Diagnostic kit for preeclampsia and diagnostic method thereof

Abstract

The invention relates to a diagnostic kit for preeclampsia, a biomarker of the preeclampsia is Syncytin-1, a detection primer group is designed according to a gene sequence of the Syncytin-1 and a human housekeeping gene beta-actin sequence, a forward primer of the Syncytin-1 is as shown in SEQ ID NO.1, a reverse primer of the Syncytin-1 is as shown in SEQ ID NO.2, a reverse primer of the Syncytin-1 is as shown in SEQ ID NO.3, and a reverse primer of the Syncytin-1 is as shown in SEQ ID NO.4. A reverse primer of the Syncytin-1 is as shown in SEQ ID NO. 2; the fluorescent probe of the Syncytin-1 is as shown in SEQ ID NO. 3; a forward primer of beta-actin is as shown in SEQ ID NO. 4; a reverse primer of the beta-actin is as shown in SEQ ID NO. 5; and a fluorescent probe of beta-actin is as shown in SEQ ID NO. 6. According to the invention, a molecular biology method is innovatively adopted to diagnose preeclampsia of the pregnant woman, so that a basis is provided for early intervention and customized treatment, and early risk assessment and intervention in an early pregnancy period become possible.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a diagnostic kit for preeclampsia and a diagnostic method thereof. Background Art

[0002] Preeclampsia (PE) refers to the occurrence of high blood pressure and proteinuria after 20 weeks of pregnancy, and may also cause symptoms such as headache, dizziness, nausea, vomiting, and upper abdominal discomfort. It is a multi-system disorder syndrome unique to pregnant women in late pregnancy and one of the important causes of maternal and perinatal illness and death worldwide. The pathogenesis of preeclampsia is not very clear. It is currently believed that its pathogenesis mainly includes genetic defects, damage to the placental microenvironment, and immune abnormalities. The three mechanisms act alone or in combination in different patients, leading to abnormal placental function. Among them, immune and genetic factors are increasingly attracting people's attention.

[0003] Syncytin-1 is mostly expressed in trophoblasts, and it acts on many aspects such as placental barrier and endocrine regulation. Syncytin-1 is closely related to cell proliferation and apoptosis. Studies have found that the concentration usually increases with the increase of gestational age and drops sharply in the late pregnancy, suggesting that it acts on the process of placental formation. However, the expression level of Syncytin-1 in maternal blood and placenta of patients with preeclampsia is lower than that in the normal group, and the expression of Syncytin-1 in the early-onset PE (gestational age of onset <34 weeks) group is lower than that in the late-onset PE (gestational age of onset ≥34 weeks) group, suggesting that Syncytin-1 is closely related to the severity of PE.

[0004] Based on this, the present invention provides a detection method for judging whether a pregnant woman is a patient with early-onset preeclampsia (gestational age of onset <34 weeks) according to the expression level of the Syncytin-1 gene. Summary of the invention

[0005] The present invention aims to solve the above problems and provides a diagnostic kit for preeclampsia. The preeclampsia biomarker is Syncytin-1. According to the gene sequence of Syncytin-1 and the sequence of human housekeeping gene β-actin, a detection primer set is designed, wherein

[0006] The forward primer of syncytin-1 is shown in SEQ ID NO.1; the reverse primer of syncytin-1 is shown in SEQ ID NO.2; the fluorescent probe of syncytin-1 is shown in SEQ ID NO.3; the forward primer of β-actin is shown in SEQ ID NO.4; the reverse primer of β-actin is shown in SEQ ID NO.5; the fluorescent probe of β-actin is shown in SEQ ID NO.6. The details are shown in the following table:

[0007] Sequence number name sequence SEQ ID NO.1 Syncytin-1-F 5'-GCGGACTTGTGGAGACAG-3' SEQ ID NO.2 Syncytin-1-R 5'-AGAGTTCAGAAACAGCTACTGG-3' SEQ ID NO.3 Syncytin-1-P 5'-CGCTCACCTTGGCTATTCAGTTGTTGC-3' SEQ ID NO.4 β-actin-F 5'-CCTCTCAGGCATGGAGTCC-3' SEQ ID NO.5 β-actin-R 5'-TTGGCGTAGAGGTCCTTCC-3' SEQ ID NO.6 β-actin-P 5'-CGGCATCCACGAGACCACCTTCAA-3'

[0008] Based on the above technical solution, the fluorescent group ROX is added to the 5' end of the Syncytin-1 fluorescent probe, and the quenching group BHQ2 is added to the 3' end of the Syncytin-1 fluorescent probe; the fluorescent group VIC is added to the 5' end of the β-actin fluorescent probe, and the quenching group BHQ2 is added to the 3' end of the β-actin fluorescent probe.

[0009] Based on the above technical solution, the diagnostic kit includes PCR reagents and quality control reagents.

[0010] Based on the above technical solution, the PCR reagent includes V Lyo-Enzyme Mix, V Lyo-Buffer, 1-5μM Syncytin-1 forward primer, 1-5μM Syncytin-1 reverse primer, 1-5μM Syncytin-1 fluorescent probe, 1-5μM β-actin forward primer, 1-5μM β-actin reverse primer, 1-5μM β-actin fluorescent probe, DEPC water is made up to 20μL, and 5μL of template is added, where the template is the nucleic acid extracted from the subject's venous blood.

[0011] Further preferably, the PCR reagent comprises V Lyo-Enzyme Mix (1X), V Lyo-Buffer (1X), 5μM Syncytin-1 forward primer, 5μM Syncytin-1 reverse primer, 5μM Syncytin-1 fluorescent probe, 5μM β-actin forward primer, 5μM β-actin reverse primer, 5μM β-actin fluorescent probe, DEPC water is made up to 20μL, and 5μL of template is added, wherein the template is the nucleic acid extracted from the subject's venous blood.

[0012] Furthermore, the quality control reagent includes a positive quality control and a negative quality control; the positive quality control has a concentration of 2×106 The synthetic plasmids were prepared by mixing equal volumes of 100 copies / mL Syncytin-1 and 100 copies / mL synthetic plasmids of β-actin; the negative quality control was TE Buffer.

[0013] Based on the same invention, the present application also provides a diagnostic method for preeclampsia, using the above-mentioned diagnostic kit, and constructing a reaction program as follows: 37°C reaction for 5 minutes, 95°C reaction for 5 minutes, 95°C reaction for 15 seconds, 60°C reaction for 30 seconds, and 45 cycles.

[0014] The specific operation was as follows: healthy pregnant women and patients with preeclampsia were selected as controls, 6 ml of fasting venous blood was drawn from the two groups of subjects, RNA was extracted using a blood RNA extraction kit, and the expression of syncytin-1 mRNA was detected by real-time fluorescence quantitative PCR. The mRNA content was calculated as △Ct target gene = Ct target gene - Ct internal reference gene, △△Ct = △Ct target gene - △Ct standard value, and the relative expression of the target gene was 2 -△△Ct Related studies have shown that the expression of Syncytin-1 in the blood of the PE group was significantly lower than that of the control group. According to experimental data, the relative expression range of Syncytin-1 mRNA in PE patients was 0.02-0.53, that is, a relative expression of Syncytin-1 mRNA of 0.02-0.53 indicates a high risk of preeclampsia, and >0.53 indicates a low risk of preeclampsia.

[0015] The present invention has the following advantages / benefits:

[0016] Traditional diagnostic methods for preeclampsia rely on clinical symptoms such as blood pressure and proteinuria, which often only manifest in the middle and late stages. At this time, the intervention effect is limited and may even cause irreversible damage. The present invention innovatively uses molecular biology methods to diagnose preeclampsia in pregnant women, thereby providing a basis for early intervention and customized treatment, making it possible to conduct early risk assessment and intervention in early pregnancy. According to the results of risk assessment, early intervention can be carried out on high-risk pregnant women, such as low-dose aspirin prevention, lifestyle guidance, etc., thereby reducing the risk and severity of preeclampsia and ensuring the safety of mother and child. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0018] Figure 1 : Experimental results of PCR reagents with different primer addition amounts of the present invention;

[0019] Figure 2 : Performance comparison result diagram between different amplification programs of the kit of the present invention;

[0020] Figure 3 : The sensitivity amplification result diagram of the kit of the present invention;

[0021] Figure 4 : Specific amplification result diagram of the kit of the present invention. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. The following embodiments are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. The experimental methods used in the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the present invention, unless otherwise specified, can be obtained from commercial channels. In addition, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0023] Example 1: Primer set for detecting syncytin-1 expression

[0024] First, based on the gene sequence of syncytin-1 and the sequence of the human housekeeping gene β-actin, a detection primer set was designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The specific sequence is as follows:

[0025]

[0026] The primers and probe plasmids shown were commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0027] Example 2: Establishment of a diagnostic kit and diagnostic method for preeclampsia

[0028] The primer composition provided in Example 1 was used to prepare the reagents.

[0029] PCR reagents include: V Lyo-Enzyme Mix (Yisheng Biotechnology (Shanghai) Co., Ltd.), V Lyo-Buffer (Yisun Biotechnology (Shanghai) Co., Ltd.), 1-5μM Syncytin-1 upstream primer, 1-5μM Syncytin-1 downstream primer, 1-5μM Syncytin-1 fluorescent probe, 1-5μM internal reference upstream primer, 1-5μM internal reference downstream primer, 1-5μM internal reference fluorescent probe, DEPC water is made up to 20μL, and 5μL of template is added, and the template is the nucleic acid extracted from the subject's venous blood.

[0030] All primers and plasmids were from Sangon Biotechnology (Shanghai) Co., Ltd.

[0031] The quality control reagents include positive quality control and negative quality control; the positive quality control is obtained by mixing equal volumes of Syncytin-1 artificial synthetic plasmid and β-actin artificial synthetic plasmid at a concentration of 1×106 copies / mL; the negative quality control is TE Buffer.

[0032] In a preferred embodiment of the present invention, the procedure of amplification detection is: reaction at 37°C for 5 min, reaction at 95°C for 5 min, reaction at 95°C for 15 s, reaction at 60°C for 30 s, and cycled 45 times.

[0033] Example 3: Optimization of the amount of primers added to the kit

[0034] The PCR reagent preparation method is as follows:

[0035] ① Preparation of PCR reagents for control group 1; V Lyo-Enzyme Mix (Yisheng Biotechnology (Shanghai) Co., Ltd.) (1X), V Lyo-Buffer (Yisun Biotechnology (Shanghai) Co., Ltd.) (1X), 5μM Syncytin-1 upstream primer, 5μM Syncytin-1 downstream primer, 2.5μM Syncytin-1 fluorescent probe, 5μM internal reference upstream primer, 5μM internal reference downstream primer, 2.5μM internal reference fluorescent probe, DEPC water to 20μL, add 5μL of the nucleic acid sample to be tested.

[0036] ② Preparation of PCR reagents for control group 2; V Lyo-Enzyme Mix (Yisheng Biotechnology (Shanghai) Co., Ltd.) (1X), V Lyo-Buffer (Yisun Biotechnology (Shanghai) Co., Ltd.) (1X), 5μM Syncytin-1 upstream primer, 5μM Syncytin-1 downstream primer, 5μM Syncytin-1 fluorescent probe, 5μM internal reference upstream primer, 5μM internal reference downstream primer, 5μM internal reference fluorescent probe, DEPC water to 20μL, add 5μL of the nucleic acid sample to be tested.

[0037] The PCR reagent prepared in Example 2 of the present invention, the prepared reagents of the control group 1 and the control group 2 were compared to compare the differences in performance.

[0038] Figure 1 Experimental results of adding different amounts of primers to PCR reagents

[0039] The results showed that the PCR reagent prepared with the primer ratio in the control group 1 was better than that in the control group 2 when the same template was amplified, with a higher plateau phase of the fluorescence curve and a relatively higher Ct value. Therefore, the primer addition amount in the control group 2 was selected as the optimal primer addition amount of the kit of the present invention.

[0040] Example 4: Optimization of kit amplification procedure

[0041] The kit amplification procedure is as follows:

[0042] ①Control group 1: 37℃ for 5 min, 95℃ for 5 min, 95℃ for 15 s, 60℃ for 30 s, and cycle 45 times.

[0043] ②Control group 2: 37℃ for 5 min, 95℃ for 5 min, 95℃ for 15 s, 55℃ for 30 s, and cycle 45 times.

[0044] The optimal PCR amplification reagent formula established in Example 3 was prepared, and the same sample to be tested was amplified according to the two amplification procedures described in Example 4 of the present invention, and the performance difference between the two procedures of control groups 1 and 2 was compared.

[0045] Figure 2 Figure 2 shows the performance comparison results between different amplification programs of the kit.

[0046] The results showed that the same sample was amplified according to the amplification procedures of control group 1 and control group 2, respectively. The Ct value of the amplification curve of control group 1 was significantly higher than that of control group 2, and the amplification effect was better. Therefore, the amplification procedure of control group 1 was selected as the optimal amplification procedure of the present invention.

[0047] Example 5: Establishment of reference range of syncytin-1 expression

[0048] Thirty patients with preeclampsia were selected as the PE group, and 30 healthy pregnant women were selected as the healthy control group. PE inclusion criteria: (1) All patients were diagnosed with preeclampsia (PE), the diagnostic criteria were all based on the "Obstetrics and Gynecology" (8th edition), and the disease occurred before 34 weeks of gestation; (2) The clinical data were complete; (3) All were singleton pregnancies.

[0049] 6 ml of fasting venous blood was drawn from the two groups of subjects, and RNA was extracted using the Kangwei Century Blood RNA Extraction Kit. DNA digestion enzymes were used during the extraction process to ensure that the product had no DNA influence. The expression of Syncytin-1 mRNA was detected by real-time fluorescence quantitative PCR. The mRNA content was calculated by △Ct target gene = Ct target gene - Ct internal reference gene, △△Ct = △Ct target gene - △Ct standard value, and the relative expression of the target gene was 2 -△△Ct .

[0050] Related studies have shown that the expression of Syncytin-1 in the blood of the PE group was significantly lower than that of the control group. According to the experimental data in Table 1, the relative expression range of Syncytin-1 mRNA in PE patients was 0.02-0.53. Pregnant women within this range have a high risk of preeclampsia. The expression level of Syncytin-1 mRNA in the blood of the subjects can be used to determine whether they are preeclampsia patients.

[0051] Table 1 is the relative expression data of Syncytin-1 mRNA in preeclampsia samples and healthy samples

[0052]

[0053]

[0054]

[0055]

[0056] Example 6 Sensitivity determination of a diagnostic kit for preeclampsia

[0057] The sample No. 1 of the control group was selected for RNA quantification after nucleic acid extraction, and the concentration was 10 4 cp / ml, the nucleic acid sample was diluted 10 times to obtain a concentration of 10 3 cp / ml, 10 2 cp / ml, 10 1 cp / ml, 10 0 cp / ml template, and perform real-time fluorescence quantitative PCR test according to Example 2.

[0058] The results are as follows Figure 1As shown, 10 1 The template detection rate of cp / ml is 100%, indicating that 10 1 copies / mL is the detection limit concentration, and the final detection limit of the primer combination of the kit is confirmed to be 1×10 1 This kit has high sensitivity and is more capable of detecting low-concentration targets, which means that changes in target gene expression can be detected at the early stage of the disease, thereby achieving early diagnosis and treatment. High sensitivity can reduce repeated experiments caused by insufficient sensitivity, thereby saving time and cost.

[0059] Example 7 Specificity test for diagnostic kit for preeclampsia

[0060] A specific experimental group for detecting the preeclampsia diagnostic kit was set up. After the nucleic acid of sample No. 1 of the control group was extracted, 5 μL of each was mixed with 1 μL of labetalol, nifedipine, nimodipine, and magnesium sulfate solution. The mixed samples were tested according to Example 2.

[0061] Figure 2 The results of the kit-specific amplification are shown in Figure 2.

[0062] The experimental results show that labetalol, nifedipine, nimodipine, and magnesium sulfate mixed samples can be detected, and the Ct value is consistent with that of unmixed samples. Labetalol, nifedipine, nimodipine, and magnesium sulfate are common drugs in the blood of patients with preeclampsia. The kit has good specificity and can accurately identify target genes. It is not affected by drugs in the sample, which can ensure the accuracy of the test results and reduce interference and false positives.

[0063] Example 8: Real sample test of the diagnostic kit for preeclampsia

[0064] 20 venous blood samples from pregnant women were randomly selected for testing, and the testing method described in Example 2 was used for testing. Samples 2, 13, 14, 16, 17, and 19 were diagnosed as high risk for preeclampsia, and the remaining 14 samples were diagnosed as low risk for preeclampsia. The 20 pregnant women were followed up and reexamined within 2 weeks, and all of them underwent blood pressure and urine protein tests. Samples 13, 16, 17, and 19 were diagnosed as preeclampsia, samples 2 and 14 did not suffer from preeclampsia, and sample 3 was diagnosed as preeclampsia. The calculation formula of clinical sensitivity of this kit is: sensitivity = A / (A+C)×100%=6 / 7*100%=85.7% (where A is the number of samples detected as positive by the kit and judged as positive by the gold standard, and C is the number of samples detected as positive by the kit and judged as negative by the gold standard), and clinical specificity is: clinical specificity = 1-A / (A+C)×100%=1-2 / 13*100%=84.6% (A is the number of patients detected as negative by the kit and positive by the gold standard, and C is the number of patients detected as negative by the kit and negative by the gold standard). The clinical sensitivity of this kit is 6 / 7*100%=85.7%, and the clinical specificity is 1-2 / 13*100%=84.6%.

[0065] Table 2 is the data table of Syncytin-1 mRNA expression in 20 pregnant women's venous blood samples

[0066]

[0067]

[0068] Table 3 is the clinical sensitivity and clinical specificity data of the kit

[0069]

[0070]

[0071] The clinical sensitivity of the test kit is 6 / 7*100%=85.7%

[0072] The clinical specificity of the test kit is 1-2 / 13*100%=84.6%

[0073] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A diagnostic kit for preeclampsia, characterized in that: The preeclampsia biomarker is Syncytin-1. According to the gene sequence of Syncytin-1 and the sequence of the human housekeeping gene β-actin, a detection primer set is designed, wherein The forward primer of Syncytin-1 is shown in SEQ ID NO.1; the reverse primer of Syncytin-1 is shown in SEQ ID NO.2; the fluorescent probe of Syncytin-1 is shown in SEQ ID NO.3; the forward primer of β-actin is shown in SEQ ID NO.4; the reverse primer of β-actin is shown in SEQ ID NO.5; and the fluorescent probe of β-actin is shown in SEQ ID NO.

6.

2. A diagnostic kit for preeclampsia according to claim 1, characterized in that: The fluorescent group ROX is added to the 5' end of the Syncytin-1 fluorescent probe, and the quenching group BHQ2 is added to the 3' end of the Syncytin-1 fluorescent probe; the fluorescent group VIC is added to the 5' end of the β-actin fluorescent probe, and the quenching group BHQ2 is added to the 3' end of the β-actin fluorescent probe.

3. A diagnostic kit for preeclampsia according to claim 1, characterized in that: The diagnostic kit comprises PCR reagents and quality control reagents.

4. A diagnostic kit for preeclampsia according to claim 3, characterized in that: The PCR reagents include V Lyo-Enzyme Mix, V Lyo-Buffer, 1-5μM Syncytin-1 forward primer, 1-5μM Syncytin-1 reverse primer, 1-5μM Syncytin-1 fluorescent probe, 1-5μM β-actin forward primer, 1-5μM β-actin reverse primer, 1-5μM β-actin fluorescent probe, DEPC water to 20μL, add 5μL template.

5. A diagnostic kit for preeclampsia according to claim 3, characterized in that: The quality control reagents include positive quality control and negative quality control; the positive quality control has a concentration of 2×10 6 The synthetic plasmids were prepared by mixing equal volumes of 100 copies / mL Syncytin-1 and 100 copies / mL synthetic plasmids of β-actin; the negative quality control was TE Buffer.

6. A method for diagnosing preeclampsia, characterized in that: The diagnostic kit according to any one of claims 1 to 5 is used, and the reaction program is constructed as follows: 37° C. for 5 min, 95° C. for 5 min, 95° C. for 15 s, 60° C. for 30 s, and cycled 45 times.

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