CN104087672A - Kit for quickly detecting number of human chromosomes 21 by multiplex real-time fluorescence quantitative PCR (polymerase chain reaction) technique - Google Patents
Kit for quickly detecting number of human chromosomes 21 by multiplex real-time fluorescence quantitative PCR (polymerase chain reaction) technique Download PDFInfo
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- CN104087672A CN104087672A CN201410333071.3A CN201410333071A CN104087672A CN 104087672 A CN104087672 A CN 104087672A CN 201410333071 A CN201410333071 A CN 201410333071A CN 104087672 A CN104087672 A CN 104087672A
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- karyomit
- necrosis factor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention discloses a kit for quickly detecting the number of human chromosomes 21 by a multiplex real-time fluorescence quantitative PCR (polymerase chain reaction) technique, which comprises amplification detection reagents, the amplification detection reagents comprise a pair of primers capable of simultaneously amplifying a certain specific repetitive sequence on the chromosome 21 and a specific repetitive sequence on the chromosome 6, two fluorescent probes for respectively detecting the amplification quantities of the specific repetitive sequences of the chromosomes 21 and 6, a pair of primers capable of simultaneously amplifying another specific repetitive sequence on the chromosome 21 and a specific repetitive sequence on the chromosome 11, and two fluorescent probes for respectively detecting the amplification quantities of the specific repetitive sequences of the chromosomes 21 and 11. The kit can determine the detection result according to Delta CT21-6 value of the chromosomes 21 and 6 and the Delta CT21-11 value of the chromosomes 21 and 11; and the two Delta CT values can be used for independently detecting the number of the chromosomes 21, and the result is more reliable.
Description
Technical field
The present invention relates to No. 21 chromosome number object test kits of a kind of multiple real time fluorescence quantifying PCR technology rapid detection people, belong to biology field.
Background technology
Mongolism (Down ' s syndrome) also referred to as mongolism, be the aneuploid disease that the modal numerical abnormalities of chromosomes of the mankind causes, newborn infant's incidence is 1/600～1/800.Infant main manifestations is serious congenital dysnoesia and various organ congenital abnormality, and most of patients life cannot be taken care of oneself.At present, this disease there is no effective methods for the treatment of, and therefore, carrying out the birth that antenatal diagnosis prevents infant is the pathogenetic main method of such disease of prevention.For a long time, classical way (the Caspersson T that amniocyte is cultivated, chromosome karyotype analysis becomes antenatal diagnosis, Zech L, Johansson C, et al.Identification of human chromosomes by DNA-binding fluorescent agents[J] .Chromosoma, 1970,30 (2): 215-227.), but the method exists time-consuming (generally wanting more than two weeks), easily cultivate unsuccessfully and can not detect the less shortcomings such as chromosomal structural aberration.Fluorescence in situ hybridization (FISH) technology is applied to antenatal quick diagnosis, without carrying out cell cultures, in 24 hours, just can show that assay becomes possible (Ho SSY, Chua C, Gole L, et al.Same-day prenatal diagnosis of common chromosomal aneuploidies using microfluidics-fluorescence in situ hybridization[J] .Prenatal Diagnosis, 2012,32 (4): 321-328. etc.); But because this technological operation is loaded down with trivial details and require to have good technology specialty, make the application of carrying out clinical samples that this technology can not be a large amount of.Therefore, should need to set up some defects and the deficiency that more quick and effective methods for prenatal diagnosis makes up above method.
Along with molecular biological development, there is the method for several rapid molecular diagnosis aneuploids at present.QF-PCR method is current a kind of most widely used method, the method can realize accurate detection (the Atef SH of aneuploid, Hafez S, Helmy S, et al.QF-PCR as a Rapid Technique for Routine Prenatal Diagnosis of Fetal Aneuploidies[J] .Pediatric Research, 2011, 70:412-412.), the main drawback of this technology is that diagnosis need to rely on pleomorphism site, and pleomorphism site may be diagnosed preferably in a certain people's group energy, and because ethnic difference may appear at the situation that in other crowd, appearance cannot be diagnosed (Mann K, Donaghue C, Fox SP, et al.Strategies for the rapid prenatal diagnosis of chromosome aneuploidy[J] .Eur J Hum Genet, 2004, 12:907-915.), and it also needs its fragment sheet degree of capillary electrophoresis analysis after pcr amplification, therefore, the expense of instrument and reagent has also limited the widespread use of the method.Multiple linking probe amplification (MLPA) technology starts to be applied to the variation of gene copy number most, be applied to afterwards diagnosis (the Slater HR of aneuploid disease, Bruno DL, Ren H, et al.Rapid, high throughput prenatal detection of aneuploidy using a novel quantitative method (MLPA) [J] .Journal of medical genetics, 2003, 40 (12): 907-912.), because this Technology Need hybridization is spent the night, the steps such as follow-up capillary electrophoresis analysis of connection and product, test the relatively loaded down with trivial details and expensive expense of subsequent analysis and affected widespread use (the Willis AS of this technology, Veyver I, Eng CM.Multiplex ligation ?dependent probe amplification (MLPA) and prenatal diagnosis[J] .Prenat Diagn, 2012, 32:315-320.).
Real time fluorescence quantifying PCR method diagnosis aneuploid does not need PCR aftertreatment or electrophoresis detection, and easy, quick, the judgement of whole operation and result can complete in 4 hours; And reading completely of data undertaken by instrument, got rid of the impact of subjective factor, other tens samples can once complete, and for carrying out large-scale Prenatal Screening, provide technical guarantee.Current research is mainly to adopt No. 21 chromosomal DSCR3 gene fragments and No. 12 chromosomal GAPDH gene fragments to carry out relative quantification (Zimmermann B, Holzgreve W, Wenzel F, et al.Novel real-time quantitative PCR test for trisomy21[J] .Clinical chemistry, 2002, 48 (2): 362-363. etc.), or adopt the distinguished sequence between other coloured differently bodies to carry out relative quantification research (Zimmermann B G, Dudarewicz L.Real-time quantitative PCR for the detection of fetal aneuploidies[M] .Prenatal Diagnosis.Humana Press, 2008:95-109, Sui builds loyalty, Zhang Huimin, Sun Xiaofang. multiple real-time quantitative PCR Kuai Suzhenduantangshi and edward's syndrome [J]. and Chinese Journal of Medical Genetics, 2010 (4): 449-452.).But, owing to adopting two pairs of primers different fragment that increases respectively, the difference of the variation of very small annealing temperature or DNA quality all can cause false negative or false-positive result, thereby inaccuracy (the Helmy SM that causes result, Ismail S, Bassiouni R, et al.Sensitivity of DCSR3/GAPDH ratio using quantitative real-time PCR in the rapid prenatal diagnosis for down syndrome[J] .Fetal Diagn Ther, 2009,25 (2): 220-223.).
Publication number is the patent of invention of CN100338227A, CN102086469A, all disclose by the distinguished sequence design different primers to coloured differently body and the method for probe and detected No. 21 chromosomal numbers, but these class methods exist foregoing may be due to the variation of annealing temperature or the difference of DNA quality, thereby cause false negative or false-positive result.On the other hand, the detection site of aforesaid method all only has one, when the genome complementation sequence producer at primer suddenlys change, may cause equally false negative or false-positive experimental result.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of No. 21 chromosome number object test kits of multiple real time fluorescence quantifying PCR technology rapid detection people of realizing two cover independent detection systems in same reaction tubes.Adopt this test kit to detect No. 21 chromosome numbers of people, result is accurate, reliable, and highly sensitive, high specificity.
No. 21 chromosome number object test kits of multiple real time fluorescence quantifying PCR technology rapid detection people of the present invention, comprise augmentation detection reagent, described augmentation detection reagent comprises 2 pairs of primers, 4 fluorescently-labeled probes of difference, the pair of primers a pair of special tumor-necrosis factor glycoproteins on No. 21 karyomit(e)s and No. 6 karyomit(e)s that can simultaneously increase wherein, another pair of primers can increase another on No. 21 karyomit(e)s and No. 11 karyomit(e)s to special tumor-necrosis factor glycoproteins simultaneously, and described amplimer and fluorescent probe are specially:
(1) simultaneously the increase primer pair of No. 21 special tumor-necrosis factor glycoproteins chr21-1 of karyomit(e) and No. 6 special tumor-necrosis factor glycoproteins chr6-1 of karyomit(e), its base sequence is respectively as shown in SEQ ID NO:1 and SEQ ID NO:2;
(2) the Taqman probe chr21-1-Probe of No. 21 special tumor-necrosis factor glycoproteins chr21-1 of karyomit(e) of specific detection, its base sequence is as shown in SEQ ID NO:3;
(3) the Taqman probe chr6-1-Probe of No. 6 special tumor-necrosis factor glycoproteins chr6-1 of karyomit(e) of specific detection, its base sequence is as shown in SEQ ID NO:4;
(4) simultaneously the increase primer pair of No. 21 special tumor-necrosis factor glycoproteins chr21-2 of karyomit(e) and No. 11 special tumor-necrosis factor glycoproteins chr11-1 of karyomit(e), its base sequence is respectively as shown in SEQ ID NO:5 and SEQ ID NO:6;
(5) the Taqman probe chr21-2-Probe of No. 21 special tumor-necrosis factor glycoproteins chr21-2 of karyomit(e) of specific detection, its base sequence is as shown in SEQ ID NO:7;
(6) the Taqman probe chr11-1-Probe of No. 11 special tumor-necrosis factor glycoproteins chr11-1 of karyomit(e) of specific detection, its base sequence is as shown in SEQ ID NO:8.
Test kit of the present invention is by a pair of common amplimer of identical DNA sequence dna position, two ends design at special tumor-necrosis factor glycoproteins (two similar sequences), avoid different primers increase the phase mutual interference of different sequence generations or the interference of other condition, thereby guaranteed the consistence of amplification efficiency; And be chosen in the DNA sequence dna position design Taqman probe with base difference so that the △ CT value of relative real-time fluorescence PCR that can be by repeated fragment accurately the relative amplification of two sequences of detection measure; On the other hand, by designing two pairs of independently detection site, effectively avoided the primer of design in complementary template sequence, to occur the situation of transgenation, realized the heavy real-time fluorescence quantitative PCR of single tube 4, and two cover detection systems can detect in same pipe simultaneously, situation without phase mutual interference occurs, and all can independently detect No. 21 chromosomal number.
In technique scheme, the special tumor-necrosis factor glycoproteins chr21-1 of described No. 21 karyomit(e)s and No. 6 special tumor-necrosis factor glycoproteins chr6-1 of karyomit(e) are two similar sequences, wherein, the base sequence of No. 21 special tumor-necrosis factor glycoproteins chr21-1 of karyomit(e) is as shown in SEQ ID NO:9, and the base sequence of No. 6 special tumor-necrosis factor glycoproteins chr6-1 of karyomit(e) is as shown in SEQ ID NO:10.5 ' the end mark CY5 of the described Taqman probe chr21-1-Probe for No. 21 special tumor-necrosis factor glycoproteins chr21-1 of karyomit(e) of specific detection, 3 ' end mark BHQ1; 5 ' the end flag F AM of the described Taqman probe chr6-1-Probe for No. 6 special tumor-necrosis factor glycoproteins chr6-1 of karyomit(e) of specific detection, 3 ' end mark BHQ1.Described Taqman probe chr21-1-Probe and Taqman probe chr6-1-Probe can detect respectively special tumor-necrosis factor glycoproteins chr21-1 on No. 21 karyomit(e)s and the special tumor-necrosis factor glycoproteins chr6-1 on No. 6 karyomit(e)s, then by the relative quantification index △ CT of Taqman probe chr21-1-Probe and Taqman probe chr6-1-Probe
21-6no. 21 chromosomal numbers of value judgement.As △ CT
21-6=Ct
cY5-Ct
fAM, represent that the sample that detects is No. 21 karyomit(e) normal specimen at=0.23～0.65 o'clock; As △ CT
21-6=Ct
cY5-Ct
fAM, represent that the sample that detects is 21-trisome caryogram sample at=-0.32～-0.05 o'clock.
In technique scheme, the special tumor-necrosis factor glycoproteins chr21-2 of described No. 21 karyomit(e)s and No. 11 special tumor-necrosis factor glycoproteins chr11-1 of karyomit(e) are two similar sequences, wherein, the base sequence of No. 21 special tumor-necrosis factor glycoproteins chr21-2 of karyomit(e) is as shown in SEQ ID NO:11, and the base sequence of No. 11 special tumor-necrosis factor glycoproteins chr11-1 of karyomit(e) is as shown in SEQ ID NO:12.The described end of the Taqman probe chr21-2-Probe5 ' for No. 21 special tumor-necrosis factor glycoproteins chr21-2 of karyomit(e) of specific detection mark ROX, 3 ' end mark BHQ1; 5 ' the end mark HEX that is used for the Taqman probe chr11-1-Probe of No. 11 special tumor-necrosis factor glycoproteins chr11-1 of karyomit(e) of specific detection, 3 ' end mark BHQ1.Described Taqman probe chr21-2-Probe and Taqman probe chr11-1-Probe can detect respectively special tumor-necrosis factor glycoproteins chr21-2 on No. 21 karyomit(e)s and the special tumor-necrosis factor glycoproteins chr11-1 on No. 11 karyomit(e)s, then by the relative quantification index △ CT of Taqman probe chr21-2-Probe and Taqman probe chr11-1-Probe
21-11no. 21 chromosomal numbers of value judgement.As △ CT
21-11=Ct
rOX-Ct
hEX, represent that the sample that detects is No. 21 karyomit(e) normal specimen at=-0.98～-0.70 o'clock; As △ CT
21-11=Ct
rOX-Ct
hEX, represent that the sample that detects is 21-trisome caryogram sample at=-1.71～-1.39 o'clock.
In fluorescent probe of the present invention, FAM refers to Fluoresceincarboxylic acid, and HEX refers to chlordene-6-methyl fluorescein, and ROX refers to carboxyl-X-rhodamine, and CY5 refers to cyanine dyes molecule 5, and BHQ1 refers to fluorescent quenching group.
Test kit of the present invention also comprises conventional in some available reagent boxes and necessary component, as positive control template, negative control template, damping fluid, enzyme liquid, dNTP, Mg
2+deng.
Compared with prior art, test kit of the present invention can be by the special tumor-necrosis factor glycoproteins on No. 21 karyomit(e)s and the relative quantification △ CT of the special tumor-necrosis factor glycoproteins on No. 6 karyomit(e)s
21-6another special tumor-necrosis factor glycoproteins on value, No. 21 karyomit(e)s and the relative quantification △ CT of the special tumor-necrosis factor glycoproteins on No. 11 karyomit(e)s
21-11the distributed area of value judges whether detection sample is normal specimen or 21-trisome caryogram sample, and two △ CT values can dually detect No. 21 chromosomal number independently, have guaranteed the reliability of result; Meanwhile, test kit of the present invention also has highly sensitive, high specificity and the advantage such as fast and convenient.
Accompanying drawing explanation
Fig. 1 is special tumor-necrosis factor glycoproteins and the position of the special tumor-necrosis factor glycoproteins on No. 6 karyomit(e)s on karyomit(e) on No. 21 karyomit(e)s, wherein, (a) being special tumor-necrosis factor glycoproteins chr21-1 on No. 21 karyomit(e)s position on No. 21 karyomit(e)s, is (b) special tumor-necrosis factor glycoproteins chr6-1 on No. 6 karyomit(e)s position on No. 6 karyomit(e)s;
Fig. 2 is the special tumor-necrosis factor glycoproteins on the special tumor-necrosis factor glycoproteins on No. 21 karyomit(e)s and No. 6 karyomit(e)s in Fig. 1, and according to the consensus primer of sequences Design and Taqman probe separately, wherein, 21-Taqman probe in figure is Taqman probe chr21-1-Probe, and the 6-Taqman probe in figure is Taqman probe chr6-1-Probe;
Fig. 3 is another special tumor-necrosis factor glycoproteins and the position of the special tumor-necrosis factor glycoproteins on No. 11 karyomit(e)s on karyomit(e) on No. 21 karyomit(e)s, wherein, (a) being another the special tumor-necrosis factor glycoproteins chr21-2 position on No. 21 karyomit(e)s on No. 21 karyomit(e)s, is (b) special tumor-necrosis factor glycoproteins chr11-1 on No. 11 karyomit(e)s position on No. 11 karyomit(e)s;
Fig. 4 is the special tumor-necrosis factor glycoproteins on the special tumor-necrosis factor glycoproteins on No. 21 karyomit(e)s and No. 11 karyomit(e)s in Fig. 3, and according to the consensus primer of sequences Design and Taqman probe separately, wherein, 21-Taqman probe in figure is Taqman probe chr21-2-Probe, and the 11-Taqman probe in figure is Taqman probe chr11-1-Probe.
Embodiment
With specific embodiment, the invention will be further described below, but the present invention is not limited to these embodiment.
Embodiment 1: adopt test kit of the present invention to detect normal specimen and 21-trisome sample to be measured
1, the composition of test kit:
The design of the selection of 1.1 target sequences and primer and probe
1) first independent detection site: selecting the special tumor-necrosis factor glycoproteins chr21-1 on No. 21 karyomit(e)s is relative real-time fluorescence quantitative PCR amplification and detection sequence with special tumor-necrosis factor glycoproteins chr6-1 on No. 6 karyomit(e)s (chr21-1 and chr6-1 are two similar sequences), as shown in Figure 1, wherein:
The base sequence of special tumor-necrosis factor glycoproteins chr21-1 on No. 21 described karyomit(e)s is: 5 '-acaggacctgaccctggctcccggccagcctcacctcccatggctttcctgcccct gtatt acctcacttccttcccctttagccatccttcgggcctcagttcaagttcacatctt caggatggt-3 ' (SEQ ID NO:9);
The base sequence of special tumor-necrosis factor glycoproteins chr6-1 on No. 6 described chromosome dyeing bodies is: 5 '-acaggacctgaccctggctcccggccagcctcatctcccatggctttcctgtccct gtact acctcgcttccttcccctttagcctcagttcaagttcacatcttcaggatggt-3 ' (SEQ ID NO:10).
At a pair of public primer of the identical base sequence site design in the both sides of No. 21 chromosomal special tumor-necrosis factor glycoproteins chr21-1 and No. 6 chromosomal special tumor-necrosis factor glycoproteins chr6-1, as shown in Figure 2, this primer pair can increase No. 21 special tumor-necrosis factor glycoproteins chr21-1 of karyomit(e) and No. 6 special tumor-necrosis factor glycoproteins chr6-1 of karyomit(e) simultaneously, in this primer pair:
Upstream primer (is used chr below
21-6-F replaces) be: 5 '-acaggacctgaccctggc-3 ' (SEQ ID NO:1);
Downstream primer (is used chr below
21-6-R replaces) be: 5 '-accatcctgaagatgtgaacttg-3 ' (SEQ ID NO:2).
The sequence site in the centre of No. 21 chromosomal special tumor-necrosis factor glycoproteins chr21-1 and No. 6 chromosomal special tumor-necrosis factor glycoproteins chr6-1 with base difference is designed for the Taqman probe chr21-1-Probe of No. 21 special tumor-necrosis factor glycoproteins chr21-1 of karyomit(e) of specific detection and the Taqman probe chr6-1-Probe of No. 6 special tumor-necrosis factor glycoproteins chr6-1 of karyomit(e) of specific detection, as shown in Figure 2, wherein:
The base sequence of Taqman probe chr21-1-Probe is:
5 '-CY5-ccctttagccatccttcgggcctcagt-BHQ1-3 ' (SEQ ID NO:3); The base sequence of Taqman probe chr6-1-Probe is:
5’-FAM-acctcgcttccttcccctttagcctca-BHQ1-3’(SEQ?ID?NO：4)。
2) second independent detection site: selecting the special tumor-necrosis factor glycoproteins chr21-2 on No. 21 karyomit(e)s is relative real-time fluorescence quantitative PCR amplification and detection sequence with special tumor-necrosis factor glycoproteins chr11-1 on No. 11 karyomit(e)s (chr21-2 and chr11-1 are two similar sequences), as shown in Figure 3, wherein:
The base sequence of special tumor-necrosis factor glycoproteins chr21-2 on No. 21 karyomit(e)s is: 5 '-gtgccattgacacaggaggacccatgcctgagaaagacttttatctgagttactga gatta agatgttttgaagctcccaagcagagggatgctggatctgctgtggaaatctggct gagcagctg cagggacagctgggggtaaag-3 ' (SEQ ID NO:11);
On No. 11 karyomit(e)s, the base sequence of special tumor-necrosis factor glycoproteins chr11-1 is:
5’-gtgccattgacacaggaggacctacgcctcagaaagacttttatctgagttaccaaggttg?agatgttttgaaagatgctagatctgggaatctggctgagcagctgctgggacagctgggggtaa?ag-3’(SEQ?ID?NO：12)。
At a pair of public primer of the identical base sequence site design in the both sides of No. 21 chromosomal special tumor-necrosis factor glycoproteins chr21-2 and No. 11 chromosomal special tumor-necrosis factor glycoproteins chr11-1, as shown in Figure 4, this primer pair can increase No. 21 special tumor-necrosis factor glycoproteins chr21-2 of karyomit(e) and No. 11 special tumor-necrosis factor glycoproteins chr11-1 of karyomit(e) simultaneously, in this primer pair:
Upstream primer (is used chr below
21-11-F replaces) be: 5 '-gtgccattgacacaggaggac-3 ' (SEQ ID NO:5);
Downstream primer (is used chr below
21-11-R replaces) be: 5 '-ctttacccccagctgtccc-3 ' (SEQ ID NO:6).
The sequence site in the centre of No. 21 chromosomal special tumor-necrosis factor glycoproteins chr21-2 and No. 11 chromosomal special tumor-necrosis factor glycoproteins chr11-1 with base difference is designed for the Taqman probe chr21-2-Probe of No. 21 special tumor-necrosis factor glycoproteins chr21-2 of karyomit(e) of specific detection and the Taqman probe chr11-1-Probe of No. 11 special tumor-necrosis factor glycoproteins chr11-1 of karyomit(e) of specific detection, as shown in Figure 4, wherein:
The base sequence of Taqman probe chr21-2-Probe is:
5 '-ROX-agccagatttccacagcagatcca-BHQ1-3 ' (SEQ ID NO:7); The base sequence of Taqman probe chr11-1-Probe is:
5’-HEX-tgctcagccagattcccagatcta-BHQ1-3’(SEQ?ID?NO：8)。
1.2 other moietys:
Hotstar-Taq enzyme, damping fluid, dATP, dTTP, dCTP and dGTP and Mg
2+all purchased from Kang Wei century bio tech ltd, Beijing.
2, the preparation of PCR reaction system:
By following table 1, prepare PCR reaction system:
Table 1:(mM represents mmol/L, and μ M represents μ mol/L)
PCR reaction system is 50uL.
3, the source of sample and processing
Sample derives from the DNA sample of determining caryogram through traditional chromosome karyotype analysis method, and DNA sample adopts the conventional DNA extraction method in laboratory to extract, and is diluted to 20ng/ μ L, and saves backup in-20 ℃ with distilled water.
4, pcr amplification program:
PCR reaction instrument is CFX96 real-time fluorescence quantitative PCR instrument.PCR response procedures is: 95 ℃ of denaturation 10min; 95 ℃ of 15sec, 60 ℃ of 1min, 40 circulations, gather fluorescent signal in 60 ℃ of annealing steps ends.
By normal specimen and 21-trisome sample to be measured, according to above-mentioned reaction system and response procedures, on quantitative real time PCR Instrument, carry out augmentation detection, record respectively CT
fAMvalue, CT
cY5value, CT
rOXvalue and CT
hEXvalue.
5, result detects and analyzes:
No. 21 karyomit(e)s and No. 6 chromosomal △ CT of normal specimen
21-6=Ct
cY5-Ct
fAM=0.53, and No. 21 karyomit(e)s and No. 6 chromosomal △ CT of 21-trisome sample
21-6=Ct
cY5-Ct
fAM=-0.09; By the △ CT value of No. 21 karyomit(e)s relatively and No. 6 karyomit(e) normal specimen and 21-trisome sample: 0.53 and-0.09, result shows No. 21 karyomit(e)s than No. 6 many copies of karyomit(e) (in theory, it is 0.5 o'clock that △ CT value differs, its copy differs 1 copy), visible, conform to the result of traditional standard detecting method (chromosome karyotype analysis), the 21-trisome sample of test is 3 No. 21 karyomit(e)s.No. 21 karyomit(e)s and No. 11 chromosomal △ CT of normal specimen
21-11=Ct
rOX-Ct
hEX=-0.98, and No. 21 karyomit(e)s and No. 11 chromosomal △ CT of 21-trisome sample
21-11=Ct
rOX-Ct
hEX=-1.58; By the △ CT value of No. 21 karyomit(e)s relatively and No. 11 karyomit(e) normal specimen and 21-trisome sample :-0.98 and-1.58, result shows No. 21 karyomit(e)s than No. 11 many copies of karyomit(e) (in theory, it is 0.5 o'clock that △ CT value differs, its copy differs 1 copy), visible, conform to the result of chromosome karyotype analysis, the 21-trisome sample of test is 3 No. 21 karyomit(e)s.
Experimental result shows, two independently detection system all to detect 21-trisome sample to be measured be 21-trisome, consistent with the result of traditional chromosome karyotype analysis judgement, therefore, test kit of the present invention can be used for the detection of clinical samples.
Embodiment 2: by test kit, method, step etc. described in embodiment 1, clinical samples is detected
Adopt test kit to test 35 routine normal specimen and 26 routine 21-trisome samples, the chromosome karyotype analysis method that all sample standard deviations source is hung oneself traditional is determined the DNA sample of caryogram.
By No. 21 karyomit(e)s and No. 6 chromosomal relative quantification △ CT
21-6value (△ CT
21-6=Ct
cY5-Ct
fAM), and No. 21 karyomit(e)s and No. 11 chromosomal relative quantification △ CT
21-11value (△ CT
21-11value=Ct
rOX-Ct
hEX) different distributions interval, can judge this sample is normal specimen or 21-trisome sample, real time fluorescent quantitative detect △ CT value and scope as follows:
No. 21 karyomit(e)s and No. 6 chromosomal △ CT of normal specimen
21-6value scope is 0.23～0.65; No. 21 karyomit(e)s and No. 6 chromosomal △ CT of 21-trisome sample
21-6value scope is-0.32～-0.05, and normal specimen and 21-trisome sample △ CT value between the two, without juxtaposition, judges that in the distribution of interval range sample to be measured is normal specimen or 21-trisome patient sample by △ CT value.
No. 21 karyomit(e)s and No. 11 chromosomal △ CT of normal specimen
21-11value scope is-0.98～-0.70; No. 21 karyomit(e)s and No. 11 chromosomal △ CT of 21-trisome sample
21-11value scope is-1.71～-1.39, and normal specimen and 21-trisome sample △ CT value between the two, without juxtaposition, judges that in the distribution of interval range sample to be measured is normal specimen or 21-trisome patient sample by △ CT value.
(karyotype result is expressed as 46, NN to the clinical normal karyotype sample detecting, and 46 represent normal chromosome number; NN represents normal sex chromosome) and 21-trisome caryogram sample (karyotype result is expressed as 47, NN, the chromosomal number of+21,47 expression, and NN represents normal sex chromosome; + 21 have represented No. 21 karyomit(e)s many) No. 21 karyomit(e)s and No. 6 chromosomal relative quantification △ CT
21-6value, and No. 21 karyomit(e)s and No. 11 chromosomal relative quantification △ CT
21-11the concrete outcome of value is in Table 2.
Table 2: Δ CT value result and the analysis of the multiple real time fluorescence quantifying of normal karyotype DNA and 21-trisome caryogram specimen dna
Experimental result shows, the detection in two sites of single tube all can detect No. 21 chromosomal number effectively, accurately, and detected result is consistent with traditional chromosome karyotype analysis method, and accuracy rate is 100%.Test kit of the present invention can carry out the detection in two independent sites to No. 21 chromosome numbers under same reaction system and reaction conditions simultaneously, and result readability is good, and analysis, testing process are simply, efficiently.
Claims (9)
1. No. 21 chromosome number object test kits of multiple real time fluorescence quantifying PCR technology rapid detection people, comprise augmentation detection reagent, it is characterized in that: described augmentation detection reagent comprises:
(1) simultaneously the increase primer pair of No. 21 special tumor-necrosis factor glycoproteins chr21-1 of karyomit(e) and No. 6 special tumor-necrosis factor glycoproteins chr6-1 of karyomit(e), its base sequence is respectively as shown in SEQ ID NO:1 and SEQ ID NO:2;
(2) the Taqman probe chr21-1-Probe of No. 21 special tumor-necrosis factor glycoproteins chr21-1 of karyomit(e) of specific detection, its base sequence is as shown in SEQ ID NO:3;
(3) the Taqman probe chr6-1-Probe of No. 6 special tumor-necrosis factor glycoproteins chr6-1 of karyomit(e) of specific detection, its base sequence is as shown in SEQ ID NO:4;
(4) simultaneously the increase primer pair of No. 21 special tumor-necrosis factor glycoproteins chr21-2 of karyomit(e) and No. 11 special tumor-necrosis factor glycoproteins chr11-1 of karyomit(e), its base sequence is respectively as shown in SEQ ID NO:5 and SEQ ID NO:6;
(5) the Taqman probe chr21-2-Probe of No. 21 special tumor-necrosis factor glycoproteins chr21-2 of karyomit(e) of specific detection, its base sequence is as shown in SEQ ID NO:7;
(6) the Taqman probe chr11-1-Probe of No. 11 special tumor-necrosis factor glycoproteins chr11-1 of karyomit(e) of specific detection, its base sequence is as shown in SEQ ID NO:8.
2. test kit according to claim 1, it is characterized in that: the special tumor-necrosis factor glycoproteins chr21-1 of described No. 21 karyomit(e)s and No. 6 special tumor-necrosis factor glycoproteins chr6-1 of karyomit(e) are two similar sequences, wherein, the base sequence of No. 21 special tumor-necrosis factor glycoproteins chr21-1 of karyomit(e) is as shown in SEQ ID NO:9, and the base sequence of No. 6 special tumor-necrosis factor glycoproteins chr6-1 of karyomit(e) is as shown in SEQ ID NO:10.
3. test kit according to claim 1, is characterized in that: 5 ' the end mark CY5 of described Taqman probe chr21-1-Probe, 3 ' end mark BHQ1.
4. test kit according to claim 1, is characterized in that: 5 ' the end flag F AM of described Taqman probe chr6-1-Probe, 3 ' end mark BHQ1.
5. test kit according to claim 1, is characterized in that: with the relative quantification index △ CT of Taqman probe chr21-1-Probe and Taqman probe chr6-1-Probe
21-6no. 21 chromosomal numbers of value judgement, described △ CT
21-6=Ct
cY5-Ct
fAM, △ CT
21-6, represent that the sample that detects is No. 21 karyomit(e) normal specimen at=0.23～0.65 o'clock; As △ CT
21-6, represent that the sample that detects is 21-trisome caryogram sample at=-0.32～-0.05 o'clock.
6. test kit according to claim 1, it is characterized in that: the special tumor-necrosis factor glycoproteins chr21-2 of described No. 21 karyomit(e)s and No. 11 special tumor-necrosis factor glycoproteins chr11-1 of karyomit(e) are two similar sequences, wherein, the base sequence of No. 21 special tumor-necrosis factor glycoproteins chr21-2 of karyomit(e) is as shown in SEQ ID NO:11, and the base sequence of No. 11 special tumor-necrosis factor glycoproteins chr11-1 of karyomit(e) is as shown in SEQ ID NO:12.
7. test kit according to claim 1, is characterized in that: 5 ' the end mark ROX of described Taqman probe chr21-2-Probe, 3 ' end mark BHQ1.
8. test kit according to claim 1, is characterized in that: 5 ' the end mark HEX of described Taqman probe chr11-1-Probe, 3 ' end mark BHQ1.
9. test kit according to claim 1, is characterized in that: with the relative quantification index △ CT of Taqman probe chr21-2-Probe and Taqman probe chr11-1-Probe
21-11no. 21 chromosomal numbers of value judgement, described △ CT
21-11=Ct
rOX-Ct
hEX, as △ CT
21-11, represent that the sample that detects is No. 21 karyomit(e) normal specimen at=-0.98～-0.70 o'clock; As △ CT
21-11, represent that the sample that detects is 21-trisome caryogram sample at=-1.71～-1.39 o'clock.
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| CN110923306A (en) * | 2019-12-11 | 2020-03-27 | 江苏圣极基因科技有限公司 | Primer, probe, kit and method for noninvasive prenatal detection of fetal 21-trisomy syndrome based on digital PCR (polymerase chain reaction) |
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