阅读说明：本技术 用于鉴定孕早期胎儿染色体性别的引物和探针、鉴定方法及试剂盒 (Primer and probe for identifying chromosome sex of fetus in early pregnancy, identification method and kit ) 是由 周英杰 刘鹏 刘学军 刘慈 于 2019-09-18 设计创作，主要内容包括：本发明涉及基因鉴定技术领域,具体公开一种用于鉴定胎儿染色体性别的引物和探针、鉴定方法及试剂盒。所述用于鉴定胎儿性别的引物和探针根据男性性别决定基因SRY设计的引物和探针以及根据Y染色体AZF区域中的a区域、b区域和c区域的基因序列分别设计的3对引物和探针。本发明设计多对引物及相应的探针,检测过程中以四个通道荧光检测的方法来判断SRY基因和Y染色体的存在,具有准确性高检测通量高以及灵敏度高的特点。(The invention relates to the technical field of gene identification, and particularly discloses a primer, a probe, an identification method and a kit for identifying the sex of a chromosome of a fetus. The primer and the probe for identifying the fetal sex are designed according to a male sex determination gene SRY and 3 pairs of primers and probes respectively designed according to gene sequences of a region a, a region b and a region c in an AZF region of a Y chromosome. The invention designs a plurality of pairs of primers and corresponding probes, judges the existence of the SRY gene and the Y chromosome by a four-channel fluorescence detection method in the detection process, and has the characteristics of high accuracy, high detection flux and high sensitivity.)

1. A primer and a probe for identifying the chromosome sex of a fetus in an early stage of pregnancy, which are characterized in that: comprises that

An upstream primer SEQ ID NO 1, a downstream primer SE Q ID NO 2 and a probe SEQ ID NO 3 which are designed according to the male sex determination gene SRY;

an upstream primer SEQ ID NO 4, a downstream primer SEQ ID NO 5 and a probe SEQ ID NO 6 which are designed according to a region a in the AZF region of the Y chromosome;

an upstream primer SEQ ID NO 7, a downstream primer SEQ ID NO 8 and a probe SEQ ID NO 9 which are designed according to a b region in an AZF region of a Y chromosome;

an upstream primer SEQ ID NO 10, a downstream primer SEQ ID NO 11 and a probe SEQ ID NO 12 which are designed according to a c region in an AZF region of a Y chromosome;

the probes SEQ ID NO 3, 6, 9 and 12 carry different fluorophores respectively.

2. The primers and probes as claimed in claim 1, wherein: also comprises a reference upstream primer SEQ ID NO 13, a downstream primer SEQ ID NO 14 and a probe SEQ ID NO 15 which are designed according to the zinc finger protein gene ZFX/ZFY;

a reference upstream primer SEQ ID NO 16, a downstream primer SEQ ID NO 17 and a probe SEQ ID NO 18 which are designed according to a region a in an AZF gene sequence of a Y chromosome;

a reference upstream primer SEQ ID NO 19, a reference downstream primer SEQ ID NO 20 and a probe SEQ ID NO 21 which are designed according to a b region in an AZF gene sequence of a Y chromosome;

a reference upstream primer SEQ ID NO 22, a downstream primer SEQ ID NO 23 and a probe SEQ ID NO 24 which are designed according to a c region in an AZF gene sequence of a Y chromosome;

the probes SEQ ID NO. 3 and 15 carry the same fluorophore, the probes SEQ ID NO. 6 and 18 carry the same fluorophore, the probes SEQ ID NO. 9 and 21 carry the same fluorophore, and the probes SEQ ID NO. 12 and 24 carry the same fluorophore.

3. The primers and probes as claimed in claim 1 or 2, characterized in that: the different fluorophores are selected from FAM, VIC, ROX and Cy 5.

4. A method for early pregnancy fetal chromosomal sex identification using the primers and probes of claim 1, comprising: the method comprises the following steps:

a. extracting free DNA from maternal peripheral blood;

b. taking the extracted free DNA as a template, adding primers designed according to a, b and c regions in SRY and AZF gene sequences and probes SEQ ID NO 1-12; performing fluorescent quantitative multiple PCR reaction in a fluorescent quantitative PCR reaction system;

c. detecting fluorescent quantitative multiplex PCR reaction by using four fluorescent detection channels corresponding to four fluorescent groups carried by the probes SEQ ID NO. 3, 6, 9 and 12 respectively, if an amplification curve is detected by using the fluorescent channel corresponding to the fluorescent group carried by the probe SEQ ID NO. 3 and at least one fluorescent channel in the three fluorescent channels corresponding to the three fluorescent groups carried by the probes SEQ ID NO. 6, 9 and 12 detects the amplification curve, the fetus contains Y chromosome; if NO fluorescence signal is detected by four fluorescence channels corresponding to four fluorophores carried by the probes SEQ ID NO. 3, 6, 9 and 12, the fetus does not contain the Y chromosome.

5. The method of early pregnancy fetal chromosomal sex identification of claim 4, wherein: the fluorescent quantitative PCR reaction system in the step b is 25ul, and comprises:

2 XPCR buffer solution 10ul,

SEQ ID NO:1 1ul，

SEQ ID NO:2 1ul，

SEQ ID NO:3 0.2ul，

SEQ ID NO:4 1ul，

SEQ ID NO:5 1ul，

SEQ ID NO:6 0.2ul，

SEQ ID NO:71ul，

SEQ ID NO:8 1ul，

SEQ ID NO:9 0.2ul，

SEQ ID NO:10 1ul，

SEQ ID NO:111ul，

SEQ ID NO:120.2ul，

2ul of Taq enzyme is added into the enzyme,

the DNA template was 2.5ul,

the balance ddH 2O;

the concentration of the primer and the probe in the PCR reaction system is 10 uM; the concentration of the DNA template is more than or equal to 0.2 ng/ul;

the fluorescent quantitative multiplex PCR reaction process in the step b is as follows:

preheating: the temperature of the mixture is 50 ℃, the time of the mixture is 2min,

pre-denaturation: the temperature is 95 ℃, the time is 5min,

denaturation: the temperature of the mixture is 95 ℃ for 15s,

annealing: the temperature of the mixture is 60 ℃, the time of the mixture is 30s,

extension: the temperature of the mixture is controlled at 72 ℃ for 30s,

the denaturation, annealing and extension are carried out for 38 cycles,

total extension: 72 ℃ for 5 min.

6. A method for early pregnancy fetal chromosomal sex identification using the primers and probes of claim 2, comprising: the method comprises the following steps:

a. extracting free DNA from maternal peripheral blood;

b. adding primers and probes SEQ ID NO:1-12 designed according to a, b and c regions in SRY and AZF gene sequences by taking the extracted free DNA as a template to obtain a fluorescent quantitative PCR reaction system A;

taking the extracted free DNA as a template, adding reference primers designed according to a, B and c regions in ZFX/ZFY and AZF gene sequences and probes SEQ ID NO:13-24 to obtain a fluorescent quantitative PCR reaction system B;

the fluorescent quantitative PCR reaction systems A and B simultaneously carry out fluorescent quantitative multiple PCR reaction;

c. detecting the fluorescent quantitative multiplex PCR reaction of the systems A and B by using fluorescent detection channels corresponding to four fluorescent groups carried by the probes SEQ ID NO. 3, 6, 9 and 12 respectively, wherein in the detection process, if 2 amplification curves are detected by the fluorescent detection channels corresponding to the fluorescent groups carried by the probe SEQ ID NO. 3 and 1 amplification curve is detected by at least one of the fluorescent detection channels corresponding to the three fluorescent groups carried by the probes SEQ ID NO. 6, 9 and 12, the fetus contains Y chromosome; if only 1 amplification curve is detected by the fluorescence detection channel corresponding to the fluorophore carried by the probe SEQ ID NO. 3, and NO amplification curve is detected by the fluorescence detection channels corresponding to the three fluorophores carried by the probes SEQ ID NO. 6, 9 and 12, the fetus does not contain the Y chromosome; in the detection process, if the fluorescence detection channel corresponding to the fluorescent group carried by the probe SEQ ID NO. 3 does not detect the amplification curve, the PCR reaction system is wrong, and the detection result is invalid.

7. The method of early pregnancy fetal chromosomal sex identification of claim 6, wherein: the fluorescent quantitative PCR reaction system A in the step b is 25ul, and comprises:

the balance ddH 2O; the fluorescent quantitative PCR reaction system B is 25ul and comprises:

the balance ddH 2O;

the concentration of the primers and the probes in the PCR reaction systems A and B is 10 uM; the concentration of the DNA template is more than or equal to 0.2 ng/ul;

the fluorescent quantitative multiplex PCR reaction process in the step b is as follows:

preheating: the temperature of the mixture is 50 ℃, the time of the mixture is 2min,

pre-denaturation: the temperature is 95 ℃, the time is 5min,

denaturation: the temperature of the mixture is 95 ℃ for 15s,

annealing: the temperature of the mixture is 60 ℃, the time of the mixture is 30s,

extension: the temperature of the mixture is controlled at 72 ℃ for 30s,

the denaturation, annealing and extension are carried out for 38 cycles,

total extension: 72 ℃ for 5 min.

8. A kit for identifying the chromosomal sex of a fetus in an early stage of pregnancy, comprising: comprising the primers and probes of any one of claims 1-3, a PCR buffer and a DNA polymerase.

Technical Field

The invention relates to the technical field of gene identification, in particular to a primer, a probe, an identification method and a kit for identifying the sex of a chromosome of a fetus.

Background

In order to reduce the incidence of sex-linked genetic diseases and improve the genetic predisposition of people and the quality of population at birth, the pregnancy fetal sex identification technology becomes one of the necessary technical means for risk assessment.

At present, prenatal identification of fetal sex is classified into invasive and noninvasive, invasive prenatal diagnosis is mainly performed by a amniocentesis technology, the method has certain danger for fetuses and pregnant women, adverse reactions such as intrauterine infection, abortion, fetal death and the like are easily caused, and the method is not easily accepted by the pregnant women and families; the noninvasive prenatal diagnosis is mainly an ultrasonic image diagnosis technology, the detection can detect the gender of the fetus or whether the fetus has malformation, but the sensitivity is low in the early pregnancy, and the identification range is limited, so that the development of a novel noninvasive method for identifying the gender or the genetic abnormality of the fetus has important clinical significance.

The discovery of fetal free DNA in the plasma of pregnant women provides a new research direction for prenatal identification, the purpose of identifying the fetal chromosomal sex is achieved by extracting the fetal free DNA in the plasma of a maternal body and analyzing specific loci of a fetal sex determining gene SRY and a fetal sex chromosome Y, and the current method for detecting the fetal free DNA comprises the following steps: nested PCR, fluorescent quantitative PCR, fluorescent in situ hybridization, high-throughput gene sequencing and the like. However, the maternal plasma contains a low amount of fetal free DNA, and the sensitivity and specificity of the currently used identification methods are affected.

At present, primers and probes are designed through male sex determination gene SRY, a single-gene and single-channel method is used, and the sex of a fetus is identified by detecting whether the SRY gene exists or not through fluorescent quantitative PCR (polymerase chain reaction), but in the early pregnancy (6-8 weeks of pregnancy), the concentration of free DNA of the fetus is low, PCR amplification failure caused by low template concentration is easy to occur, repeated DNA extraction and detection are needed for many times, and the identification difficulty and identification cost are increased. If a plurality of sites of the SRY gene and the specific region of the Y chromosome are detected at the same time, the traditional fluorescent quantitative PCR method has low detection flux, is inconvenient to set internal references and is complex to operate, and the application of the fluorescent quantitative PCR method is greatly limited.

Disclosure of Invention

Aiming at the problems of low detection sensitivity, easy failure of template amplification and high identification cost, low detection flux, difficult internal reference setting, complex operation and the like of the conventional method for identifying the sex of the chromosome of the fetus by using fetal free DNA in the plasma of a pregnant woman after 6 weeks of gestation, the invention provides a primer and a probe for identifying the sex of the chromosome of the fetus, an identification method and a kit.

In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:

a primer and probe for identifying the sex of chromosome in early pregnancy includes

An upstream primer SEQ ID NO 1, a downstream primer SEQ ID NO 2 and a probe SEQ ID NO 3 which are designed according to the male sex determination gene SRY;

an upstream primer SEQ ID NO 4, a downstream primer SEQ ID NO 5 and a probe SEQ ID NO 6 which are designed according to a region a in an AZF gene sequence of a Y chromosome;

an upstream primer SEQ ID NO 7, a downstream primer SEQ ID NO 8 and a probe SEQ ID NO 9 which are designed according to a b region in an AZF gene sequence of a Y chromosome;

an upstream primer SEQ ID NO 10, a downstream primer SEQ ID NO 11 and a probe SEQ ID NO 12 which are designed according to a c region in an AZF gene sequence of a Y chromosome;

the probes SEQ ID NO 3, 6, 9 and 12 carry different fluorophores respectively.

Compared with the prior art, the primers and the probes for identifying the sex of the fetus at the early stage of pregnancy provided by the invention comprise four pairs of different primers and corresponding probes, the four primers can be subjected to PCR amplification in the same fluorescence quantitative RCR reaction system at the same time, multiple fluorescence quantitative PCR of the same reaction system can be realized, the four probes carry different fluorescent groups, and the existence of the SRY gene and the Y chromosome is judged by a four-channel fluorescence detection method in the detection process.

Also comprises a reference upstream primer SEQ ID NO 13, a downstream primer SEQ ID NO 14 and a probe SEQ ID NO 15 which are designed according to the zinc finger protein gene ZFX/ZFY;

a reference upstream primer SEQ ID NO 16, a downstream primer SEQ ID NO 17 and a probe SEQ ID NO 18 which are designed according to a region a in an AZF gene sequence of a Y chromosome;

a reference upstream primer SEQ ID NO 19, a reference downstream primer SEQ ID NO 20 and a probe SEQ ID NO 21 which are designed according to a b region in an AZF gene sequence of a Y chromosome;

a reference upstream primer SEQ ID NO 22, a downstream primer SEQ ID NO 23 and a probe SEQ ID NO 24 which are designed according to a c region in an AZF gene sequence of a Y chromosome;

the probes SEQ ID NO. 3 and 15 carry the same fluorophore, the probes SEQ ID NO. 6 and 18 carry the same fluorophore, the probes SEQ ID NO. 9 and 21 carry the same fluorophore, and the probes SEQ ID NO. 12 and 24 carry the same fluorophore.

The invention carries out the fluorescent quantitative PCR amplification on the template according to the reference primer designed by the zinc finger protein gene ZFX/ZFY and the probe SEQ ID NO:13-15 to judge the effectiveness of the multiple fluorescent quantitative PCR reaction system, and carries out the fluorescent quantitative PCR amplification on the template as the further reference of the existence of Y chromosome by combining the primer respectively designed according to the AZF gene sequences a, b and c and the probe SEQ ID NO:16-24, thereby realizing the strict quality control of the steps of the sample collection, the extraction and the amplification of the extracted free DNA and further ensuring the accuracy of the result.

Preferably, the different fluorescent groups are selected from FAM, VIC, ROX and Cy5, i.e., the different fluorescent groups carried by the probes SEQ ID NO. 3, 6, 9 and 12 can be selected from any one of FAM, VIC, ROX and Cy 5.

The invention also provides a method for identifying the chromosome sex of a fetus in an early pregnancy period by using the primer and the probe, which at least comprises the following steps:

a. extracting free DNA from maternal peripheral blood;

b. taking the extracted free DNA as a template, adding primers designed according to a, b and c regions in SRY and AZF gene sequences and probes SEQ ID NO 1-12; performing fluorescent quantitative multiple PCR reaction in a fluorescent quantitative PCR reaction system;

c. detecting fluorescent quantitative multiplex PCR reaction by using four fluorescent detection channels corresponding to four fluorescent groups carried by the probes SEQ ID NO. 3, 6, 9 and 12 respectively, if an amplification curve is detected by using the fluorescent channel corresponding to the fluorescent group carried by the probe SEQ ID NO. 3 and at least one fluorescent channel in the three fluorescent channels corresponding to the three fluorescent groups carried by the probes SEQ ID NO. 6, 9 and 12 detects the amplification curve, the fetus contains Y chromosome; if NO fluorescence signal is detected by four fluorescence channels corresponding to four fluorophores carried by the probes SEQ ID NO. 3, 6, 9 and 12, the fetus does not contain the Y chromosome.

Compared with the prior art, the identification method provided by the invention has the advantages of simple operation, high detection flux and high real-time fluorescence monitoring sensitivity, adopts a multiple fluorescence quantitative PCR technology, judges the existence of SRY genes and Y chromosomes by a four-channel fluorescence detection method, and has the detection success rate of 100% and the accuracy rate of over 100% under the conditions of not increasing the blood collection amount, not prolonging the detection time and not changing the blood collection gestational period.

Preferably, the fluorescent quantitative PCR reaction system in step b is 25ul, and comprises:

the concentration of the primer and the probe in the PCR reaction system is 10 uM; the concentration of the DNA template is more than or equal to 0.2 ng/ul;

the fluorescent quantitative multiplex PCR reaction process in the step b is as follows:

preheating: the temperature of the mixture is 50 ℃, the time of the mixture is 2min,

pre-denaturation: the temperature is 95 ℃, the time is 5min,

denaturation: the temperature of the mixture is 95 ℃ for 15s,

annealing: the temperature of the mixture is 60 ℃, the time of the mixture is 30s,

extension: the temperature of the mixture is controlled at 72 ℃ for 30s,

the denaturation, annealing and extension are carried out for 38 cycles,

total extension: 72 ℃ for 5 min.

The invention also provides another method for identifying the chromosome sex of a fetus in an early pregnancy by using the primer and the probe, which at least comprises the following steps:

a. extracting free DNA from maternal peripheral blood;

b. adding primers and probes SEQ ID NO:1-12 designed according to a, b and c regions in SRY and AZF gene sequences by taking the extracted free DNA as a template to obtain a fluorescent quantitative PCR reaction system A;

taking the extracted free DNA as a template, adding reference primers designed according to a, B and c regions in ZFX/ZFY and AZF gene sequences and probes SEQ ID NO:13-24 to obtain a fluorescent quantitative PCR reaction system B;

the fluorescent quantitative PCR reaction systems A and B simultaneously carry out fluorescent quantitative multiple PCR reaction;

c. detecting the fluorescent quantitative multiplex PCR reaction of the systems A and B by using fluorescent detection channels corresponding to four fluorescent groups carried by the probes SEQ ID NO. 3, 6, 9 and 12 respectively, wherein in the detection process, if 2 amplification curves are detected by the fluorescent detection channels corresponding to the fluorescent groups carried by the probe SEQ ID NO. 3 and 1 amplification curve is detected by at least one of the fluorescent detection channels corresponding to the three fluorescent groups carried by the probes SEQ ID NO. 6, 9 and 12, the fetus contains Y chromosome; if only 1 amplification curve is detected by the fluorescence detection channel corresponding to the fluorophore carried by the probe SEQ ID NO. 3, and NO amplification curve is detected by the fluorescence detection channels corresponding to the three fluorophores carried by the probes SEQ ID NO. 6, 9 and 12, the fetus does not contain the Y chromosome; in the detection process, if the fluorescence detection channel corresponding to the fluorescent group carried by the probe SEQ ID NO. 3 does not detect the amplification curve, the PCR reaction system is wrong, and the detection result is invalid.

Compared with the prior art, the identification method provided by the invention has the advantages of simple operation, high detection flux and high real-time fluorescence monitoring sensitivity, adopts a two-tube two-system multiple fluorescence quantitative PCR technology, adopts a four-channel fluorescence detection method, and simultaneously sets four pairs of reference primers and probes to judge the existence of SRY genes and Y chromosomes, so that the detection success rate reaches 100% and the accuracy rate reaches 100% under the conditions of not increasing the blood collection amount, not prolonging the detection time and not changing the blood collection pregnancy period.

Preferably, the fluorescent quantitative PCR reaction system a in step b is 25ul, and includes:

the balance ddH 2O; the fluorescent quantitative PCR reaction system B is 25ul and comprises:

the concentration of the primers and the probes in the PCR reaction systems A and B is 10 uM; the concentration of the DNA template is more than or equal to 0.2 ng/ul;

the fluorescent quantitative multiplex PCR reaction process in the step b is as follows:

preheating: the temperature of the mixture is 50 ℃, the time of the mixture is 2min,

pre-denaturation: the temperature is 95 ℃, the time is 5min,

denaturation: the temperature of the mixture is 95 ℃ for 15s,

annealing: the temperature of the mixture is 60 ℃, the time of the mixture is 30s,

extension: the temperature of the mixture is controlled at 72 ℃ for 30s,

the denaturation, annealing and extension are carried out for 38 cycles,

total extension: 72 ℃ for 5 min.

The invention also provides a kit for identifying the sex of chromosomes of a fetus in an early pregnancy period, which specifically comprises the primers and the probe as described in any one of claims 1 to 3, a PCR buffer solution and a DNA polymerase.

The kit can be used for rapidly detecting the sex of the chromosome of the fetus at the early pregnancy (6-8 weeks) so as to judge the sex of the fetus, has the advantages of simple and rapid detection method, high detection success rate and high accuracy of detection results, and realizes rapid diagnosis of the sex of the fetus at the pregnancy.

Drawings

FIG. 1A is a detection signal of FAM channel when the fetus detected in example 1 of the present invention is male;

FIG. 1B is a VIC channel detection signal when the fetus detected in example 1 of the present invention is male;

FIG. 1C is a signal of the ROX channel when the fetus examined in example 1 of the present invention is male;

FIG. 1D is a graph showing the detection signal of Cy5 channel when the fetus detected in example 1 of the present invention is male;

FIG. 2A is a detection signal of FAM channel when the fetus detected in example 1 of the present invention is female;

FIG. 2B is a signal of VIC channel detection when the fetus detected in example 1 is female;

FIG. 2C is a signal of the ROX channel when the fetus detected in example 1 of the present invention is female;

FIG. 2D is a graph showing the detection signal of Cy5 channel when the fetus detected in example 1 of the present invention is female;

FIG. 3A is a detection signal of FAM channel when the fetus detected in example 2 of the present invention is male;

FIG. 3B is a VIC channel detection signal when the fetus detected in example 2 is male;

FIG. 3C is a signal of the ROX channel when the fetus detected in example 2 of the present invention is male;

FIG. 3D is a graph showing a detection signal of Cy5 channel when the fetus detected in example 2 of the present invention is a male;

FIG. 4A is a detection signal of FAM channel when the fetus detected in example 2 of the present invention is female;

FIG. 4B is a VIC channel detection signal when the fetus detected in example 2 is female;

FIG. 4C is a signal of the ROX channel when the fetus detected in example 2 of the present invention is female;

FIG. 4D is a signal of the Cy5 channel when the fetus detected in example 2 of the present invention is female;

FIG. 5A is a detection signal of FAM channel for detecting a positive control in example 2 of the present invention;

FIG. 5B is a detection signal of the VIC channel for detecting the positive control in example 2 of the present invention;

FIG. 5C is a detection signal of the ROX channel for detecting the positive control in example 2 of the present invention;

FIG. 5D is a graph showing the detection signal of Cy5 channel for detecting a positive control in example 2 of the present invention;

FIG. 6A is a detection signal of a FAM channel for detecting a negative control in example 2 of the present invention;

FIG. 6B is a detection signal of a VIC channel for detecting a negative control in example 2 of the present invention;

FIG. 6C is a detection signal of ROX channel for detecting negative control in example 2 of the present invention;

FIG. 6D is a graph showing the detection signal of Cy5 channel for detecting a negative control in example 2 of the present invention;

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.