CN101092647A - Nucleic acid film tape and kit for diagnosing alpha mediterranean anemia - Google Patents
Nucleic acid film tape and kit for diagnosing alpha mediterranean anemia Download PDFInfo
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Abstract
This invention relates to test kit and test paper with oligonucleotide probes for diagnosing alpha-thalassemia. The test paper comprises a base, and specific oligonucleotide probes immobilized on the base. The oligonucleotide probes comprise: specific oligonucleotide probes for detecting mutation of alpha-globin gene, specific oligonucleotide probes for detecting deletion of alpha-globin gene, and base sequence complementary with the above sequences. The test kit comprises specific primer for amplifying alpha-globin gene or transcript, and specific oligonucleotide probes for detecting mutation/deletion of alpha-globin gene. The test kit and the test paper have such advantages as low cost, rapid detection, and stable results. The test kit and the test paper can be used in detection of mutation/deletion of alpha-globin gene, and prenatal diagnosis of severe thalassemia (alphaTalpha) caused by point mutation, and do not have error diagnosis.
Description
Technical field
The present invention relates to thalassemic diagnostic reagent, relate in particular to a kind of diagnosing alpha-thalassemic nucleic acid film bar and test kit.
Background technology
Thalassemia (Thalassemia, poor hereinafter to be referred as ground) is because patient certain or some globin chain synthesis rates reduce, and causes some peptide chains to lack, and other peptide chains are too much relatively, the imbalance of quantity of peptide chains resulted occurs, the hemolytic anemia that causes.Ground poorly mainly contains two types, is respectively synthetic the subtracting of α-Zhu Danbai chain and lacks the α-Di Zhonghaipinxue and synthetic the subtracting of beta-globin that cause and lack the β-thalassemia that causes.
α-ground is poor to be one of modal single gene inheritance disease in the world.This disease is occurred frequently in the wide geographic area from Italy of Mediterranean Sea bank, Greece, Malta, Cyprus to Southeast Asian countries.Provinces and cities such as the southern Guangxi of China, Guangdong, Hainan, Hong Kong, Taiwan and surrounding area thereof also are this sick hotspots, 29 provinces 900,000 human hemoglobins in the whole nation are sick, and the survey showed that, the poor genetic flaw carrying rate in α-ground is 2.64% among the crowd, and wherein Guangxi, Guangdong, Jiangxi, Sichuan sickness rate are respectively up to 14.95%, 4.11%, 2.60%, 1.92%.
α-the ground of China is poor to be mainly on genotype-α
3.7,-α
4.2With--
SEAThis three kinds of absence types and α
CSα, α
QSα, α
WSThese three kinds of mutants of α, also having three kinds of more rare mutation types in addition is α
30α, α
31α, α
59α according to the various combination of these several genotype in individuality, can show as silent oscillation, standard type, Hemoglobin H disease (HbH disease) and these four kinds of phenotypes of Hb Bart ' s fetus edema syndromes.Relation between them sees the following form:
Table 1: poor clinical manifestation in α-ground and genotypic relation
| The clinical manifestation type | Main possible genotype | Clinical manifestation |
| Silent oscillation | -α/α α, α Tα/α α | Clinical asymptomatic |
| Standard type | --/α α ,-α/-α, α Tα/-α, α Tα/α Tα | Asymptomatic clinically, but hematological examination can present the reduction of mean corpuscular volume (MCV) and mean corpuscular hemoglobin |
| The HbH disease | The α of--/-,--/α Tα, | Chronic hemolytic anemia, most with hepatosplenomegaly and slight jaundice, the minority severe patient also has bone to change and special anemic face. |
| Hb Bar t ' s fetus edema syndromes | --/-- | The fetus hydrosarca, hepatosplenomegaly, four limbs are short and small, and belly swells because of ascites is arranged.More than gestation death or premature labor during 30～40 weeks, and the premature infant is promptly dead in half an hour in postpartum. |
Annotate: go up table-α and represent-α
3.7Or-α
4.2,--expression--
SEA, α
Tα represents α
CSα, α
QSα, α
WSα, α
30α, α
31α or α
59A kind of among the α.
Though above silent oscillation and standard type be non-evident sympton clinically, after man and wife's marriage of carrying the poor silent oscillation in α-ground and standard type (--/α α) gene respectively, 1/4 the offspring that may produce Hemoglobin H disease is just arranged; And two all be Mr. and Mrs' combination of standard type (--/α α), its fetus just have 1/4 may be Hb Bart ' s fetus edema syndromes.The poor serious public health problem of China that become in α-ground.
And except preventing, also do not have other truly feasible method to effect a radical cure to Hemoglobin H disease at present, be exactly the method for unique feasible poor prenatal and postnatal care in α-ground so the method for pre-marital/antenatal diagnosis screens out the Hemoglobin H disease Disease-causing gene.
The poor screening method in traditional ground mainly contains: routine blood test mensuration, erythrocyte fragility detection, hemoglobin electrophoresis etc., the common deficiency of these methods is to carry out poor preliminaryly examination to the patient, can't make a definite diagnosis patient's genotype, and fail to pinpoint a disease in diagnosis easily lightly poor, cause the severe of future generation poor child's in ground birth.
In the zone of carrying out poor pre-marital, the antenatal detection in α-ground, still have the heavy poor youngster's in α-ground birth at present, its major cause is in Chinese population, cause the poor genetic flaw in α-ground except three kinds of common α-Zhu Danbai gene large fragment deletions, also having quite a few is the α-Zhu Danbai transgenation, and the common α-Zhu Danbai transgenation of Chinese population is α
CS, α
QSAnd α
WS, also having small part in addition is α
30α, α
31α, α
59These three kinds of mutation types of α.The poor detection in present existing α-ground is most of at three kinds of absence types, and can not suddenly change by check point, causes and fails to pinpoint a disease in diagnosis.Like this, in antenatal diagnosis, just might be to contain the poor heavyly of point mutation--/α
Tα (represents α
Tα among the α
CSα, α
QSα, α
WSα, α
30α, α
31α or α
59α's is a kind of) the fetus mistaken diagnosis becomes lightly poor--/α α, thus cause poor youngster's birth heavyly, cause huge family burden and social cost.
Application number is that 02117287.0 Chinese patent application discloses and is used to diagnose thalassemic DNA chip and preparation method thereof, a kind of poor DNA chip of clinical diagnosis α-type and β-type ground that is used for is provided, chip is by a substrate and be fixed in suprabasil probe and form, at the sequence of hitherto known α-Zhu Danbai genetically deficient and beta-globin gene mutation, according to the oligonucleotide probe of these sudden changes of diagnosis of the principle design of dna sequence dna specific recognition.The weak point of this method is to detect α genetically deficient, and can not detect the α transgenation, easily causes and fails to pinpoint a disease in diagnosis, and causes the birth of defect baby.And if detect α transgenation (with reference to detecting the poor principle in β-ground on this patent) with this method principle, mutant probe and normal probe are in the same position of slide, just distinguish by the color difference, the sample of heterozygous mutation is exactly a mixture colors like this, bad judgement; The unicity of adding this method signal is relatively poor, and often with spurious response, being difficult to sometimes distinguish when mixed signal occurring is with spurious response or heterozygous mutation.And detect needs with this method and buy expensive laser scanner, the general hospital laboratory of China does not also have such economic condition.This has significant limitation with regard to causing this method product in the popularization of China.
Application number is reagent and the application thereof that 02103839.2 Chinese patent application discloses a kind of diagnosing alpha-thalassemia, utilizes this reagent can detect the disappearance and the sudden change of the α-Zhu Danbai gene of multiple α-Di Zhonghaipinxue disease.Its technical scheme is disappearance or sudden change for utilizing round pcr amplification α-Zhu Danbai gene through electrophoretic analysis α-Zhu Danbai gene, and this method causes EB to pollute easily.
Up to now, not having still that publication or bibliographical information have is the nucleic acid hybridization film bar that substrate and mutator gene are positioned at oligonucleotide probe middle part with the nylon membrane at α-Di Zhonghaipinxue disease.
Summary of the invention
The object of the present invention is to provide a kind of diagnosing alpha-thalassemic nucleic acid film bar.
Another object of the present invention is to provide a kind of diagnosing alpha-thalassemic test kit
The 3rd purpose of the present invention is to provide a kind of absence type α-Di Zhonghaipinxue and mutant ' alpha '-thalassemic method of detecting simultaneously.
For achieving the above object, the present invention adopts following technical scheme:
A kind of diagnosing alpha-thalassemic nucleic acid film bar comprises a substrate and is fixed in described suprabasil specific oligonucleotide probe, and specific oligonucleotide probe comprises:
1) be used to detect the specific oligonucleotide probe of α-Zhu Danbai transgenation, its base sequence is shown in SEQ ID NO:1-6;
2) be used to detect the specific oligonucleotide probe of α-Zhu Danbai genetically deficient, its base sequence is shown in SEQ ID NO:12-14;
Above-mentioned all sequences can be replaced by its complementary base sequences thereof.
Described specific oligonucleotide probe also comprises:
1) the pairing normal control specific oligonucleotide probe of α-Zhu Danbai mutator gene, its base sequence is shown in SEQ ID NO:7-11;
2) the pairing normal control specific oligonucleotide probe of α-Zhu Danbai missing gene, its base sequence is shown in SEQ ID NO:15;
Above-mentioned all sequences can be replaced by its complementary base sequences thereof.
A kind of diagnosing alpha-thalassemic test kit, described test kit comprises:
1) primer of specific amplification α-Zhu Danbai gene or transcript, its base sequence is shown in SEQID NO:16-22;
2) be used to detect the specific oligonucleotide probe of α-Zhu Danbai transgenation, its base sequence is shown in SEQ ID NO:1-6;
3) be used to detect the specific oligonucleotide probe of α-Zhu Danbai genetically deficient, its base sequence is shown in SEQ ID NO:12-14;
Above-mentioned all sequences can be replaced by its complementary base sequences thereof.
Described test kit also comprises:
1) the pairing normal control specific oligonucleotide probe of α-Zhu Danbai mutator gene, its base sequence is shown in SEQ ID NO:7-11;
2) the pairing normal control specific oligonucleotide probe of α-Zhu Danbai missing gene, its base sequence is shown in SEQ ID NO:15;
Above-mentioned all sequences can be replaced by its complementary base sequences thereof.
Described oligonucleotide probe is fixed in the substrate.
Described oligonucleotide probe is fixed on the nylon membrane, can also be fixed in other substrate, for example nitrocellulose filter, polypropylene film, sheet glass, silica gel wafer, micro magnetic bead etc.
Described primer mark vitamin H can also carry out radioactivity, fluorescence mode marks such as (as CY5, CY3, TAMRA).
A kind of absence type α-Di Zhonghaipinxue and mutant ' alpha '-thalassemic method of detecting simultaneously may further comprise the steps:
1) blood sampling extracts the human gene group DNA;
2) be template with step 1) gained DNA, utilize base sequence such as SEQ ID NO:
Primer shown in the 16-22 carries out pcr amplification;
3) above-mentioned PCR product be fixed on the suprabasil specific oligonucleotide probe hybridization that is used to detect the specific oligonucleotide probe of α-Zhu Danbai transgenation and is used to detect α-Zhu Danbai genetically deficient;
4) wash film and colour developing.
Described pcr amplification system is: 10 * buffer, 2.5 μ L
Primer A1 5pmol
Primer A2 5pmol
Primer A3 5pmol
Primer A4 5pmol
Primer A5 5pmol
Primer A6 5pmol
Primer A7 5pmol
Template 4μL(100-200ng)
dNTPs 5nmol(each)
TaqE 2.5U
Add sterilized water to 25 μ L.
Described pcr amplification condition is:
Described hybridization temperature is 44-50 ℃.
Described hybridization temperature the best is 46-48 ℃.
The present invention utilizes primer and the α-Zhu Danbai genetically deficient specific oligonucleotide probe, α-Zhu Danbai transgenation specific oligonucleotide probe of making nucleic acid molecular hybridization technology by special specific amplification α-Zhu Danbai gene of design or transcript.The quadruple pcr amplification product can be with the hybridization of α-Zhu Danbai genetically deficient specific oligonucleotide probe, α-Zhu Danbai transgenation specific oligonucleotide probe hybridization again.Above-mentioned probe stationary can be to 3 kinds of α-Zhu Danbai absence type gene (α on same film bar
3.7,-α
4.2,--
SEA) detect, and detect 6 kinds of α-Zhu Danbai mutated genes (α simultaneously
CSα, α
QSα, α
WSα, α
30α, α
31α or α
59α), be about to clinical multinomial detection and merge into 1 detection, not only reduced patient's economical load, and provide more comprehensive reference information for clinical diagnosis.In antenatal diagnosis, avoided to contain the poor heavyly of point mutation--/α
Tα (represents α
Tα among the α
CSα, α
QSα, α
WSα, α
30α, α
31α or α
59α's is a kind of) the fetus mistaken diagnosis becomes lightly poor--the possibility of/α α, thus avoid poor youngster's birth heavyly, alleviate huge family burden and social cost.
Description of drawings
Fig. 1 blood sample A detected result figure.
Fig. 2 blood sample B detected result figure.
Fig. 3 blood sample C detected result figure.
Fig. 4 blood sample D detected result figure.
Fig. 5 blood sample E detected result figure.
Fig. 6 blood sample E detected result figure.
Fig. 7 blood sample E detected result figure.
Fig. 8 blood sample E detected result figure.
The drawing reference numeral explanation:
1, CSN: the base of α 2-globin the 142nd amino acids correspondence is normal;
2, QSN: the base of α 2-globin the 125th amino acids correspondence is normal;
3, WSN: the base of α 2-globin the 129th amino acids correspondence is normal;
4,59N: the base of α 2-globin the 59th amino acids correspondence is normal;
5,30/31N: the base of the 30th of α 2-globin and the 31st amino acids correspondence is normal;
6, α α: the α-Zhu Danbai gene does not have above-mentioned three kinds of disappearances;
7 ,-α
3.7: α-Zhu Danbai gene-α
3.7The type disappearance;
8 ,-α
4.2: α-Zhu Danbai gene-α
4.2The type disappearance;
9, CSM: the base mutation of α 2-globin the 142nd amino acids correspondence;
10, QSM: the base mutation of α 2-globin the 125th amino acids correspondence;
11, WSM: the base mutation of α 2-globin the 129th amino acids correspondence;
12,59M: the base mutation of α 2-globin the 59th amino acids correspondence;
13,30M: the base mutation of α 2-globin the 30th amino acids correspondence;
14,31M: the base mutation of α 2-globin the 31st amino acids correspondence;
15 ,-
SEA: the α-Zhu Danbai gene--
SEAThe type disappearance.
Embodiment
1, the design of probe
For a α-Zhu Danbai gene base mutation detection site, design two groups of probes, i.e. one group and wild-type base sequence coupling, another group and mutant base sequence coupling.11 sudden changes in 6 mutational sites and the principle of design of normal probe: in the sequence of one section about 14-25 base of both sides, mutational site intercepting, the mutational site roughly is positioned at probe middle (generally one of them end also has 4 more than the base).The principle of design of 4 probes of disappearance and normal control: contrast 4 kinds of pcr amplification sequences, select one section sequence that is different from (also not being complementary to) other 3 PCR products as probe in amplification PCR products, this probe is generally 14-25 base.
In addition, the probe design on same film bar has also been considered following two problems: 1) the no serious secondary structure of probe sequence itself reduces self palindrome and self pairing structure as far as possible.2) the probe hybridization temperature unanimity on same film bar.The consistence of hybridization temperature does not also have the method can be by calculating at present, and this need be by testing designed probe, and on the basis of test conditioning redesign probe, the probe that can on a film bar, use with this final repeatedly acquisition.
Take into full account the homogeneity of polymorphism, secondary structure and hybridization temperature, screen from specificity and two aspects of sensitivity, 3 specific oligonucleotide probes that detect α-Zhu Danbai genetically deficient among the present invention have been determined at last, shown in SEQ ID NO:12-14; Article 1, the pairing normal control specific oligonucleotide probe of α-Zhu Danbai missing gene, its base sequence is shown in SEQ ID NO:15; Be used to detect totally 6 of the specific oligonucleotide probes of α-Zhu Danbai transgenation, its base sequence such as SEQ ID NO:1-6; Totally 5 of the pairing normal control specific oligonucleotide probes of α-Zhu Danbai mutator gene, its base sequence such as SEQ IDNO:7-11.
The dna sequence dna of table 1 probe
| Sequence numbering | The probe title | Base length (bp) | Sequence |
| SEQ ID NO：1 | CSM | 19 | 5’TACCGTCAAGCTGGAGCCT 3’ |
| SEQ ID NO：2 | QSM | 17 | 5’CCTCCCCGGACAAGTTC 3’ |
| SEQ ID NO：3 | WSM | 17 | 5’CTGCGGTGCAGGCCTCC 3’ |
| SEQ ID NO：4 | 30M | 18 | 5’GGCCCTGAGGTGAGGCTC 3’ |
| SEQ ID NO：5 | 31M | 18 | 5’GCCCTGGAGAAGTGAGGC 3’ |
| SEQ ID NO：6 | 59M | 19 | 5’GGGCCACGACAAGAAGGTG 3’ |
| SEQ ID NO：7 | CSN | 19 | 5’TACCGTTAAGCTGGAGCCT 3’ |
| SEQ ID NO：8 | QSN | 17 | 5’CCTCCCTGGACAAGTTC 3’ |
| SEQ ID NO：9 | WSN | 17 | 5’CTGCGGTGCACGCCTCC 3’ |
| SEQ ID NO：10 | 30/31N | 18 | 5’GCCCTGGAGAGGTGAGGC 3’ |
| SEQ ID NO：11 | 59N | 19 | 5’GGGCCACGGCAAGAAGGTG 3’ |
| SEQ ID NO：12 | -- SEA | 17 | 5’TCGGTCGTCCCCACTGT 3’ |
| SEQ ID NO：13 | -α 3.7 | 16 | 5’GACCAACGTCAAGGCC 3’ |
| SEQ ID NO：14 | -α 4.2 | 18 | 5’TGCCTGGGTTCTCTCTGT 3’ |
| SEQ ID NO：15 | αα | 16 | 5’ACTGCCTGCTGGTGAC 3’ |
2, the making of nucleic acid film bar
Finish after oligonucleotide probe synthetic, oligonucleotide probe is fixed on the nylon membrane.Earlier nylon membrane is cut into certain size, and the probe title of printed grid and corresponding grid thereon, activated liquid pre-treatment is after 30 minutes, the activated surface carboxyl; Simultaneously, will be diluted to suitable concentration with probe dilution liquid at the probe that 5 ' or 3 ' end has an amino labeled after, with spot sample device each probe points is added on the corresponding grid.The carboxyl of the amino of probe end and film surface active reacts and generates stable covalent linkage, thus with probe stationary on nylon membrane.At last, handle nylon membrane with the liquid of blockading, seal the not surperficial carboxyl of bonding probes, nucleic acid hybridization film bar completes.
The array order of table 2 nucleic acid hybridization film bar point sample
| CSN | QSN | WSN | 59N | 30/31N | αα | -α 3.7 | -α 4.2 |
| CSM | QSM | WSM | 59M | 30M | 31M | -- SEA |
Annotate: 30M, 31M are normal control with 30/31N simultaneously,--
SEA,-α
3.7,-α
4.2Be normal control with α α simultaneously.
According to α-Di Zhonghaipinxue disease gene order data, with 4 to (totally 7, have one be two pairs shared) specific, have different lengths and Tm value and contain biotin labeled primer amplification α-Zhu Danbai gene fragment at 5 ' end.The nucleotide sequence of these 7 primers is seen SEQ ID NO.16-22.The amplified production of SEQ ID NO.16 and SEQ ID NO.22 can with--
SEAWhether probe hybridization is used for test sample and exists--
SEADisappearance; The amplified production of SEQ ID NO.17 and SEQ ID NO.20 can with-α
4.2Whether probe hybridization is used for test sample and exists-α
4.2Disappearance; The amplified production of SEQ ID NO.18 and SEQ ID NO.21 can with-α
3.7Whether probe hybridization is used for test sample and exists-α
3.7Disappearance; The amplified production of SEQ ID NO.18 and SEQ IDNO.19 can with α α probe hybridization, two genomes that are used for test sample whether all lacked (if two genomes all give birth to above-mentioned three kinds of disappearances, then α α probe does not develop the color).Simultaneously; when α α probe develops the color; this PCR product also may with CSN, CSM, QSN, QSM, WSN, WSM, 30/31N, 30M, 31M, 59N, these 11 probe hybridizations of 59M, these 11 probes whether develop the color depends on this sample whether exist in corresponding site the sudden change or the normal gene sequence.
The α-Zhu Danbai gene PCR primed DNA sequence of table 3 different lengths
| Sequence numbering | The primer title | Base length (bp) | Sequence |
| SEQ ID NO：16 | A1 | 18 | 5’TGTTCTCAGTATTGGAGG 3’ |
| SEQ ID NO：17 | A2 | 17 | 5’GAGCATTGGTGGTCATG 3’ |
| SEQ ID NO：18 | A3 | 16 | 5’CCCACCCTCCCCCTCG 3’ |
| SEQ ID NO：19 | A4 | 19 | 5’GGTCTTTGAATAAAGTCTG 3’ |
| SEQ ID NO：20 | A5 | 17 | 5’TGAGTAAGGGCCTGGGG 3’ |
| SEQ ID NO：21 | A6 | 18 | 5’GAGTGCTTTGAGGATGCA 3’ |
| SEQ ID NO：22 | A7 | 19 | 5’CTCACACTCATCACCCAGG 3’ |
Annotate: A1 and A7 amplification gene type be--
SEALength is: 1306bp.
A2 and A5 amplification gene type are-α
4.2Length is: 1645bp.
A3 and A4 amplification gene type are α α; Length is: 1826bp.
A3 and A6 amplification gene type are-α
3.7Length is: 2051bp.
α
CSα, α
QSα, α
WSα, α
30α, α
31α, α
59The gene information in six sites of α is included in the amplified production α α of A3 and A4.
The detection of embodiment 3 sample DNAs to be checked
One, tests with instrument and reagent
1, test instrument
PCR instrument, DNA electrophoresis apparatus, molecular hybridization case, shaking table
2, test reagent
(1)3％H2O2
(2) 20 * SSC (pH7.0): get NaCl 175.3g, Trisodium Citrate 88.2g, it is molten to 1000mL to add H2O, transfers pH to 7.0, autoclaving with pH meter.
(3) 10%SDS (pH7.0): 20g SDS is added H2O 180mL dissolving, transfer pH to 7.0, be settled to 200mL at last with HCl.
(4) A liquid (2 * SSC, 0.1%SDS, pH7.4): get 20 * SSC 100mL, 10%SDS 10mL,
It is molten to 1000mL to add H2O, transfers pH to 7.4.
(5) (pH7.4): get 20 * SSC 25mL, 10%SDS 10mL, it is molten to 1000mL to add H2O for 0.5 * SSC, 0.1%SDS, transfers pH to 7.4 for B liquid.
(6) C liquid (the 0.1M Trisodium Citrate, pH5.4): get Trisodium Citrate 14.7g, be dissolved in 450mL water, transfer pH to 5.0, be settled to 500mL at last with dense HCl.
(7) strepto-affinity element-peroxidase (streptavidin-POD)
(8) (TMB) of tetramethyl benzidine
Two, the detection of sample DNA to be checked
1, the preparation of sample DNA to be checked
Get five parts of blood samples (being designated as A, B, C, D, E, F, G and H respectively), use the Qiagen whole blood to extract whole blood DNA rapid extraction test kit extracting human gene group DNA from anticoagulated whole blood of test kit or Yaneng Biotechnology (Shenzhen) Co., Ltd..
2, the pcr amplification of goal gene
Get eight of PCR reaction solutions, carry out mark on tube wall, and in 5000rpm centrifugal 2 seconds, (it is about 100～200ng) to contain genomic dna, and this moment, reaction totally was 25 μ L then to add the testing sample DNA4 μ L that extracted respectively.
The pcr amplification system is: 10 * buffer, 2.5 μ L
Primer A1 5pmol
Primer A2 5pmol
Primer A3 5pmol
Primer A4 5pmol
Primer A5 5pmol
Primer A6 5pmol
Primer A7 5pmol
Template 4μL(100-200ng)
dNTPs 5nmol(each)
TaqE 2.5U
Add sterilized water to 25 μ L.
Increase by following condition:
The amplification PCR fragment includes the testing gene fragment, and has biotin labeling at PCR product 5 ' end.
3, hybridization
Get the 15mL plastic centrifuge tube, indicate sample number into spectrum to be checked, put into the nucleic acid hybridization film bar (by embodiment 1 preparation) that indicates sample number into spectrum to be checked equally, add A liquid 5-8mL and PCR product, lid is screwed on.Plastics tubing is put into 10 minutes (guaranteeing that the hybridization solution liquid level is positioned under the water-bath liquid level fully) of boiling water heating, take out and tighten lid, put into 48 ℃ of hybridization casees hybridization 1.5～4 hours.Get a 50mL plastics tubing, add 40mL B liquid and in hybridization case or incubator, be preheated to 48 ℃.
4, wash film
Take out the film bar, move in the 50mL pipe that preheating B liquid is housed, in 48 ℃ of jog washings 15 minutes.By aforesaid operations, can remove non-specific hybridization to greatest extent.
5, colour developing
With strepto-affinity element-peroxidase (streptavidin-POD) solution of 1: 2000 of A liquid preparation.Earlier the film bar was soaked 30 minutes with this streptavidin-POD solution room temperature jog, discard streptavidin-POD solution; Wash film twice with A liquid chamber temperature jog again, each 5 minutes; Washed film 1～2 minute with C liquid chamber temperature then, (colour developing liquid needs fresh preparation to cofabrication colour developing liquid.Be sequentially added into 19mL 0.1M Trisodium Citrate, 1mL TMB, 10 μ L 3%H
2O
2, formulated.)。The film bar is soaked in the colour developing liquid, and the lucifuge colour developing was that visible spot manifests in 10～15 minutes.The Streptavidin (streptavidin) that is marked with peroxidase (POD) is held the vitamin H specific combination that has with PCR product 5 ', and issues biochemical color reaction in the existence of hydrogen peroxide and brown substrate TMB (tetramethyl benzidine).Like this, there is the film bar correspondent probe position of hybridization product will manifest blue spot.
The detected result of eight duplicate samples is seen Figure of description.
6, the result judges
Can judge the type of transgenation by the colour developing result of film bar, also can further after reading apparatus is read, carry out Computer Analysis, directly provide results of hybridization, and the result is preserved and statistical study signal and background.
Blood sample A α 2-globin gene does not have sudden change, does not have disappearance as can be seen from Figure 1.
Sudden change has taken place in the base of blood sample B α 2-globin the 142nd amino acids correspondence as can be seen from Figure 2, and genotype is α α/α
CSα.
Sudden change has taken place in the base of blood sample C α 2-globin the 125th amino acids correspondence as can be seen from Figure 3, and the α-Zhu Danbai gene-
SEAType disappearance, genotype be--
SEA/ α
QSα.
Sudden change has taken place in the base of blood sample D α 2-globin the 129th amino acids correspondence as can be seen from Figure 4, and the α-Zhu Danbai gene-
3.7The type disappearance, genotype is-α
3.7/ α
WSα.
Sudden change has taken place in the base of blood sample E α 2-globin the 59th amino acids correspondence as can be seen from Figure 5, and α-Zhu Danbai gene-α
4.2The type disappearance, genotype is-α
4.2/ α
59α.
Sudden change has taken place in the base of blood sample F α 2-globin the 30th amino acids correspondence as can be seen from Figure 6, and genotype is α/α
30α.
Sudden change has taken place in the base of blood sample G α 2-globin the 31st amino acids correspondence as can be seen from Figure 7, and genotype is--
SEA/ α
31α.
As can be seen from Figure 8 blood sample H α-Zhu Danbai gene simultaneously-
SEAType disappearance and-α
4.2Type disappearance, genotype be--
SEA/-α
3.7
Diagnosing alpha of the present invention-thalassemic nucleic acid hybridization film bar and test kit cost of manufacture are low, and its detection speed is fast, detected result stable, can detect the α-Zhu Danbai transgenation when detecting α-Zhu Danbai genetically deficient.
SEQUENCE LISTING
＜110〉Yaneng Biotechnology (Shenzhen) Co., Ltd.
＜120〉diagnosing alpha-thalassemic nucleic acid film bar and test kit
<160>22
<170>PatentIn version 3.3
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taccgtcaag ctggagcct 19
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cctccccgga caagttc 17
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ctgcggtgca ggcctcc 17
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ggccctgagg tgaggctc 18
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gccctggaga agtgaggc 18
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gggccacgac aagaaggtg 19
<210>7
<211>19
<212>DNA
＜213〉artificial sequence
<400>7
taccgttaag ctggagcct 19
<210>8
<211>17
<212>DNA
＜213〉artificial sequence
<400>8
cctccctgga caagttc 17
<210>9
<211>17
<212>DNA
＜213〉artificial sequence
<400>9
ctgcggtgca cgcctcc 17
<210>10
<211>18
<212>DNA
＜213〉artificial sequence
<400>10
gccctggaga ggtgaggc 18
<210>11
<211>19
<212>DNA
＜213〉artificial sequence
<400>11
gggccacggc aagaaggtg 19
<210>12
<211>17
<212>DNA
＜213〉artificial sequence
<400>12
tcggtcgtcc ccactgt 17
<210>13
<211>16
<212>DNA
＜213〉artificial sequence
<400>13
gaccaacgtc aaggcc 16
<210>14
<211>18
<212>DNA
＜213〉artificial sequence
<400> 14
tgcctgggtc ctctctgt 18
<210> 15
<211> 16
<212> DNA
＜213〉artificial sequence
<400> 15
actgcctgct ggtgac 16
<210> 16
<211> 18
<212> DNA
＜213〉artificial sequence
<400> 16
tgttctcagt attggagg 18
<210> 17
<211> 17
<212> DNA
＜213〉artificial sequence
<400> 17
gagcattggt ggtcatg 17
<210> 18
<211> 16
<212> DNA
＜213〉artificial sequence
<400> 18
cccaccctcc ccctcg 16
<210> 19
<211> 19
<212> DNA
＜213〉artificial sequence
<400>19
ggtctttgaa taaagtctg 19
<210>20
<211>17
<212>DNA
＜213〉artificial sequence
<400>20
tgagtaaggg cctgggg 17
<210>21
<211>18
<212>DNA
＜213〉artificial sequence
<400>21
gagtgctttg aggatgca 18
<210>22
<211>19
<212>DNA
＜213〉artificial sequence
<400>22
ctcacactca tcacccagg 19
Claims (8)
1. diagnosing alpha-thalassemic nucleic acid film bar comprises a substrate and is fixed in described suprabasil specific oligonucleotide probe, it is characterized in that described specific oligonucleotide probe comprises:
1) be used to detect the specific oligonucleotide probe of α-Zhu Danbai transgenation, its base sequence is shown in SEQ ID NO:1-6;
2) be used to detect the specific oligonucleotide probe of α-Zhu Danbai genetically deficient, its base sequence is shown in SEQ ID NO:12-14;
Above-mentioned all sequences can be replaced by its complementary base sequences thereof.
2. diagnosing alpha according to claim 1-thalassemic nucleic acid film bar is characterized in that described specific oligonucleotide probe also comprises:
1) the pairing normal control specific oligonucleotide probe of α-Zhu Danbai mutator gene, its base sequence is shown in SEQ ID NO:7-11;
2) the pairing normal control specific oligonucleotide probe of α-Zhu Danbai missing gene, its base sequence is shown in SEQ ID NO:15;
Above-mentioned all sequences can be replaced by its complementary base sequences thereof.
3. diagnosing alpha-thalassemic test kit is characterized in that described test kit comprises:
1) primer of specific amplification α-Zhu Danbai gene or transcript, its base sequence is shown in SEQID NO:16-22;
2) be used to detect the specific oligonucleotide probe of α-Zhu Danbai transgenation, its base sequence is shown in SEQ ID NO:1-6;
3) be used to detect the specific oligonucleotide probe of α-Zhu Danbai genetically deficient, its base sequence is shown in SEQ ID NO:12-14;
Above-mentioned all sequences can be replaced by its complementary base sequences thereof.
4. diagnosing alpha according to claim 2-thalassemic test kit is characterized in that described test kit also comprises:
1) the pairing normal control specific oligonucleotide probe of α-Zhu Danbai mutator gene, its base sequence is shown in SEQ ID NO:7-11;
2) the pairing normal control specific oligonucleotide probe of α-Zhu Danbai missing gene, its base sequence is shown in SEQ ID NO:15;
Above-mentioned all sequences can be replaced by its complementary base sequences thereof.
5. according to claim 3 or 4 described diagnosing alphas-thalassemic test kit, it is characterized in that described oligonucleotide probe is fixed in the substrate.
6. according to claim 3 or 4 described diagnosing alphas-thalassemic test kit, it is characterized in that described oligonucleotide probe is fixed on the nylon membrane.
7. according to claim 3 or 4 described diagnosing alphas-thalassemic test kit, it is characterized in that described primer mark vitamin H.
8. detect absence type α-Di Zhonghaipinxue and mutant ' alpha '-thalassemic method simultaneously, may further comprise the steps:
A) blood sampling extracts the human gene group DNA;
B) be template with step 1) gained DNA, utilize the primer of claim 3 described specific amplification α-Zhu Danbai gene or transcript to carry out pcr amplification;
C) above-mentioned PCR product be fixed on suprabasil right and want 3 or 4 described specific oligonucleotide probe sequence hybridizations;
D) wash film and colour developing.
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| CNB2007100742035A CN100557033C (en) | 2007-04-25 | 2007-04-25 | Diagnosing alpha-thalassemic nucleic acid film bar and test kit |
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