CN101701248A

CN101701248A - SYBR GREEN I real-time fluorescence quantitative PCR detection kit of Y chromosome microdeletion

Info

Publication number: CN101701248A
Application number: CN200910106346A
Authority: CN
Inventors: 汪朝晖
Original assignee: SHENZHEN BORUI XIANGHUI BIO-TECHNOLOGY Co Ltd
Current assignee: SHENZHEN BORUI XIANGHUI BIO-TECHNOLOGY Co Ltd
Priority date: 2009-03-23
Filing date: 2009-03-23
Publication date: 2010-05-05

Abstract

The invention discloses an SYBR GREEN I real-time fluorescence quantitative PCR detection kit of Y chromosome microdeletion. The kit can detect the gene deletions of a sY86 locus of a Y chromosome AZFa subregion, a sY127 locus of a Y chromosome AZFb subregion and a sY255 locus of a Y chromosome AZFc subregion by utilizing real-time fluorescence quantitative PCR and has convenient operation and stable detection result.

Description

The micro-deleted SYBR Green of Y chromosome I real-time fluorescence quantitative PCR detection kit

Technical field

The present invention relates to the chromosome deletion detection range, relate in particular to the micro-deleted real-time fluorescence quantitative PCR detection kit of a kind of Y chromosome.

Background technology

Have 15% the couple at child-bearing age to suffer from infertility in the world wide, wherein the factor of male sterility accounts for 50%, in this male sterility patient of 50%, causes unusually owing to genetics more than 35%.Therefore Y chromosome is micro-deleted to be one of main genetics factor that causes male sterility, detects that Y chromosome is micro-deleted to have an important clinical reference value.1976, Tieplolo and Zuffardi found that the azoospermia patient has Y chromosome long-armed (Yq11) disappearance, thus this position of title be no sperm factor (azoospermia factor, AZF).Think that at present the gene of control sperm growth is positioned at the euchromatic region in the long-armed distally of Y chromosome, this position has or not sperm factor AZF, now clearly have 3 spermatogenesis positions (AZFa, AZFb, AZFc) at least, lay respectively at near-end, centre and the far-end of Yq11.Y chromosome is micro-deleted to be occurred on a plurality of genes relevant with AZF on the Y chromosome.Though because the choice criteria difference of each laboratory detected object, recall rate has bigger difference, the deletion frequency in each district is basicly stable: AZFc accounts for 79% of total miss rate, and AZFb accounts for 9%, and AZFa+b accounts for 6%, and AZFa accounts for 3%, and AZFa+b+c accounts for 3%.The micro-deleted of these zones will cause the spermatogeny obstacle, and be few smart, weak essence, even do not have essence.

The STS site mode chart in AZFa, AZFb, 3 zones of AZFc as shown in Figure 1.

Test kit among the present invention is respectively at 3 STS sites wherein, be that the special primer of sY86, sY127 and sY255 design detects carrying out SYBR Green I real-time fluorescence quantitative PCR, sY86, sY127 and sY2553 are provided the positive control sample in a site simultaneously, guarantee the accuracy of detected result.

Y chromosome is micro-deleted to be a kind of common chromosome abnormalty disease, mainly is the unbalanced result of chromosome segregation in the sexual cell reduction division process.The micro-deleted of Y chromosome can also can take place by the eupyrene sperm after fertilization from getting off with micro-deleted sperm transmission normally in this disease in embryo development procedure.Along with the progress of modern assisted reproductive, (intracytoplasmic sperm injection, ICSI) technology is considered to the revolution in the male infertility treatment to sperm injection in the ovum endochylema.Yet this technology may be injected into the sperm that carries karyomit(e) deformity, disappearance or transgenation in the ooecium slurry, makes ovum fertilization, thereby above-mentioned various hereditary defectes are passed to the next generation.Therefore, in order to determine whether to need treatment, the clinician advises all sperm concentrations＜5 * 10 ⁶The male sex of/mL will carry out the micro-deleted molecular diagnosis of Y chromosome in principle.Since 1999, European andrology association and European molecular genetic experiment Quality Control coorporative network (EMQN) were published the micro-deleted molecular diagnosis guide of Y chromosome, and objective quality evalution experimental technique are provided for improving quality of diagnosis.The lab guide of latest edition is according to the achievement of holding " Best Practice Meeting " in October, 2003 in Florence, revises to form on the basis of version in 1999.For the micro-deleted examination of Y chromosome, the guide of version in 1999 is proved to be accurately, sensitivity and easy handling.According to the latest Progress of Y chromosome sequence and micro-deleted mechanism, the guide of version in 1999 can satisfy the needs of accurate diagnosis, facts have proved it is very effective and suitable.

The micro-deleted molecular detecting method of Y chromosome mainly contains Southern blot blot hybridization, polymerase chain reaction (polymerase chain reaction, PCR) in conjunction with the electrophoresis detection technology etc., wherein conventional PCR electrophoretic technique is the most frequently used, a simplest and effective means in each research at present.Yet the huge obstacle that influences conventional round pcr clinical application is the amplified production pollution problems, and its result is prone to false negative and false positive.Although take to optimize the PCR condition and set up contrast safety assurance a plurality of strict quality control measures such as (as using women DNA as negative control), the amplification of PCR still may be failed.

Chinese patent 200410043918.0 discloses sY87 among the micro-deleted gene STS of specific amplification Y chromosome and two pairs of primer sequences of sY143; Chinese patent 200710074317.X discloses 30 primers of the micro-deleted gene A ZF of specific amplification Y chromosome and 2 primers of the peculiar sry gene of the specific amplification male sex.More than two patents all be to adopt conventional PCR method to carry out amplified reaction, the weak point of this method is to adopt electrophoresis detection, easily causes the cross infection of PCR product, has increased the possibility of false positive results.

Summary of the invention

For addressing the above problem, the object of the present invention is to provide a kind ofly stablize, SYBR Green I real-time fluorescence quantitative PCR detection kit that the efficient detection Y chromosome is micro-deleted.

For achieving the above object, the present invention adopts following technical scheme:

The micro-deleted SYBR Green of a kind of Y chromosome I real-time fluorescence quantitative PCR detection kit, described test kit comprises:

The primer of specific amplification Y chromosome AZFa subprovince sY86 sequence, its base sequence is shown in SEQID NO:1 and 2;

SY86 site, Y chromosome AZFa subprovince positive control sample: the primer with sequence shown in SEQ ID NO:1 and the SEQ ID NO:2 is to insert the recombinant plasmid of son structure as positive reference material to the amplified production that obtains;

The primer of specific amplification Y chromosome AZFb subprovince sY127 sequence, its base sequence is shown in SEQ ID NO:3 and 4;

SY127 site, Y chromosome AZFb subprovince positive control sample: the primer with sequence shown in SEQ ID NO:3 and the SEQ ID NO:4 is to insert the recombinant plasmid of son structure as positive reference material to the amplified production that obtains;

The primer of specific amplification Y chromosome AZFc subprovince sY255 sequence, its base sequence is shown in SEQ ID NO:5 and 6;

SY255 site, Y chromosome AZFc subprovince positive control sample: the primer with sequence shown in SEQ ID NO:5 and the SEQ ID NO:6 is to insert the recombinant plasmid of son structure as positive reference material to the amplified production that obtains.

Described test kit further comprises: SYBR Green I fluorescence dye.

Described test kit further comprises: MgCl ₂, dNTPs and PCR reaction buffer.

Described MgCl ₂The reaction final concentration is 4mM.

Described dNTPs reaction final concentration is 0.2mM.

Second purpose of the present invention is to provide the application of SYBR Green I real-time fluorescence quantitative PCR in the micro-deleted detection of Y chromosome.

Real-time fluorescence quantitative PCR (real time fluorescence quantitative PCR) is a kind of new technique of gene detection, thereby it realizes starting template quantitatively and is qualitatively analyzed by the real-time detection to each circulation products fluorescent signal in the pcr amplification reaction.This technology has improved detection specificity on the PCR basis; Amplification is with analysis is integrated, convenient and simple for operation, quick automatically; The stopped pipe operation need not electrophoresis, can reduce PCR product pollution possibility; Experimental result is to detect in real time the gained performance graph, and is reliable etc. than end point determination.Based on above advantage, the real-time fluorescence quantitative PCR technology presents the trend that replaces conventional round 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.SYBR Green I has many good qualities in the real-time context of detection of nucleic acid, because it combines with all double-stranded DNA, needn't special customization because template is different, and therefore the program versatility that designs is good, and price is relatively low.Utilize fluorescence dye can indicate the character of double-stranded DNA fusing point, can discern amplified production and primer dimer, thereby can distinguish non-specific amplification, can also realize monochromatic multiple assay further by the melting curve analysis.In addition, owing to a PCR product can combine with polymolecular dyestuff, so the sensitivity of SYBR Green I is very high.But because SYBR Green I combines with all double-stranded DNA, therefore the false positive that is caused by the amplified production of primer dimer, strand secondary structure and mistake can influence quantitative accuracy.Can help to reduce the influence of non-special product by the variation of fluorescence behind the measurement elevated temperature.Come the homogeneity of assay products to help to analyze the quantitative result that obtains by SYBRGreen I by melting curve.

The present invention provides the micro-deleted SYBR Green of Y chromosome I real-time fluorescence quantitative PCR detection kit first, and this test kit has following advantage:

1) lack detection time, speed is fast, and high-throughput can be finished the quantitative analysis of 96 samples at 4-6 hour.

2) operating process is simple, from the PCR reaction, finishes amplification and The real time measure exactly in the system of sealing, greatly reduces contamination of heavy, has therefore also just reduced the probability of result error.

3) specification of quality that specimen dna is obtained is lower, no matter is paraffin organization or flesh tissue, can both obtain the ideal detected result.

4) fluorescence quantitative PCR method result's interpretation: have the sample of amplification curve to be positive sample more than threshold line, interpretation as a result is simple, clear and definite, directly perceived, also can carry out quantitative analysis to the result if need.

That 5) detects is highly sensitive, and the fluorescent PCR detection technique is that to combine round pcr, fluorescent mark technology, laser technology, digital imaging technology be the technology of one, so its detection sensitivity is very high.

6) utilize fluorescence dye can indicate the character of double-stranded DNA fusing point, analyze identification specificity product and non-specific product by melting curve, the influence of the non-specific product that reduction primer dimer, strand secondary structure and wrong amplification cause, the accuracy of assurance detected result.

Description of drawings

Fig. 1 is the STS site mode chart in AZFa, AZFb, 3 zones of AZFc;

Fig. 2 is the FQ-PCR figure in sY86 site, positive control sample Y chromosome AZFa subprovince, sY127 site, Y chromosome AZFb subprovince, sY255 site, Y chromosome AZFc subprovince;

Fig. 3, Fig. 4, Fig. 5 are three clinical FQ-PCR figure that normally educated male sex's sample;

Fig. 6, Fig. 7, Fig. 8 are the FQ-PCR figure of three male gene group dna samples to be detected;

Fig. 9 is the pUCm-T-sY86 plasmid map;

Figure 10 is the pUCm-T-sY127 plasmid map;

Figure 11 is the pUCm-T-sY255 plasmid map.

The drawing reference numeral explanation: S represents sample, and P represents positive control, and N represents negative control.

Embodiment

One, material

1, instrument:

The real-time fluorescence quantitative PCR instrument, pipettor, whizzer.

2, design of primers:

The present invention has designed 3 pairs of amplification AZF STSs sequences that the PCR primer is special respectively, and primer sequence is as follows.

The tabulation of table 1 primer

The primer title	Sequence number	The amplification object
The primer title	Sequence number	The amplification object	??sY86-F??sY86-R	??SEQ?ID?NO：1??SEQ?ID?NO：2	Specific amplification Y chromosome AZFa subprovince sY86 sequence
??sY127-F??sY127-R	??SEQ?ID?NO：3??SEQ?ID?NO：4	Specific amplification Y chromosome AZFb subprovince sY127 sequence	??sY86-F??sY86-R	??SEQ?ID?NO：1??SEQ?ID?NO：2
??sY127-F??sY127-R	??SEQ?ID?NO：3??SEQ?ID?NO：4		??sY255-F??sY255-R	??SEQ?ID?NO：5??SEQ?ID?NO：6	Specific amplification Y chromosome AZFc subprovince sY255 sequence

3, positive control recombinant plasmid: pUCm-T-sY86, pUCm-T-sY127 and pUCm-T-sY255.

4, reagent:

10 * PCR buffer, MgCl ₂, Taq enzyme, dNTPs, 1000 * SYBR Green I fluorescence dye.

Two, method

1, human genome DNA's extraction

From human anticoagulation cirumferential blood white corpuscle, extract genomic dna with Proteinase K cracking and phenol-chloroform extraction process or commercial reagent box, extract three clinical male gene group DNA, three male gene group DNA to be detected and women's genomic dnas of normally having educated respectively.

2, fluorescent quantitative PCR reaction

Get the PCR reaction tubes, on tube wall, carry out mark, add fluorescent quantitation reaction solution 9.75 μ L respectively and (comprise 10 * PCR Buffer, 2.5 μ L (no Mg ²⁺), 25mM MgCl ₂4 μ L, 10mM dNTPs0.5 μ L, 10 μ M upstream primers, 0.5 μ L, 10 μ M downstream primers, 0.5 μ L, 10 * SYBR Green I1.25 μ L, 5U/ μ L enzyme 0.5 μ L), the sample genomic dna 0.625 μ L (containing the about 25ng of genomic dna) that has extracted adds sterilized water to 25 μ L at last and reacts total system.This process needs in operation on ice, and each sample is done 3 pcr amplifications simultaneously and detected, and each the detection all set up one group of positive control and one group of negative control (women's genomic dna) simultaneously.

Fluorescent quantitation is reflected on ABI 7300 detectors and carries out, and increases by following condition:

Fs: 95 ℃ of 15min;

Subordinate phase: 94 ℃ of 30sec, 57 ℃ of 45sec (phosphor collection), 72 ℃ of 30sec, 35 circulations;

Phase III (melting curve): 95 ℃ of 15sec, 60 ℃ of 1min, 95 ℃ of 15sec, 60 ℃ of 15sec.

Three, the result judges

Read detected result, the threshold setting principle is with the vertex of threshold line just above normal negative control product amplification curve, and the result shows that feminine gender is as the criterion, or can adjust according to instrument noise situation.

(1) sample: Ct value≤30.0, typical amplification curve appears, represent that then the sample Y chromosome does not have micro-deleted in this site; If no typical amplification curve represents that then the sample Y chromosome has taken place micro-deleted in this site.

(2) positive control: Ct value≤30.0 typical amplification curve occurs, as shown in Figure 2.

(3) negative control: no Ct value and do not have amplification curve.

From Fig. 3, Fig. 4, Fig. 5 as can be seen, three clinical male sex's sample standard deviations of normally having educated amplify typical positive amplification curve, and these three male sex are not taken place by the micro-deleted of Y chromosome.

From Fig. 6, Fig. 7, Fig. 8 as can be seen, three male gene group dna samples to be detected also all amplify typical positive amplification curve, confirm that the micro-deleted gene of Y chromosome does not take place these three male sex yet.

SEQUENCE?LISTING

＜110〉Shenzhen Smartshining Biotechnologies Co., Ltd.

＜120〉the micro-deleted gene SYBR of Y chromosome GreenI real-time fluorescence quantitative PCR detection kit

<130>P10922

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<170>PatentIn?version?3.3

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Claims

1. the micro-deleted SYBR Green of Y chromosome I real-time fluorescence quantitative PCR detection kit is characterized in that described test kit comprises:

2. the micro-deleted SYBR Green of Y chromosome according to claim 1 I real-time fluorescence quantitative PCR detection kit is characterized in that described test kit further comprises: SYBR GreenI fluorescence dye.

3. the micro-deleted SYBR Green of Y chromosome according to claim 1 and 2 I real-time fluorescence quantitative PCR detection kit is characterized in that described test kit further comprises: MgCl ₂, dNTPs and PCR reaction buffer.

4. the micro-deleted SYBR Green of Y chromosome according to claim 3 I real-time fluorescence quantitative PCR detection kit is characterized in that described MgCl ₂The reaction final concentration is 4mM.

5. the micro-deleted SYBR Green of Y chromosome according to claim 3 I real-time fluorescence quantitative PCR detection kit is characterized in that, described dNTPs reaction final concentration is 0.2mM.

6.SYBR the application of Green I real-time fluorescence quantitative PCR in the micro-deleted detection of Y chromosome.