CN115449549A - Kit for screening immunodeficiency diseases of newborn and preparation method thereof - Google Patents
Kit for screening immunodeficiency diseases of newborn and preparation method thereof Download PDFInfo
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- 238000012216 screening Methods 0.000 title claims abstract description 22
- 208000029462 Immunodeficiency disease Diseases 0.000 title claims abstract description 12
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 238000003908 quality control method Methods 0.000 claims abstract description 22
- 239000000126 substance Substances 0.000 claims abstract description 11
- 108091034117 Oligonucleotide Proteins 0.000 claims abstract description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 10
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims abstract description 7
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims abstract description 7
- 238000012408 PCR amplification Methods 0.000 claims abstract description 7
- 239000007853 buffer solution Substances 0.000 claims abstract description 7
- 239000000463 material Substances 0.000 claims abstract description 5
- 239000003623 enhancer Substances 0.000 claims abstract description 4
- 210000004027 cell Anatomy 0.000 claims description 14
- 210000004369 blood Anatomy 0.000 claims description 12
- 239000008280 blood Substances 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 11
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- YMZMTOFQCVHHFB-UHFFFAOYSA-N 5-carboxytetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=C(C(O)=O)C=C1C([O-])=O YMZMTOFQCVHHFB-UHFFFAOYSA-N 0.000 claims description 3
- 108091033409 CRISPR Proteins 0.000 claims description 3
- 238000010354 CRISPR gene editing Methods 0.000 claims description 3
- 238000005516 engineering process Methods 0.000 claims description 3
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- 230000002452 interceptive effect Effects 0.000 claims description 3
- 239000012744 reinforcing agent Substances 0.000 claims description 3
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 claims description 3
- 238000003198 gene knock in Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 11
- 230000003321 amplification Effects 0.000 abstract description 7
- 238000009826 distribution Methods 0.000 abstract description 7
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 7
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- 238000000605 extraction Methods 0.000 abstract description 5
- 230000001360 synchronised effect Effects 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 7
- 201000010717 Bruton-type agammaglobulinemia Diseases 0.000 description 4
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- 238000003745 diagnosis Methods 0.000 description 4
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- 208000036364 Normal newborn Diseases 0.000 description 1
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Abstract
The invention discloses a kit for screening immunodeficiency diseases of newborns and a preparation method thereof, wherein the kit comprises an oligonucleotide sequence combination, a negative quality control material, a positive quality control material, necessary reagents Mg2+, dNTPs, DNA polymerase, PCR buffer solution, a PCR enhancer and the like for real-time fluorescent PCR amplification. This application is through design general primer Tag, can be in multiple fluorescence amplification system, the amplification efficiency of balanced each primer pair, establish the detection to DeltaTREC and DeltaKREC, deltaTREC and DeltaKREC index that this application detected, compare in ration TREC and KREC copy number, it is less to receive sample homogeneity and nucleic acid extraction efficiency's influence, detect advantage with low costs (the quantitative standard substance of the known concentration of synchronous detection is not required), distribution of DeltaTREC and DeltaKREC in the neonate crowd is normal simultaneously, better predictability has, more be suitable for extensive screening application.
Description
Technical Field
The invention belongs to the technical field of kits, and particularly relates to a kit for screening immunodeficiency diseases of newborns and a preparation method thereof.
Background
The T cell receptor excision loop (TREC) is a circular DNA fragment generated by base rearrangement in the process of forming a T Cell Receptor (TCR), does not replicate along with the division of T cells, stably exists in the cells, can indirectly reflect the initial T lymphocyte quantity output by thymus, can be used as a screening and diagnosis index of T lymphocyte immunodeficiency accompanied by reduction of the T cell quantity, is used for auxiliary diagnosis or screening of diseases such as Primary immunodeficiency diseases (CID) of newborns, can be used as a screening and diagnosis index of B lymphocyte immunodeficiency accompanied by reduction of the B cell quantity, and is used for auxiliary diagnosis or screening of diseases such as X-linked agammaglobulinemia (XLA) of newborns.
Screening means generally adopted in the prior art are all based on a quantitative mode of a fluorescent quantitative PCR platform, and the TREC and KREC copy numbers in dry blood spots of newborns of specific sizes are quantitatively detected by referring to external quantitative markers, and the method takes the copy number corresponding to a certain percentile as a positive judgment threshold value according to the distribution of TREC and KREC copy number concentrations in the newborn population, but the method has the following defects: firstly, positive and negative are judged by quantifying absolute copy number, the requirements on the uniformity of a sample and the stability of nucleic acid extraction treatment are high, in other words, the heterogeneity of blood in dry blood spots, and the instability of different nucleic acid extraction methods or extraction processes can influence the detection result; secondly, the accuracy of the quantitative result depends on external quantitative standards, but TREC and KREC have no universal standard substances for calibration, so even if the same set of scheme is used, even the same detection product is used, a laboratory is generally recommended to establish or adjust the positive judgment threshold of the current laboratory according to the results of thousands of newborn samples before formal screening is carried out; third, the distribution of copy number quantification results in the population is abnormal and unbalanced, which is not favorable for the estimation of statistically based ratio thresholds, and thus, using thresholds established in earlier studies, it is still necessary to continuously adjust the distribution in formal screening tests.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the kit for screening the immunodeficiency diseases of the newborn and the preparation method thereof, and the kit has the advantages of better predictability, suitability for large-scale screening, higher specificity and stable quality for the detection of TREC and KREC, strong accessibility and easiness in industrial production.
In order to achieve the purpose, the invention provides the following technical scheme: the kit comprises an oligonucleotide sequence combination, a negative quality control product, a positive quality control product, necessary reagents Mg2+, dNTPs for real-time fluorescent PCR amplification, DNA polymerase, PCR buffer solution, a PCR enhancer and the like.
Preferably, the combination of oligonucleotide sequences is:
(01)GCGTGACCTACTGACTC；
(02)GCGTGATGCATCGTACC；
(03)GCGTGACCTACTGACTCTGCTGACACCTCTGGTTTTTGTAA；
(04)GCGTGATGCATCGTACCACTCGTGAGAACGGTGAATGAAG；
(05)CCCACTCCTGTGCACGGTGATGCAT；
(06)GCGTGACCTACTGACTCAGCTCAGCGCCCATTACGTT；
(07)GCGTGATGCATCGTACCAGGAGCCAGCTCTTACCCTAGAGT；
(08)GTGCACAGTGTGCGCTGCCAACC；
(09)GCGTGACCTACTGACTCGGCCATATTACTGACCCTCTACAGA；
(10)GCGTGATGCATCGTACCGAAAGATCAAAGCCTTGCAGGTAAT；
(11)ACCACCTGCCTTTATCCTGCAATACCA。
preferably, the sequence 01 and the sequence 02 are universal primer Tag sequences, the sequences 03, 04, 06 and 07 are target-specific sequences connected with the universal primer Tag sequences respectively, the sequences 05, 08 and 11 are fluorescent probe sequences and detect TREC, KREC and B2M genes respectively, the sequence 05 is designed at a TREC junction and spans two sides of the junction, and the sequence 06 is designed at a KREC junction and spans two sides of the junction.
Preferably, the fluorescent reporter groups of the sequences 05, 08 and 11 can be selected from FAM, HEX, VIC, ROX, JOE, CY3, CY5, CY5.5, TET, 5-TAMRA and the like without interfering with each other, and the fluorescent reporter groups of the sequences 05, 08 and 11 can be selected from BHQ0, BHQ1, BHQ2, BHQ3, MGB, dabcyl, TAMRA and the like according to the matching of the fluorescent reporter groups.
Preferably, the method comprises the following steps:
the first step is as follows: preparing oligonucleotide sequence combination, negative quality control substances, positive quality control substances, necessary reagents Mg2+, dNTPs for real-time fluorescent PCR amplification, DNA polymerase, PCR buffer solution, PCR reinforcing agent and the like for later use;
the second step is that: mixing the positive quality control product with commercial red blood cells and plasma in a ratio by using a 293T cell line, and preparing into a dry blood tablet;
the third step: the negative quality control product is prepared into a dry blood spot tablet after being mixed with commercial red blood cells and blood plasma according to a proportion by using a 293T cell line subjected to gene knock-in;
the fourth step: the 293T cell line into which the gene in the third step is knocked in can knock the TREC characteristic sequence and the KREC characteristic sequence into the 293T cell line by using CRISPR technology;
the fifth step: the materials are placed in a reagent kit.
Preferably, the TREC characteristic sequence in the fifth step is:
GCTCTGAAAGGCAGAAAGAGGGCAGCCCTCTCCAAGGCAAAATGGGGCTCCTGTGGGGAACAGAGGGGTGCCTCTGTCAACAAAGGTGATGCCACATCCCTTTCAACCATGCTGACACCTCTGGTTTTTGTAAAGGTGCCCACTCCTGTGCACGGTGATGCATAGGCACCTGCACCCCGTGCCTAAACCCTGCAGCTGGCACGGGCCCTGTCTGCTCTTCATTCACCGTTCTCACGAGTTGCAATAAGTTCAGCCCTCCATGTCACACTGTGTTTTCCATCCTGGGGAGTGTTTCACAGCTATCCCAAGCCCCACGCTGA。
preferably, the KREC characteristic sequence in the fifth step is:
TGGAAATTTTCACTAATGAGATATCATAATGAATGTGAATTTTATTTCTGAAATCTCTAATAAATCAGTCTTCTCCCTGGTTTTCCCAGCTCAGCGCCCATTACGTTTCTGTTCTCTTTCCCTTAGTGGCATTATTTGTATCACTGTGCACAGTGTGCGCTGCCAACCTGCTGCCCGTGCAGAAACTCTAGGGTAAGAGCTGGCTCCTGGAGTCCCACCCAGGCTGCGTGTCCCTCACAGTCTGCTCTGTGTCTATGTGTGTGTGTTGGGGGGATATTATTGGACAATTCAAGGGAGGCT。
compared with the prior art, the invention has the following beneficial effects:
according to the application, by designing the universal primer Tag, the amplification efficiency of each primer pair can be balanced in a multiplex fluorescence amplification system, and the detection of the Delta TREC and the Delta KREC is established, compared with the quantitative TREC and KREC copy number, the indexes of the Delta TREC and the Delta KREC detected by the application have the advantages of being less influenced by the sample uniformity and the nucleic acid extraction efficiency and low in detection cost (the quantitative standard substance with known concentration does not need to be synchronously detected), and meanwhile, the distribution of the Delta TREC and the Delta KREC in newborn crowds is normal, so that the application has better predictability and is more suitable for large-scale screening application;
the probe is designed at the connecting point of TREC and KREC, so that the probe has higher specificity on the detection of TREC and KREC;
compared with the preparation by adopting newborn umbilical cord blood and old person blood, the 293T cell line using gene editing has the advantages of stable quality, strong accessibility and the like, and is easy for industrial production.
Drawings
FIG. 1 is a schematic diagram showing the amplification result of the negative control substance of the present invention;
FIG. 2 is a schematic diagram showing the amplification result of the positive quality control substance of the present invention;
FIG. 3 is a graph showing the distribution of Δ TREC in a 2453 sample of dried blood spots of a newborn infant of the present invention;
FIG. 4 is a graph showing the distribution of Δ KREC in a 2453 sample of dried blood spots of a newborn infant of the present invention;
FIG. 5 is a schematic diagram of the present invention for differentiating between normal neonatal populations, SCID cases, and XLA cases.
Detailed Description
All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a technical scheme that: a kit for screening immunodeficiency diseases of newborns and a preparation method thereof are disclosed, wherein the kit comprises an oligonucleotide sequence combination, a negative quality control product, a positive quality control product, necessary reagents Mg2+, dNTPs, DNA polymerase, PCR buffer solution, PCR enhancer and the like for real-time fluorescent PCR amplification.
Wherein the combination of oligonucleotide sequences is:
(01)GCGTGACCTACTGACTC；
(02)GCGTGATGCATCGTACC；
(03)GCGTGACCTACTGACTCTGCTGACACCTCTGGTTTTTGTAA；
(04)GCGTGATGCATCGTACCACTCGTGAGAACGGTGAATGAAG；
(05)CCCACTCCTGTGCACGGTGATGCAT；
(06)GCGTGACCTACTGACTCAGCTCAGCGCCCATTACGTT；
(07)GCGTGATGCATCGTACCAGGAGCCAGCTCTTACCCTAGAGT；
(08)GTGCACAGTGTGCGCTGCCAACC；
(09)GCGTGACCTACTGACTCGGCCATATTACTGACCCTCTACAGA；
(10)GCGTGATGCATCGTACCGAAAGATCAAAGCCTTGCAGGTAAT；
(11)ACCACCTGCCTTTATCCTGCAATACCA。
the sequence 01 and the sequence 02 are universal primer Tag sequences, the sequences 03, 04, 06 and 07 are target-specific sequences connected with the universal primer Tag sequences respectively, the sequences 05, 08 and 11 are fluorescent probe sequences and detect TREC, KREC and B2M genes respectively, the sequence 05 is designed at a TREC junction and spans two sides of the junction, the sequence 06 is designed at the KREC junction and spans two sides of the junction, the amplification efficiency of each primer pair can be balanced by adding the universal primer Tag sequences at the 5' end of the target-specific primers, the stability of the system for detection of Delta TREC and Delta KREC can be improved, the sequence 05 is designed at the TREC junction and spans two sides of the junction, genomic nucleic acid can be effectively distinguished, the detection specificity of TREC can be improved, the sequence 06 is designed at the KREC junction and spans two sides of the junction, the genomic nucleic acid can be effectively distinguished, and the detection specificity of TREC can be improved.
Wherein, the fluorescent reporter groups of the sequences 05, 08 and 11 can be selected from FAM, HEX, VIC, ROX, JOE, CY3, CY5, CY5.5, TET, 5-TAMRA and the like without interfering with each other, and the fluorescent reporter groups of the sequences 05, 08 and 11 can be selected from BHQ0, BHQ1, BHQ2, BHQ3, MGB, dabcyl, TAMRA and the like according to the matching of the fluorescent reporter groups.
The method comprises the following steps:
the first step is as follows: preparing oligonucleotide sequence combination, negative quality control substances, positive quality control substances, necessary reagents Mg2+, dNTPs for real-time fluorescent PCR amplification, DNA polymerase, PCR buffer solution, PCR reinforcing agent and the like for later use;
the second step is that: the positive quality control product is prepared into dry blood tablets by mixing a 293T cell line with commercial red blood cells and plasma in proportion;
the third step: the negative quality control product is prepared into a dry blood spot tablet after being mixed with commercial red blood cells and blood plasma according to a proportion by using a 293T cell line with a knocked-in gene;
the fourth step: the 293T cell line into which the gene in the third step is knocked in can knock the TREC characteristic sequence and the KREC characteristic sequence into the 293T cell line by using CRISPR technology;
the fifth step: the materials are placed in a reagent kit.
Wherein, the TREC characteristic sequence in the fifth step is as follows:
GCTCTGAAAGGCAGAAAGAGGGCAGCCCTCTCCAAGGCAAAATGGGGCTCCTGTGGGGAACAGAGGGGTGCCTCTGTCAACAAAGGTGATGCCACATCCCTTTCAACCATGCTGACACCTCTGGTTTTTGTAAAGGTGCCCACTCCTGTGCACGGTGATGCATAGGCACCTGCACCCCGTGCCTAAACCCTGCAGCTGGCACGGGCCCTGTCTGCTCTTCATTCACCGTTCTCACGAGTTGCAATAAGTTCAGCCCTCCATGTCACACTGTGTTTTCCATCCTGGGGAGTGTTTCACAGCTATCCCAAGCCCCACGCTGA。
wherein, the KREC characteristic sequence in the fifth step is as follows:
TGGAAATTTTCACTAATGAGATATCATAATGAATGTGAATTTTATTTCTGAAATCTCTAATAAATCAGTCTTCTCCCTGGTTTTCCCAGCTCAGCGCCCATTACGTTTCTGTTCTCTTTCCCTTAGTGGCATTATTTGTATCACTGTGCACAGTGTGCGCTGCCAACCTGCTGCCCGTGCAGAAACTCTAGGGTAAGAGCTGGCTCCTGGAGTCCCACCCAGGCTGCGTGTCCCTCACAGTCTGCTCTGTGTCTATGTGTGTGTGTTGGGGGGATATTATTGGACAATTCAAGGGAGGCT。
preparing a primer and probe mixed solution:
| sequence of | Final concentration |
| 01 | 5μM |
| 02 | 5μM |
| 03 | 1μM |
| 04 | 1μM |
| 05 | 0.5μM |
| 06 | 1μM |
| 07 | 1μM |
| 08 | 0.5μM |
| 09 | 1μM |
| 10 | |
| 11 | 0.5μM |
Negative and positive quality controls (as described above) are prepared.
The screening sensitivity is 100% and the specificity is more than 99.5% by differentiating and detecting normal newborn population, SCID case and XLA case (as shown in figure 5).
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (4)
1. A kit for screening immunodeficiency diseases of newborns, which is characterized in that: the kit comprises an oligonucleotide sequence combination, a negative quality control product, a positive quality control product, necessary reagents Mg2+, dNTPs for real-time fluorescent PCR amplification, DNA polymerase, PCR buffer solution, a PCR enhancer and the like;
the oligonucleotide sequences are combined as follows:
(01)GCGTGACCTACTGACTC；
(02)GCGTGATGCATCGTACC；
(03)GCGTGACCTACTGACTCTGCTGACACCTCTGGTTTTTGTAA；
(04)GCGTGATGCATCGTACCACTCGTGAGAACGGTGAATGAAG；
(05)CCCACTCCTGTGCACGGTGATGCAT；
(06)GCGTGACCTACTGACTCAGCTCAGCGCCCATTACGTT；
(07)GCGTGATGCATCGTACCAGGAGCCAGCTCTTACCCTAGAGT；
(08)GTGCACAGTGTGCGCTGCCAACC；
(09)GCGTGACCTACTGACTCGGCCATATTACTGACCCTCTACAGA；
(10)GCGTGATGCATCGTACCGAAAGATCAAAGCCTTGCAGGTAAT；
(11)ACCACCTGCCTTTATCCTGCAATACCA；
the sequence 01 and the sequence 02 are universal primer Tag sequences, the sequences 03, 04, 06 and 07 are target specific sequences connected with the universal primer Tag sequences respectively, the sequences 05, 08 and 11 are fluorescent probe sequences and detect TREC, KREC and B2M genes respectively, the sequence 05 is designed at a TREC junction and spans two sides of the junction, and the sequence 06 is designed at a KREC junction and spans two sides of the junction;
the neonatal immunodeficiency disease screening method comprises the following steps:
the first step is as follows: preparing oligonucleotide sequence combination, negative quality control substances, positive quality control substances, necessary reagents Mg2+, dNTPs for real-time fluorescent PCR amplification, DNA polymerase, PCR buffer solution, PCR reinforcing agent and the like for later use;
the second step: mixing the positive quality control product with commercial red blood cells and plasma in a ratio by using a 293T cell line, and preparing into a dry blood tablet;
the third step: the negative quality control product is prepared into a dry blood spot tablet after being mixed with commercial red blood cells and blood plasma according to a proportion by using a 293T cell line subjected to gene knock-in;
the fourth step: the 293T cell line into which the gene of the third step is knocked in can knock the TREC characteristic sequence and the KREC characteristic sequence into the 293T cell line by using a CRISPR technology;
the fifth step: the materials are placed in a reagent box.
2. The kit for screening immunodeficiency diseases in neonates according to claim 1, wherein said kit comprises: the fluorescent reporter groups of the sequences 05, 08 and 11 can be selected from FAM, HEX, VIC, ROX, JOE, CY3, CY5, CY5.5, TET, 5-TAMRA and the like without interfering with each other, and the fluorescent reporter groups of the sequences 05, 08 and 11 can be selected from BHQ0, BHQ1, BHQ2, BHQ3, MGB, dabcyl, TAMRA and the like according to the matching property of the fluorescent reporter groups.
3. The method for preparing a kit for screening immunodeficiency diseases of newborn according to claim 1, characterized in that: the TREC characteristic sequence in the fifth step is as follows:
GCTCTGAAAGGCAGAAAGAGGGCAGCCCTCTCCAAGGCAAAATGGGGCTCCTGTGGGGAACAGAGGGGTGCCTCTGTCAACAAAGGTGATGCCACATCCCTTTCAACCATGCTGACACCTCTGGTTTTTGTAAAGGTGCCCACTCCTGTGCACGGTGATGCATAGGCACCTGCACCCCGTGCCTAAACCCTGCAGCTGGCACGGGCCCTGTCTGCTCTTCATTCACCGTTCTCACGAGTTGCAATAAGTTCAGCCCTCCATGTCACACTGTGTTTTCCATCCTGGGGAGTGTTTCACAGCTATCCCAAGCCCCACGCTGA。
4. the kit for screening immunodeficiency diseases of newborn infants and the preparation method thereof according to claim 1 are characterized in that: the KREC characteristic sequence in the fifth step is as follows:
TGGAAATTTTCACTAATGAGATATCATAATGAATGTGAATTTTATTTCTGAAATCTCTAATAAATCAGTCTTCTCCCTGGTTTTCCCAGCTCAGCGCCCATTACGTTTCTGTTCTCTTTCCCTTAGTGGCATTATTTGTATCACTGTGCACAGTGTGCGCTGCCAACCTGCTGCCCGTGCAGAAACTCTAGGGTAAGAGCTGGCTCCTGGAGTCCCACCCAGGCTGCGTGTCCCTCACAGTCTGCTCTGTGTCTATGTGTGTGTGTTGGGGGGATATTATTGGACAATTCAAGGGAGGCT。
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Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009027568A1 (en) * | 2007-08-02 | 2009-03-05 | Fundación Reina Mercedes Para La Investigación Sanitaria | Primers, method and set of tools for determining the functionality of the human thymus |
| CN102102124A (en) * | 2010-06-17 | 2011-06-22 | 深圳市疾病预防控制中心 | Multiplex fluorescence PCR (Polymerase Chain Reaction) detection method and detection kit for typhoid/paratyphoid saimonella |
| CN103173533A (en) * | 2012-11-20 | 2013-06-26 | 无锡联合利康临床检验所有限公司 | Real-time fluorescence quantitative PCR (polymerase chain reaction) kit for quantitatively detecting TRECs and KRECs genes and application thereof |
| CN103173534A (en) * | 2012-11-20 | 2013-06-26 | 无锡联合利康临床检验所有限公司 | Real-time fluorescence quantitative PCR (polymerase chain reaction) kit for quantitatively detecting TRECs gene and application thereof |
| CN105274231A (en) * | 2015-11-02 | 2016-01-27 | 上海领检科技有限公司 | Real-time fluorescent quantitative PCR (polymerase chain reaction) kit for one-step quantitative detection of KRECs gene and its application |
| CN105274230A (en) * | 2015-11-02 | 2016-01-27 | 上海领检科技有限公司 | Real-time fluorescent quantitative PCR (polymerase chain reaction) kit for one-step quantitative detection of TRECs and KRECs genes and its application |
| CN106119413A (en) * | 2016-07-01 | 2016-11-16 | 浙江省疾病预防控制中心 | A kind of HIV (human immunodeficiency virus) multiple fluorescence PCR detection reagent box and detection method |
| CN108998511A (en) * | 2018-07-31 | 2018-12-14 | 朱智 | It is a kind of detect TRECs and KRECs gene real-time fluorescence quantitative PCR kit and its application |
| WO2019044952A1 (en) * | 2017-08-30 | 2019-03-07 | 国立研究開発法人国立成育医療研究センター | Method for evaluating trec or krec level, particles used in said method, and use thereof |
| CN110408713A (en) * | 2019-05-16 | 2019-11-05 | 重庆医科大学附属儿童医院 | A kind of kit and method detecting newborn TREC and KREC |
| CN114250319A (en) * | 2020-09-22 | 2022-03-29 | 厦门大学 | Kit for detecting multiple nucleic acids of sexually transmitted infection |
-
2022
- 2022-10-27 CN CN202211322242.3A patent/CN115449549B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009027568A1 (en) * | 2007-08-02 | 2009-03-05 | Fundación Reina Mercedes Para La Investigación Sanitaria | Primers, method and set of tools for determining the functionality of the human thymus |
| CN102102124A (en) * | 2010-06-17 | 2011-06-22 | 深圳市疾病预防控制中心 | Multiplex fluorescence PCR (Polymerase Chain Reaction) detection method and detection kit for typhoid/paratyphoid saimonella |
| CN103173533A (en) * | 2012-11-20 | 2013-06-26 | 无锡联合利康临床检验所有限公司 | Real-time fluorescence quantitative PCR (polymerase chain reaction) kit for quantitatively detecting TRECs and KRECs genes and application thereof |
| CN103173534A (en) * | 2012-11-20 | 2013-06-26 | 无锡联合利康临床检验所有限公司 | Real-time fluorescence quantitative PCR (polymerase chain reaction) kit for quantitatively detecting TRECs gene and application thereof |
| CN105274231A (en) * | 2015-11-02 | 2016-01-27 | 上海领检科技有限公司 | Real-time fluorescent quantitative PCR (polymerase chain reaction) kit for one-step quantitative detection of KRECs gene and its application |
| CN105274230A (en) * | 2015-11-02 | 2016-01-27 | 上海领检科技有限公司 | Real-time fluorescent quantitative PCR (polymerase chain reaction) kit for one-step quantitative detection of TRECs and KRECs genes and its application |
| CN106119413A (en) * | 2016-07-01 | 2016-11-16 | 浙江省疾病预防控制中心 | A kind of HIV (human immunodeficiency virus) multiple fluorescence PCR detection reagent box and detection method |
| WO2019044952A1 (en) * | 2017-08-30 | 2019-03-07 | 国立研究開発法人国立成育医療研究センター | Method for evaluating trec or krec level, particles used in said method, and use thereof |
| CN108998511A (en) * | 2018-07-31 | 2018-12-14 | 朱智 | It is a kind of detect TRECs and KRECs gene real-time fluorescence quantitative PCR kit and its application |
| CN110408713A (en) * | 2019-05-16 | 2019-11-05 | 重庆医科大学附属儿童医院 | A kind of kit and method detecting newborn TREC and KREC |
| CN114250319A (en) * | 2020-09-22 | 2022-03-29 | 厦门大学 | Kit for detecting multiple nucleic acids of sexually transmitted infection |
Non-Patent Citations (1)
| Title |
|---|
| 国家药品监督管理局医疗器械监督管理司: "《医疗机械生产质量管理规范检查指南》", 中国医药科技出版社, pages: 293 * |
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