CN103074416B

CN103074416B - A kind of method for detecting five numerical abnormalities of chromosomes

Info

Publication number: CN103074416B
Application number: CN201210204204.8A
Authority: CN
Inventors: 吴勇; 徐冰
Original assignee: NINGBO HEALTH GENE TECHNOLOGIES Co Ltd
Current assignee: NINGBO HEALTH GENE TECHNOLOGIES CO., LTD.
Priority date: 2012-06-20
Filing date: 2012-06-20
Publication date: 2017-12-08
Anticipated expiration: 2032-06-20
Also published as: CN103074416A

Abstract

The present invention relates to a kind of method for detecting five numerical abnormalities of chromosomes.For detecting five chromosome number purpose aneuploidy including 21,13,18, X and Y chromosome simultaneously, including：(1) seven genes, are chosen respectively on five chromosome, and 38 pairs of primers are devised for 38 selected gene；(2), combined using above primer and carry out multiplex PCR and electrocapillary phoresis；(3), electrophoresis result is analyzed using software.

Description

A kind of method for detecting five numerical abnormalities of chromosomes

Technical field

The present invention relates to a kind of detection method, and specifically, the present invention relates to one kind to detect five numerical abnormalities of chromosomes Method.

Background technology

Trisomy 21 syndrome be also known as Down syndrome or mongolism category autosome distortion, be in children's chromosomal disorder most Common one kind, incidence about 1/ (600~800) in baby living, maternal age is bigger, and this sick incidence of disease is higher.About 60% suffers from Youngster is miscarriage of dying young in fetus early stage.Trisomy 21 syndrome can cause to include learning disorder, disturbance of intelligence and deformity etc..In addition, 13rd, 18, the aneuploidy of X and Y chromosome causes pa pottery Cotard, Edward's syndrome, Ke Laien Fils spy comprehensive respectively Simulator sickness and Turner's syndrome, hereditary disease caused by these non-multiple chromosomes can make human body growth error to some extent and go out Existing other lesions.Once infant is born, huge pain will be brought to family, also bring white elephant to society.

At present, the means for being clinically used to detect Down syndrome mainly extract pregnancy serum, detect in maternal serum Pregnancy-associated plasma protein (PAPP-A), free hCGB subunits (early stage two) or A type fetoprotein (AFP) and fine hair rush property The index of glandular hormone (HCG) and FE3 (uE3) (mid-term three), when with reference to pregnant woman's expected date of childbirth, body weight, age and taking a blood sample Pregnant week, calculate the danger coefficient of " Tang Shi ", can so find 80% Tang Shi and the body of part 18 3, but can not be true Whether ill examine fetus.Tang Shi examinations index should carry out amniocentesis inspection or fine hair inspection, Ran Houjin beyond normal pregnant woman Row fetal cell chromosome karyotype analysis.And the shortcomings that chromosome karyotype analysis is that time-consuming (more than one week), technical requirements phase To higher, charge is also higher.

Molecule diagnoses, also known as gene diagnosis, is a kind of using high-tech means detection DNA and RNA, and then disclose gene Structure and the technology of expression.The technology is quickly grown at present, and application field is quite varied.Our method application multiplex PCR and The method of electrocapillary phoresis, there is the technical advantages such as sensitive, quantitative, quick, high flux.Our invention can provide for clinical obstetricses A kind of superior molecular diagnosis method.

The content of the invention

The invention provides a kind of 38 human gene sites of synchronous amplification, (site of male 38, women 30 One site) multiplex PCR scheme, and can synchronously detect 13,18,21, the method for five numerical abnormalities of chromosomes of X and Y and the party The purposes of method.

According to the first aspect of the invention, a kind of method for detecting five numerical abnormalities of chromosomes, for detecting bag simultaneously Include 21,13,18, five chromosome number purpose aneuploidy including X and Y chromosome, including：(1), in five chromosome It is upper to choose seven genes respectively, devise 38 pairs of primers (table 1) for 38 selected gene；(2), utilize with Upper primer combination carries out multiplex PCR and electrocapillary phoresis；(3), electrophoresis result is analyzed using software.

Preferably, the chromosome number purpose aneuploidy refer to different from normal human chromosome number purpose increase or Person lacks.

Preferably, step (1) also includes choosing three gene conducts on other chromosomes outside this five chromosomes Compare (CK).

Preferably, the pcr template in the step (2) is human genome DNA.

Preferably, several genes of same chromosome are tried one's best positioned at the different zones of the chromosome；Design clip size Between 100bp and 400bp, the difference of adjacent two clip size is generally 3~10bp.

According to another aspect of the present invention, a kind of purposes using preceding method, described method are applied to using invasive The pre-natal diagnosis for the clinical sample for needing to detect is obtained with noninvasive two kinds of means.

Preferably, described invasive means obtain amniotic fluid or embryo villi theca cell by amniocentesis.

Preferably, described non-invasive means screen maternal blood by flow cytometer and obtain fetal cell.

By adopting the above-described technical solution, the present invention has following beneficial effect：

The present inventor passes through in-depth study, and development ＆ production combines PCR and the " human chromosome of electrocapillary phoresis advantage first Body aneuploidy multiple molecular detection kit " (hereinafter referred to as " kit ").The method that the kit uses molecule diagnosis, no Only sensitivity and accuracy rate are high, take short (8 hours), and can detect 5 kinds of different congenital genetic disorders simultaneously, and expense is also relative It is cheap.This is exactly the diagnostic means of current urgent clinical needs, has good market application foreground.

Table 1, human chromosomal aneuploidy analyze each site and primer sequence table

Brief description of the drawings

The analysis of cases of Fig. 1 chromosomal aneuploidies.

Chromosome number purpose quantitative analysis in Fig. 2 normal specimens.

Chromosome number purpose quantitative analysis in Fig. 3 normal specimens and case sample.

Embodiment

Embodiments of the invention are as follows：

First, DNA is extracted

Sample DNA extraction uses Chelex-100 (BioRad) method.

1.1 amniotic fluid and its cell culture

1ml amniotic fluid or 200 μ l cell cultures are taken, into 1.5ml centrifuge tubes, add the μ l pure water of people 500, acutely vibration, room Temperature is lower to place 15 minutes.13,000rpm centrifugations 3 minutes, remove supernatant, collect precipitation.The Chelex- of 200 μ l 5% are added in precipitation 100 solution (5%Chelex-100 is suspension, using shake well is preceding wanted, makes Chelex-100 particles suspend), in oscillator On vibrate repeatedly, be put into 56 DEG C be incubated more than 30 minutes.Vibrated after taking-up, 100 DEG C are incubated 8 minutes, after vibration, 13,000rpm Centrifugation 3 minutes, supernatant expands for PCR, or puts 4 DEG C and save backup.

1.2 fetus fine hair membrane tissues

The tissue block of soya bean size is taken, is put into 1.5ml centrifuge tubes, pure water is added and washes once, then add 200 μ l 5% Chelex-100 and 5 μ l 5mg/ml Proteinase K, be put into 56 DEG C be incubated more than 3 hours.Vibration is taken out, 100 DEG C are incubated 8 points Clock, vibration, 13000rpm are centrifuged 3 minutes, and supernatant expands for PCR or puts 4 DEG C and saves backup.

2nd, kit forms (PCR reagent, 100 secondary responses)

The composition of this kit includes：

H without DNA enzymatic/RNase₂O, 1000 μ l

10 × PCR buffer solutions, 220 μ l

5 × PCR primer mixture, 440 μ l

MgCl₂, 440 μ l, 25mM

Taq archaeal dna polymerases, 55 μ l

Solution X, 220 μ l

DNA is compareed, 100 μ l, 20ng/ μ l

3rd, experimentation

3.1PCR reaction：

3.1.1 reagent and sample (table 2) are added in 96 hole sample panels (PCR plate) in proportion

Table 2, PCR reaction systems

PCR reaction reagents	Amount/reaction
		10 × PCR buffer solutions	2μl
25mM MgCl₂	4μl
		5 × PCR primer mixture	4μl
HotStart Taq archaeal dna polymerases	0.7μl
		Solution X	2μl
DNA profiling	50~100ng
		H without DNA enzymatic/RNase₂O	Add to 20 μ l
Cumulative volume	20μl

3.1.2 thermal cycle reaction (table 3) is carried out by temperature below

Table 3, PCR programs

Step	Temperature	Time
			1	94℃	3min
2	94℃	30s
			3	60℃	30s
4	70℃	1min
			5	N/A	Repeat 2-4 steps 34 time (totally 35 times)
6	70℃	1min
			7	4℃	Continue：Until collecting PCR primer

3.2 electrocapillary phoresis (by taking GeXP genetic analyzers as an example)

3.2.1 GeXP samples (table 4) are prepared

Table 4, GeXP loading systems

GeXP samples	Amount/hole
		The upper foreign buffer solutions of GeXP	38.75μl
DNA fragmentation size criteria -400	0.25μl

PCR primer	1μl
		Cumulative volume	40μl
Mineral oil	1 drop

3.2.2 separating liquid is prepared：About 250 μ l separating liquids are added on 96 hole separating liquid plates in an appropriate number of hole.

3.2.3GenomeLab GeXP genetic analyzers electrocapillary phoresis separation sample

(see GenomeLab GeXP genetic analyzers specification)

4th, data analysis

4.1 data export

" File " → " file of Export Fragments/Genotypes As " → preservation CSV forms.

4.2 data analysis

This kit includes 38 pairs of primers, and its amplified production size is between 151 and 372bp.Wherein 35 pairs of primers are put down 13,18,21, X and Y chromosome are distributed in, every chromosome seven is to primer.Other 3 pairs of primer amplified fragments are respectively 150bp (Chr.11, RRM1), 239bp (Chr.11/16, β-Globin) and 282bp (Chr.10, RNaseP), as control.

4.2.1 the comparison in sample

By taking XY samples as an example, 35 genes that we expand 35 pairs of primers are divided into seven groups, and each group includes 13 respectively, 18,21, X and Y chromosome on a gene.When analyzing the relative signal of some gene, we are calculated corresponding to the gene The peak area (Peak Area) at GeXP peaks accounts for the summation of five gene peak areas of group.Seven gene peaks on each chromosome Area accounting value can average, and calculate variation value (Stdev).The advantages of this computational methods is farthest to subtract The influence that small pieces piecewise analysis baseline (Baseline) is brought；Its shortcoming is such as trisomy 21 when running into three body samples, and No. 21 are dyed Gene peak area accounting increase on body, while gene peak area accounting will have and reduce to some extent on other chromosomes, this Kind, which reduces, to be obscured with situation during corresponding chromosome monosomy.Conversely, when running into monomer sample.Such as X monomers, X dyeing Gene peak area accounting on body reduces, while gene peak area accounting will have and increase to some extent on other chromosomes, this Kind increase may be obscured with situation during corresponding chromosome trisomy.This just needs us further to analyze.

4.2.2 the comparison of sample room

The seven gene peak area accounting values on each chromosome relatively drawn in above sample can be with normal control pair The accounting value answered is compared.In addition, when analyzing the relative signal of gene, except by peak area value corresponding to each gene divided by Outside crt gene (RRM1, β-Globin and RNaseP) peak area value, also need each gene peak and normal reference sample Each gene peak is compared.We introduce such computational methods：

X represents unknown DNA sample to be analyzed, X_G1-2, X_G3-4And X_G5-71-2 groups, 3-4 groups and 5-7 groups are represented respectively The peak area of gene, X_RRM1, X_β-GlobinAnd X_RNasePFor the peak area of each crt gene,；Ctrl represents normal comparison DNA sample Product (X sexes corresponding with Ctrl are consistent).

There are seven log values on each chromosome corresponding to seven genes；Log values between coloured differently body can carry out difference Significance analysis is to analyze chromosome number purpose aneuploidy.

Herein, normal control is particularly important.This normal reference sample needs to meet certain requirement, i.e., with dividing Sample is analysed from similar tissue samples, extracts DNA with same method, while enter performing PCR and GeXP analyses.With it is normal right When product are compared in the same old way, the principle of same gender comparison must be followed.

5th, sample analysis

The present inventor using kit a collection of fetus chorionic cells sample is analyzed (table 5) analysis result with MLPA results are coincide.The kit is working properly, shows as：Peak number meets design；Peak type is normal (Fig. 1)；Chromosome (13,18 With No. 21 chromosomes) on each gene primer it is working properly, quantitative purpose (Fig. 2) can be reached；Sex recall rate reaches 100%；Each chromosomal aneuploidy case can clearly detect (Fig. 3).

The analysis of cases of Fig. 1 chromosomal aneuploidies.DNA sample (50-100ng) is illustrated after multiplex PCR, GeXP capillarys The result of electrophoretic analysis is successively from top to bottom：Normal female DNA sample；X chromosome monomer；No. 13 chromosome trisomies；No. 18 Chromosome trisomy and No. 21 chromosome trisomies.Chromosomal aneuploidy can be detected by quantitative analysis.Such as pass through observation The relative altitude of each chromosome specific peak can clearly illustrate the difference and chromosomal aneuploidy or sex pair of peak height in black surround Should；Chromosomal aneuploidy can be more accurately shown additionally by the relative peak area for comparing and calculating each chromosome specific peak (see " embodiment 4, data analysis ")

Chromosome number purpose quantitative analysis in Fig. 2 normal specimens.Each base on each chromosome (13,18 and No. 21 chromosome) Because the Relative copy number of (primer pair) represents quantitative result, wherein 50% sample is located in cylindricality box；95% sample position In in error line.(n=205, SD=0.07~0.24)

Chromosome number purpose quantitative analysis in Fig. 3 normal specimens and case sample.Cylindricality box represents each in normal specimens The Relative copy number of each gene (primer pair) on chromosome (13,18,21, X and Y chromosome).50% sample is located at cylindricality In box；95% sample is located in error line.The statistics of sex chromosome comes from two gender groups (n=102 and 77, SD= 0.05~0.09).Horizontal line represents in case sample each gene (primer on each chromosome (13,18,21, X and Y chromosome) It is right) Relative copy number average value.

Table 5, chromosomal aneuploidy sample analysis result.

Chromosomal aneuploidy	Statistics numbers
		Normally	179
X monomers	22
		13 3 bodies	8
18 3 bodies	4
		Trisomy 21	12
XXY	1
		XYY	1
21 monomers	4
		It is total	231

Obviously, the above embodiment of the present invention is only intended to clearly illustrate example of the present invention, and is not pair The restriction of embodiments of the present invention.For those of ordinary skill in the field, may be used also on the basis of the above description To make other various forms of changes and variation.Here all embodiments can not be exhaustive.It is every to belong to this hair Row of the obvious changes or variations that bright technical scheme is amplified out still in protection scope of the present invention.

Claims

A kind of 1. multiple gene detection kit for detecting five numerical abnormalities of chromosomes, it is characterised in that：Be included in 21,13, 18th, on five chromosomes including X and Y chromosome 38 genes forward and reverse primer, it is specific as shown in table 1,

Table 1, human chromosomal aneuploidy analyze each site and primer sequence table