SEQ ID NO:8 and the SEQ ID NO:9.

3. according to the test kit of claim 1 or 2, wherein said test kit also is included in the fluorescence dye that is used for the quantitative PCR product in the PCR reaction process, preferred SYBR GI fluorescence dye.

4. according to the test kit of claim 1 or 2, wherein said test kit also is included in the TaqMan probe that is used for the quantitative PCR product in the PCR reaction process, preferred pin is the probe that comprises the oligonucleotide sequence shown in the SEQ ID NO:10 to the TaqMan probe of IP-10, TaqMan probe at granzyme B is the probe that comprises the oligonucleotide sequence shown in the SEQ ID NO:11, is the probe that comprises the oligonucleotide sequence shown in the SEQ ID NO:12 at the TaqMan probe of HPRT.

5. diagnostic reagent that is used to detect immunological rejection after the renal transplantation, described diagnostic reagent comprise be used for detecting the cell transplanted kidney repel protein marker IP-10 expression one couple of PCR primers and be used for detecting second pair of PCR primer that the cell transplanted kidney repels the expression of protein marker granzyme B, forward in the described one couple of PCR primers and reverse primer are respectively the oligonucleotide sequences shown in SEQ ID NO:4 and the SEQ ID NO:5, forward in described second pair of PCR primer and reverse primer are respectively the oligonucleotide sequences shown in SEQ ID NO:6 and the SEQ ID NO:7, and preferred described cell is the urine cell.

6. according to the diagnostic reagent of claim 5, wherein said diagnostic reagent also comprises the 3rd pair of PCR primer that is used for detecting cell reference gene HPRT, and forward in described the 3rd pair of PCR primer and reverse primer are respectively the oligonucleotide sequences shown in SEQ ID NO:8 and the SEQ ID NO:9.

7. according to the diagnostic reagent of claim 5 or 6, wherein said diagnostic reagent also is included in the fluorescence dye that is used for the quantitative PCR product in the PCR reaction process, preferred SYBR GI fluorescence dye.

8. according to the diagnostic reagent of claim 5 or 6, wherein said diagnostic reagent also is included in the TaqMan probe that is used for the quantitative PCR product in the PCR reaction process, preferred pin is the probe that comprises the oligonucleotide sequence shown in the SEQ ID NO:10 to the TaqMan probe of IP-10, TaqMan probe at granzyme B is the probe that comprises the oligonucleotide sequence shown in the SEQ ID NO:11, is the probe that comprises the oligonucleotide sequence shown in the SEQ ID NO:12 at the TaqMan probe of HPRT.

9. the method for the expression of IP-10 and granzyme B in the vitro detection urine cell, this method comprise use according in the claim 1 to 4 any one test kit and according to any one diagnostic reagent in the claim 5 to 8, come the expression of quantitative IP-10 and granzyme B by PCR method.

10. according to the method for claim 9, wherein said urine cell is isolating from renal transplantation patient's urine.