CN114517229A

CN114517229A - Application of serum exosome RNA in preparation of pregnancy diabetic giant screening or early diagnosis reagent

Info

Publication number: CN114517229A
Application number: CN202210159716.0A
Authority: CN
Inventors: 文娟; 马锦琪; 章乐; 李鹰; 李欣; 袁迎第
Original assignee: Nanjing Maternity and Child Healthcare Hospital
Current assignee: Nanjing Maternity and Child Healthcare Hospital
Priority date: 2022-02-22
Filing date: 2022-02-22
Publication date: 2022-05-20
Anticipated expiration: 2042-02-22
Also published as: CN114517229B

Abstract

The invention discloses an application of serum exosome RNA in preparing a reagent for screening or early diagnosing gestational diabetic giant children. Application of a reagent for detecting mRNA GDF3 and lncRNA AC006064.4 from serum exosomes in preparation of a screening or early diagnosis reagent for pregnant women with huge gestational diabetes and high risk of babies. The invention utilizes the expression levels of mRNA GDF3 and lncRNA AC006064.4 from serum exosomes, combines the age of pregnant women, fasting blood sugar and 2h blood sugar, and has the specificity of 1.00 and the sensitivity of 0.55 for early diagnosis of gestational diabetes mellitus giant children. Therefore, the reagents with the two indexes can be used for auxiliary early diagnosis of the gestational diabetic giant child, provide support for a clinician to accurately predict the occurrence risk of the giant child and timely adopt a more individualized prevention and treatment scheme, and further reduce the poor pregnancy outcome to the maximum extent.

Description

Application of serum exosome RNA in preparation of pregnancy diabetic giant screening or early diagnosis reagent

Technical Field

The invention belongs to the field of obstetrical molecular biology, and particularly relates to application of serum exosome RNA in preparation of a pregnancy diabetic giant screening or early diagnosis reagent.

Background

Giant infants refer to infants with birth weights up to or exceeding 4000 grams, and are the most common poor outcome of pregnancy in Gestational Diabetes Mellitus (GDM). According to the recent statistics of the international diabetes union 2019, more than 15% of the live births worldwide are affected by hyperglycemia during pregnancy, of which 83.6% are GDM. In recent years, with the improvement of the living standard of China and the implementation of a 'comprehensive second-child' policy, more and more pregnant women with excessive weight or high age have increasingly developed GDM incidence, so that the incidence of GDM-related large children is also sharply increased, and the health of mothers and infants is seriously threatened. GDM giant children are in asymmetric metabolism, and are characterized in that the fat weight of the whole body is obviously increased, the volume of other tissues/organs is not obviously increased, the growth and development of the infants are influenced, and the occurrence risk of obesity or diabetes in the childhood adolescence and after-adulthood is greatly increased. The well-known "origin of health and disease development (DOHaD)" theory holds that the nutritional and fertility environments (e.g., intrauterine hyperglycemic environment, etc.) of the first 1000 days of life are critical to the health impact of an individual throughout their lifetime. Therefore, the early diagnosis, the early intervention and the timely and reasonable treatment have very important meanings for clinically controlling the occurrence of GDM giant children, reducing the mother-infant complications and delaying the development of GDM filial generation into obesity or diabetes.

Exosomes (Exosomes) are used as stable intercellular information communication carriers, and are vesicles formed by endocytosis of normal or pathological intracellular multivesicular bodies, and are fused with cell membranes and secreted outside the cells to form a group of vesicular vesicles with double-layer membrane structures, and the diameter of the vesicular vesicles is 30-150 nm. After the secretory cells are stimulated, exosomes released by stress can enter the receptor cells through the modes of near secretion, membrane fusion, ligand-receptor combination, phagocytosis and the like, and release a plurality of wrapped abnormally expressed bioactive substances, such as mRNA, non-coding RNA (including lncRNA), proteins and the like, so that the biological functions of the receptor cells are influenced, and the exosomes further participate in various physiological and pathological processes, namely, the exosomes play a role in information transfer between the secretory cells and the receptor cells. Research shows that the placenta trophoblast-derived specific exosome can be detected in pregnant mother plasma as early as 6 weeks of gestation, and the concentration of the placenta-derived exosome is continuously increased along with the progress of pregnancy; for GDM pregnant women in early, middle and late pregnancy, the concentration of placenta-derived exosomes is obviously higher than that of normal pregnant women in the same period, and the process that the placenta releases exosomes to the maternal circulation is prompted to be regulated by blood sugar.

The exosome is protected by a double-layer membrane structure, and the content such as lncRNA and the like wrapped by the exosome can be prevented from being degraded by ribonuclease, so that the exosome can be widely detected in various body fluids such as peripheral blood, cord blood, amniotic fluid, breast milk, saliva and the like, and can be used as a biomarker for good disease occurrence and development. However, no research report on exosome RNA as a large biomarker has been found at present. The mRNA GDF3 and lncRNA AC006064.4 from exosome sources are used for assisting in diagnosing the GDM giant baby, so that not only can early diagnosis and early intervention be better performed on the GDM giant baby, but also the test result is more sensitive and specific. The above shows the great potential of serum exosome-derived mRNA GDF3 and lncRNA AC006064.4 in early diagnosis and screening of GDM giant infants.

Disclosure of Invention

The invention aims to provide application of mRNA GDF3 and lncRNA AC006064.4 derived from serum exosomes, in particular application of the mRNA GDF3 and lncRNA AC006064.4, and particularly application of the mRNA GDM in giant child screening or early diagnosis. Researches prove that mRNA GDF3 and lncRNA AC006064.4 derived from serum exosomes can be used for auxiliary early diagnosis of GDM giant children, and the detection result has high sensitivity and good specificity. Therefore, the mRNA GDF3 and lncRNA AC006064.4 derived from serum exosomes can be used for screening and early diagnosis of GDM pregnant women at high risk.

The purpose of the invention can be realized by the following technical scheme:

application of a reagent for detecting mRNA GDF3 and lncRNA AC006064.4 from serum exosomes in preparation of a screening or early diagnosis reagent for pregnant women with huge gestational diabetes and high risk of babies.

Preferably, the reagent for detecting the mRNA GDF3 and the lncRNA AC006064.4 derived from the serum exosomes is a real-time fluorescent quantitative PCR detection reagent for quantitatively detecting the mRNA GDF3 and the lncRNA AC006064.4 derived from the serum exosomes.

Preferably, the reagent for detecting mRNA GDF3 and lncRNA AC006064.4 derived from serum exosomes comprises real-time fluorescent quantitative PCR specific primers for detecting mRNA GDF3 and lncRNA AC 006064.4.

As a preferred choice of the invention, the real-time fluorescent quantitative PCR specific primer for detecting mRNA GDF3 is shown as SEQ ID NO.3 and SEQ ID NO. 4; the real-time fluorescent quantitative PCR specific primers for detecting the lncRNA AC006064.4 are shown as SEQ ID NO.5 and SEQ ID NO. 6.

A screening or early diagnosis kit for pregnant women with high risk of gestational diabetes mellitus and giant children comprises reagents for detecting mRNA GDF3 and lncRNA AC006064.4 derived from serum exosomes.

Preferably, the kit comprises a real-time fluorescent quantitative PCR detection reagent for quantitatively detecting mRNA GDF3 and lncRNA AC006064.4 derived from serum exosomes.

As a preferred mode of the invention, the kit comprises a real-time fluorescent quantitative PCR specific primer for detecting mRNA GDF3 and lncRNA AC 006064.4.

As a preferable mode of the invention, the kit comprises specific primers for real-time fluorescent quantitative PCR for detecting mRNA GDF3 shown in SEQ ID NO.3 and SEQ ID NO.4 and specific primers for real-time fluorescent quantitative PCR for detecting IncRNA AC006064.4 shown in SEQ ID NO.5 and SEQ ID NO. 6.

As a preferred aspect of the present invention, the kit comprises:

(1) a reagent for reverse transcription of mRNA GDF3 and LncRNA AC015961.2 into cDNA using total RNA isolated and purified from serum exosomes as a template, comprising a reverse transcription buffer, deoxynucleotides in the base triphosphate, RNase inhibitor, MMLV reverse transcriptase and random primers for mRNA GDF3 and LncRNA AC 015961.2;

(2) the reagents used for real-time quantitative PCR of cDNA comprise a real-time fluorescent quantitative PCR specific primer for detecting mRNA GDF3, a real-time fluorescent quantitative PCR specific primer for detecting lncRNA AC006064.4, a U6snRNA internal reference specific PCR primer, a real-time fluorescent quantitative SYBR dye and enzyme-free water.

mRNA GDF3 real-time fluorescent quantitative PCR specific primers:

the forward primer is 5'-AAAAGGAGAGCAGCCATCCC-3' (SEQ ID NO.3)

The negative primer is 5'-CTTGGGGGCAATGATCCACT-3' (SEQ ID NO.4)

lncRNA AC006064.4 real-time fluorescent quantitative PCR specific primers:

the forward primer is 5'-GGGAGATTCAGTGTGGTGGG-3' (SEQ ID NO.5)

The negative primer is 5'-CTTTGTCAAGCTCATGGCCC-3' (SEQ ID NO.6)

U6snRNA internal reference specific PCR primers:

the forward primer is 5'-ATTGGAACGATACAGAGAAGATT-3' (SEQ ID NO.7)

The negative primer was 5'-GGAACGCTTCACGAATTTG-3' (SEQ ID NO. 8).

Has the advantages that:

the applicant collects 3 cases of umbilical vein blood of GDM giant children and normal control (table 1), and separates exosome to extract RNA; the result of screening differential RNA by using a high-throughput sequencing technology shows that the RNA expression profiles in two groups of umbilical vein blood exosomes have obvious difference. According to the principle of higher expression abundance, large difference between groups and small difference between groups, 5 mRNAs (NUTM1, GDF3, C17orf105, PROM1, NFE2L1),10 lncRNAs (AC073912.1, IL10RB-DT 19, lnc-FAM49B-4:1, lnc-ZFHX3-7:1, AC006064.4, lnc-HPS6-1:1, lnc-SEC11A-2:1, lnc-ZA 3-3:2, lnc-TNFSF 13-2: 1, lnc-SOX B-1: 1) and 10 circRNAs (CAP _ B, circ _ B, GDC _ B, GDF _ B, GDE _ B, GDF _ DT 2L-DT 19, GDN _ S685 _ 19, I, GDN _ S685 _ S2-2, GDN-1, I-2, 2-I, 2-I, GDC-I, and GDC-I-II, and the results were selected, and the results were confirmed in the primary group, and the blood was shown in the test, and the test, the blood was performed on the subjects showed significant increase in the subjects showed that the expression of the blood of the subjects showed significant expression of the subjects showed significant increase in the GDC-C-pK 2 group, GDC-C685-S685 group, GDC B, GDC 685-C B, GDC B group, GDC 685-2, GDC-2-C B, GDC B group, GDC B, GDC-2, GDC B group, GDC B, and the blood group, and the test, the test showed significant increase in the test, the test was increased in the test was shown in the test, the test was in the test, the test was shown in the test group, the test was confirmed in the test subjects showed a test group, the test subjects showed a test subjects showed significant increase in the test subjects showed a test subjects, while lnc-ZFHX3-7:1 expression was significantly reduced in the GDM large group (figure 1). We then analyzed the expression of differential RNAs (GDF3, PROM1, AC006064.4, lnc-HPS6-1:1, lnc-ZFHX3-7:1, and circ _0014635) in peripheral blood exosomes at 24-28 weeks gestation. And constructing an ROC curve, and evaluating the prediction effect of the expression of the genes on the GDM giant child occurrence risk. The results showed that only GDF3 expression and AC006064.4 expression in peripheral blood exosomes had better predictive effect (fig. 2, GDF3 expression: AUC ═ 0.78, 95% CI ═ 0.63-0.94, P ═ 0.002; AC006064.4 expression: AUC ═ 0.74, 95% CI ═ 0.58-0.90, P ═ 0.009). Subsequently, maternal characteristics of GDM cohorts and controls were compared in the validation set and we found that maternal age, fasting plasma glucose and 2h plasma glucose were significantly elevated in GDM cohorts (table 2). Therefore, maternal age, fasting plasma glucose, 2h plasma glucose, GDF3 expression and AC006064.4 expression in peripheral blood exosomes can be used as predictors of macrosomia. By analyzing the prediction ability of the different combinations of the above predictors for GDM giant children, it was found that a model comprising maternal age, fasting plasma glucose, 2h plasma glucose, GDF3 expression and AC006064.4 expression in peripheral blood exosomes had the best prediction ability (AUC ═ 0.86) (table 3 and fig. 3). The probability of occurrence of giant children is calculated by drawing a nomogram by using the predictive model formula as shown in fig. 4. The method extracts total RNA by separating exosomes from GDM pregnant woman serum, adopts fluorescent quantitative PCR to detect the expression of mRNA GDF3 and lncRNA AC006064.4, combines the age, fasting blood sugar and 2h blood sugar of the pregnant woman, predicts the occurrence risk of the giant, screens high risk groups, and makes early and rapid noninvasive diagnosis on the GDM giant. The invention has good specificity for GDM giant children, the specificity can reach 1.00, the sensitivity can reach 0.55, the expression levels of mRNA GDF3 and lncRNA AC006064.4 can be detected only by extracting RNA from serum exosomes, the operation is simple, and the stability is good. The method can be used for the early diagnosis of GDM giant infants, can be used for the large-scale screening of GDM pregnant women and the prediction of occurrence risk of the GDM giant infants, and provides powerful technical support for the early diagnosis and prediction of the GDM giant infants, so that a more personalized prevention and treatment scheme is adopted in time, the occurrence rate of the GDM giant infants is reduced to the maximum extent, poor pregnancy outcome is reduced, and the method has profound clinical significance and popularization.

Drawings

Figure 1. verification of differentially expressed mRNAs, lncRNAs and circRNAs by qPCR. P <0.01, P <0.001.

FIG. 2 Receiver Operating Characteristic (ROC) curves for GDF3 and AC006064.4 expression in peripheral blood exosomes.

FIG. 3 Receiver Operating Characteristic (ROC) curves for different models. Model 1: age of pregnant woman; model 2: age of pregnant woman + fasting blood sugar +2h blood sugar; model 3: GDF3 expression + AC006064.4 expression (peripheral blood exosomes); model 4: age of pregnant woman + fasting blood glucose +2h blood glucose + GDF3 expression + AC006064.4 expression (peripheral blood exosomes).

FIG. 4 is an alignment chart of risk of developing GDM macrosomia. The predictor factors included maternal age, fasting plasma glucose, 2h plasma glucose, expression of GDF3 and AC006064.4 in peripheral blood exosomes.

Detailed Description

The following examples are intended to further illustrate the invention without limiting it.

EXAMPLE 1 Collection of samples and working up of sample data

The applicant collected a large amount of peripheral blood samples of pregnant women of 24-28 gestational weeks and umbilical cord blood samples at the time of delivery (samples for research are collected at the same period, and sampling, subpackaging and storage conditions are uniform) from a maternal-child health care institute of Nanjing city from 1 month to 12 months in 2020, and selected 46 samples meeting the following standards as experimental samples by collating sample data.

1.24-28 pregnant women who are diagnosed with GDM by OGTT when collecting peripheral blood in gestational weeks;

2. pregnant women who born a newborn with a weight of more than 4000g after the term of the above-mentioned study subjects were defined as an experimental group (23 cases);

3. the weight of the newborn born by the study object after the full term does not exceed 4000g, the pregnant women matched with the age, BMI and gestational week of the experimental group are defined as a control (23 cases), and the conditions of the demographic data, clinical data and the like of the samples are collected systematically;

4. and collecting 5mL of peripheral blood and cord blood of an individual to be detected by adopting a blood procoagulant tube. Centrifuging at 1000rpm for 6min after blood collection, and storing serum in EP tube at-80 deg.C. GDM macrogol and control were used as screening set (table 1) for 3 pairs, and validation set (table 2) for 20 pairs.

Example 2 isolation of exosomes in serum, extraction and purification of RNA in exosomes

1. Separation of exosomes in serum: 100ul of Total Exosome Isolation Reagent was added to 500ul of each serum sample, vortexed and reacted at 4 ℃ for 30 min. Centrifugation was carried out at 10000g for 10min at room temperature. Exosomes were deposited at the bottom of the EP tube and resuspended in 200 ul PBS (a commercially available exosome isolation kit was selected);

2. extracting and purifying RNA in exosome (selecting a commercial exosome RNA separating and purifying kit):

2.1 extraction of RNA from exosomes: adding 200ul of 2X Denaturing Solution, mixing, incubating on ice for 5min, adding 400ul of acid-Phenol: Chloroform, and vortexing for 60 s. Centrifuging at 12000g for 10min at room temperature to obtain supernatant containing RNA;

2.2 purification of RNA: 300ul of the supernatant was pipetted into an enzyme-free EP tube, 375ul of absolute ethanol was added, and the two were mixed well. Adding the mixed solution into a filter column, centrifuging for 15s at 10000g, and pouring the mixed solution in a collecting pipe. 700ul of Wash Solution 1 was added and centrifuged at 10000g for 15s at room temperature, and the mixture in the collection tube was decanted. 500ul of Wash Solution 2/3 was added and centrifuged at 10000g for 15s at room temperature, and the procedure was repeated. The filter column was put into a collection tube and centrifuged at 10000g for 1 min. The filter column was put into a new collection tube, 35ul of Solution was added, and the mixture was centrifuged at 10000g for 30s at room temperature to obtain purified RNA.

Example 3 selection of GDM giant predictor

1. Extracting exosomes and isolating RNA from 3 pairs of GDM giant and normal control (screening set) umbilical vein blood; differential RNA was screened using high throughput sequencing technology. According to the principle of higher expression abundance, large difference between groups and small difference between groups, 5 mRNAs (NUTM1, GDF3, C17orf105, PROM1, NFE2L1),10 lncRNAs (AC073912.1, IL10RB-DT:19, lnc-FAM49B-4:1, lnc-ZFHX3-7:1, AC006064.4, lnc-HPS6-1:1, lnc-SEC11A-2:1, lnc-ZA 3-3:2, lnc-TNFSF B-2:1, lnc-SOX6-1:1) and 10 circRNAs (circ _0012756, circ _0041183, circ _0065086, circ _0014635, circ _0075695, circ _0074153, rc _0068824, umbilical vein 0066837, venous 0066837 _0066837, GDM 0066837-0066837, and venous 0066837-0066837 pairs of umbilical cord blood were selected Child vs. normal control) exosomes, quantitative results showed that GDF3, PROM1, AC006064.4, lnc-HPS6-1:1, and circ _0014635 were significantly increased in GDM macrohypothermia, whereas lnc-ZFHX3-7:1 were significantly decreased in GDM macrohypothermia (fig. 1). GDF3 inhttps://www.ebi.ac.uk/The ID number of (A) is ENST00000329913.4, the cDNA sequence is shown in SEQ ID NO.1, lncRNA AC006064.4 is shown inhttps://www.ebi.ac.uk/The ID number of (1) is ENST00000602946.1, and the cDNA sequence is shown in SEQ ID NO. 2.

2. Expression of differential RNAs (GDF3, PROM1, AC006064.4, lnc-HPS6-1:1, lnc-ZFHX3-7:1, and circ _0014635) in peripheral blood exosomes at 24-28 weeks of gestation was analyzed. And constructing an ROC curve, and evaluating the prediction effect of the expression of the genes on the GDM giant child occurrence risk. The results showed that only GDF3 expression and AC006064.4 expression in peripheral blood exosomes had better predictive effect (GDF3 expression: AUC 0.78, 95% CI 0.63-0.94, P0.002; AC006064.4 expression: AUC 0.74, 95% CI 0.58-0.90, P0.009) (fig. 2).

3. In the validation set comparing the maternal characteristics of GDM cohort and control, we found that maternal age, fasting plasma glucose and 2h plasma glucose were significantly elevated in GDM cohort (table 2). Therefore, maternal age, fasting plasma glucose, 2h plasma glucose, GDF3 expression and AC006064.4 expression in peripheral blood exosomes can be used as predictors of macrosomia.

Example 4 reverse transcription and real-time quantification methods were used to detect mRNA GDF3 and lncRNA AC006064.4

1. Kit for preparing mRNA GDF3 and lncRNA AC006064.4 for GDM giant screening or early diagnosis (50 reactions), comprising:

1.1 enzyme-free Water 10ml

1.2 random reverse transcription primer (1uM) 50ul

1.35 XRT buffer 200ul

1.4 base triphosphate deoxynucleotides (10mM) 100ul

1.5 RNase inhibitor (40M/ul) 50ul

1.6 MMLV reverse transcriptase (200M/ul) 50ul

1.7 SYBR Premix Ex Taq 1000ul

1.8 real-time fluorescent quantitative PCR specific primer (10uM) for mRNA GDF3 (30 ul)

The forward primer is 5'-AAAAGGAGAGCAGCCATCCC-3'

The negative primer is 5'-CTTGGGGGCAATGATCCACT-3'

1.9 LncRNA AC006064.4 real-time fluorescent quantitative PCR specific primer (10uM) 30ul

The forward primer is 5'-GGGAGATTCAGTGTGGTGGG-3'

The negative primer is 5'-CTTTGTCAAGCTCATGGCCC-3'

1.10U 6snRNA internal reference specific PCR primer (10uM) 60ul

The forward primer is 5'-ATTGGAACGATACAGAGAAGATT-3'

The negative primer was 5'-GGAACGCTTCACGAATTTG-3'.

mRNA GDF3 and lncRNA AC006064.4 reverse transcription:

2.1 System for the first step of reverse transcription reaction comprises random reverse transcription primer (1uM)1ul, RNA sample 2ug, enzyme free water added to 12 ul. The condition of the first step of reverse transcription is 60 ℃ for 5 min;

2.2 the system of the second step of the reverse transcription reaction comprises 4ul of 5 Xreverse transcription buffer solution, 2ul of base triphosphate deoxynucleotide (10mM), 1ul of RNase inhibitor (40M/ul), 1ul of MMLV reverse transcriptase (200M/ul), and 12ul of PCR product of the first step. The second step of reverse transcription is performed at 25 deg.C for 5min, 42 deg.C for 60min, and 70 deg.C for 5 min.

3. Real-time quantitative PCR: firstly, the reverse transcription product is diluted by 5 times and mixed evenly. The reaction system (20ul) for mRNA GDF3 expression was as follows: SYBR Premix Ex Taq 10ul, GDF3 specific primer (10uM)0.5ul, cDNA product 1ul, enzyme free water added to 20 ul. Real-time fluorescent quantitative PCR reaction program: 95 ℃ for 3min, 40 cycles (95 ℃ for 10s, 60 ℃ for 30 s). The reaction system (20ul) for lncRNA AC006064.4 expression was as follows: SYBR Premix Ex Taq 10ul, AC006064.4 specific primer (10uM)0.5ul, cDNA product 1ul, enzyme free water added to 20 ul. Real-time fluorescent quantitative PCR reaction program: 95 ℃ for 3min, 40 cycles (95 ℃ for 10s, 60 ℃ for 30 s).

4. 2^-△△CtMeasurement of the index: the experimental data adopts a relatively quantitative analysis method, U6 is used as an internal reference gene, and the data is analyzed by R software.The analysis finds that: compared with the control group, the expression of mRNA GDF3 and lncRNA AC006064.4 in 20 cases of pregnant woman serum exosomes with GDM giant infants is obviously up-regulated. Meanwhile, ROC analysis shows that the expression levels of mRNA GDF3 and lncRNA AC006064.4 in serum exosomes are combined with the age, fasting blood glucose and 2h blood glucose of pregnant women, the Area Under the ROC Curve (AUC) for early diagnosis of GDM giant children can reach 0.86, the specificity can reach 1.00, and the sensitivity can reach 0.55. Therefore, the mRNA GDF3 and lncRNA AC006064.4 derived from serum exosomes can be better used for early diagnosis of GDM giant children.

The detection method can separate enough exosomes for detecting the expression level of mRNA GDF3 and lncRNA AC006064.4 by only 500ul of serum, which indicates that the method has better operability.

TABLE 1 basic clinical information for screening samples

Table 2 verification of basic clinical information of samples

TABLE 3 prediction of GDM macrosomia by different models

Sequence listing

<110> Nanjing city women and children health care hospital

Application of <120> serum exosome RNA in preparation of pregnancy diabetic giant child screening or early diagnosis reagent

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<211> 1236

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<213> human (Homo sapiens)

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aagaatatgt ctttctccaa tttctgggct tagataaggc gccttcaccc cagaagttcc 180

aacctgtgcc ttatatcttg aagaaaattt tccaggatcg cgaggcagca gcgaccactg 240

gggtctcccg agacttatgc tacgtaaagg agctgggcgt ccgcgggaat gtacttcgct 300

ttctcccaga ccaaggtttc tttctttacc caaagaaaat ttcccaagct tcctcctgcc 360

tgcagaagct cctctacttt aacctgtctg ccatcaaaga aagggaacag ttgacattgg 420

cccagctggg cctggacttg gggcccaatt cttactataa cctgggacca gagctggaac 480

tggctctgtt cctggttcag gagcctcatg tgtggggcca gaccacccct aagccaggta 540

aaatgtttgt gttgcggtca gtcccatggc cacaaggtgc tgttcacttc aacctgctgg 600

atgtagctaa ggattggaat gacaaccccc ggaaaaattt cgggttattc ctggagatac 660

tggtcaaaga agatagagac tcaggggtga attttcagcc tgaagacacc tgtgccagac 720

taagatgctc ccttcatgct tccctgctgg tggtgactct caaccctgat cagtgccacc 780

cttctcggaa aaggagagca gccatccctg tccccaagct ttcttgtaag aacctctgcc 840

accgtcacca gctattcatt aacttccggg acctgggttg gcacaagtgg atcattgccc 900

ccaaggggtt catggcaaat tactgccatg gagagtgtcc cttctcactg accatctctc 960

tcaacagctc caattatgct ttcatgcaag ccctgatgca tgccgttgac ccagagatcc 1020

cccaggctgt gtgtatcccc accaagctgt ctcccatttc catgctctac caggacaata 1080

atgacaatgt cattctacga cattatgaag acatggtagt cgatgaatgt gggtgtgggt 1140

aggatgtcag aaatgggaat agaaggagtg ttcttagggt aaatctttta ataaaactac 1200

ctatctggtt tatgaccact tagatcgaaa tgtcaa 1236

<210> 2

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<213> human (Homo sapiens)

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ggttgagcac agggtacttt attgatggta catgacaagg tgcggctccc taggcccctc 60

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tgagtgtggc agggactccc cagcagtgag ggtctctctc ttcctcttgt gctcttgctg 180

gggctggtgg tccaggggtc ttactccttg gaggccatgt gggccatgag cttgacaaag 240

tggtcgttga gggcaatgcc agccccagcg tcaaaggtgg aggagtgggt gtcgctgttg 300

aagtcagagg agaccacctg gtgctcagtg tagcccagga tgcccttgag ggggccctcc 360

gacgcctgct tcaccacctt cttgatgtca tcatatttgg caggtttttc tagacggcag 420

gt 422

<210> 3

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aaaaggagag cagccatccc 20

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<213> Artificial Sequence (Artificial Sequence)

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cttgggggca atgatccact 20

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gggagattca gtgtggtggg 20

<210> 6

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ctttgtcaag ctcatggccc 20

<210> 7

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

attggaacga tacagagaag att 23

<210> 8

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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ggaacgcttc acgaatttg 19

Claims

1. Application of a reagent for detecting mRNA GDF3 and lncRNA AC006064.4 from serum exosomes in preparation of a screening or early diagnosis reagent for pregnant women with huge gestational diabetes and high risk of babies.

2. The use according to claim 1, characterized in that the reagent for detecting serum exosome-derived mRNA GDF3 and lncRNA AC006064.4 is a real-time fluorescent quantitative PCR detection reagent for quantitatively detecting serum exosome-derived mRNA GDF3 and lncRNA AC 006064.4.

3. The use according to claim 2, characterized in that the reagents for detecting serum exosome-derived mRNA GDF3 and lncRNA AC006064.4 comprise real-time fluorescent quantitative PCR specific primers for detecting mRNA GDF3 and lncRNA AC 006064.4.

4. The use according to claim 3, characterized in that the real-time fluorescent quantitative PCR specific primers for detecting mRNA GDF3 are shown in SEQ ID No.3 and SEQ ID No. 4; the real-time fluorescent quantitative PCR specific primers for detecting the lncRNA AC006064.4 are shown as SEQ ID NO.5 and SEQ ID NO. 6.

5. A screening or early diagnosis kit for pregnant women with high risk of gestational diabetic giant children is characterized by comprising reagents for detecting mRNA GDF3 and lncRNA AC006064.4 derived from serum exosomes.

6. The use according to claim 5, characterized in that the kit comprises real-time fluorescent quantitative PCR detection reagents for quantitatively detecting serum exosome-derived mRNA GDF3 and lncRNA AC 006064.4.

7. The use according to claim 6, wherein the kit comprises real-time fluorescent quantitative PCR specific primers for detection of mRNA GDF3 and IncRNA AC 006064.4.

8. The use according to claim 7, characterized in that the kit comprises the real-time fluorescent quantitative PCR specific primers shown in SEQ ID No.3 and SEQ ID No.4 for detecting mRNA GDF3 and the real-time fluorescent quantitative PCR specific primers shown in SEQ ID No.5 and SEQ ID No.6 for detecting IncRNA AC 006064.4.

9. The use according to claim 8, characterized in that said kit comprises: