Diagnostic kit for detecting preeclampsia by qPCR (quantitative polymerase chain reaction) method and diagnostic method thereof

Abstract

The invention relates to a diagnostic kit for detecting preeclampsia by a qPCR (quantitative polymerase chain reaction) method, which is characterized in that a preeclampsia biomarker is PPAP-A, a detection primer group is provided according to a gene sequence of the PPAP-A and a human housekeeping gene beta-actin sequence, a forward primer of the PPAP-A is shown as SEQ ID NO.1, a reverse primer of the PPAP-A is shown as SEQ ID NO.2, a reverse primer of the PPAP-A is shown as SEQ ID NO.3, and a reverse primer of the PPAP-A is shown as SEQ ID NO.4. A reverse primer of the PPAP-A is as shown in SEQ ID NO. 2; a fluorescent probe of the PPAP-A 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 technical field of biological detection, and in particular to a diagnostic kit for detecting preeclampsia by qPCR method and a diagnostic method thereof. Background Art

[0002] Preeclampsia (PE) is a pregnancy complication that usually occurs after 20 weeks of pregnancy and is mainly characterized by the appearance of high blood pressure and protein in the urine. Its mechanism of occurrence is not fully understood, but it is related to factors such as placental maldevelopment and abnormal maternal immune response. Symptoms of preeclampsia include high blood pressure (usually blood pressure over 140 / 90 mmHg), protein in the urine, edema of the hands, feet or face, and possible headaches, blurred vision, upper abdominal pain, nausea or vomiting.

[0003] Preeclampsia poses significant risks to the health of both the mother and the fetus. For the mother, severe preeclampsia may cause organ damage, especially to the liver and kidneys, and may even develop into eclampsia, which manifests as seizures and is life-threatening. In addition, the risk of placental abruption increases, which may lead to bleeding and fetal hypoxia. In the long term, women who experience preeclampsia are also at increased risk of future cardiovascular disease. For the fetus, preeclampsia may lead to growth restriction because insufficient blood flow to the mother affects the nutrient supply to the fetus. In addition, in order to protect the health of the mother and baby, an early cesarean section may be required, increasing the risk of premature birth. The fetus may also be in distress due to lack of oxygen, affecting its health and development. Therefore, preeclampsia is a pregnancy complication that requires great attention, and timely monitoring and intervention are key to ensuring the safety of mother and baby.

[0004] Pregnancy-Associated Plasma Protein A (PAPP-A) is a protein produced by the placenta and the mother during pregnancy, and its main function is to regulate the activity of growth factors. Studies have shown that there is a certain relationship between PAPP-A and preeclampsia, and changes in its expression at different stages of pregnancy may be associated with the risk of preeclampsia. In normal pregnancy, the level of PAPP-A is usually high in early pregnancy (such as 11-14 weeks in the first trimester), and gradually increases as pregnancy progresses, especially in the second and third trimesters. However, in patients with preeclampsia, the level of PAPP-A often becomes abnormal, usually manifested as low levels of PAPP-A in early pregnancy. This low level of PAPP-A may be related to factors such as placental dysplasia and abnormal maternal immune response to the placenta, thereby increasing the risk of preeclampsia.

[0005] In addition, the study also found that low levels of PAPP-A may be associated with fetal growth restriction and placental insufficiency, which further increase the risk of preeclampsia. Therefore, monitoring changes in the level of pregnancy-related serum protein A, especially in early pregnancy, may help to identify high-risk pregnancies early and take appropriate intervention measures to reduce the risk of preeclampsia and its related complications.

[0006] Based on this, this patent provides a detection method for determining whether a pregnant woman in the early stages of pregnancy (11-14 weeks) suffers from preeclampsia based on the expression level of the PAPP-A gene. Summary of the invention

[0007] The present invention aims to solve the above problems and provides a diagnostic kit for detecting preeclampsia by qPCR, wherein the preeclampsia biomarker is PPAP-A, and a detection primer set is provided according to the gene sequence of PPAP-A and the sequence of human housekeeping gene β-actin, wherein The forward primer of PPAP-A is shown in SEQ ID NO.1; the reverse primer of PPAP-A is shown in SEQ ID NO.2; the fluorescent probe of PPAP-A 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.

[0008] SEQ ID NO.1: 5'-CCCTCCCTGCCTTCTGAC-3' (PPAP-AF); SEQ ID NO.2: 5'-CCTGCCAGTTGGTGAAGC-3' (PPAP-AR); SEQ ID NO.3: 5'-AGCAGACTCCAGCCATTCCATTATCATTCA-3' (PPAP-AP); SEQ ID NO.4: 5'- GCCAGGTCATCACCATTGG-3' (β-actin-F); SEQ ID NO.5: 5'-CAGAGCCGGGAGACAGTC-3' (β-actin-R); SEQ ID NO.6: 5'-CCACTCACCCAGGAAGGAAGGCT-3' (β-actin-P); Based on the above technical solution, the fluorescent group VIC is added to the 5' end of the PPAP-A fluorescent probe, and the quenching group BHQ1 is added to the 3' end of the PPAP-A fluorescent probe; the fluorescent group ROX 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 10×Hifair® V Lyo-Enzyme Mix, 4×Hifair® V Lyo-Buffer, 1-5μM PPAP-A upstream primer, 1-5μM PPAP-A downstream primer, 1-5μM PPAP-A fluorescent probe, 1-5μM β-actin upstream primer, 1-5μM β-actin downstream primer, 1-5μM β-actin fluorescent probe, DEPC water is filled to 20μL, and 5μL of template is added, and the template is the nucleic acid extracted from the subject's venous blood.

[0011] Further preferably, the PCR reagents include 10×Hifair® V Lyo-Enzyme Mix (1X), 4×Hifair® V Lyo-Buffer (1X), 5μM PPAP-A upstream primer, 5μM PPAP-A downstream primer, 5μM PPAP-A fluorescent probe, 5μM β-actin upstream primer, 5μM β-actin downstream primer, 5μM β-actin fluorescent probe, DEPC water is made up to 20μL, and 5μL of template is added, and the template is 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 is obtained by mixing equal volumes of PPAP-A artificial synthetic plasmid and β-actin artificial synthetic plasmid at a concentration of 1×106 copies / mL; the negative quality control is TE Buffer.

[0013] Based on the same invention, the present application also provides a diagnostic method for detecting preeclampsia by qPCR, characterized in that the diagnostic kit as described in any one of claims 1 to 5 is used, and the reaction program is constructed as follows: 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.

[0014] The present invention has the following advantages / benefits: The innovative use of molecular biology methods to diagnose preeclampsia in pregnant women helps to detect and treat the disease in a timely manner. Doctors can assess and manage the disease more effectively, thereby developing targeted treatment plans, including drug therapy and lifestyle adjustments, providing a basis for early intervention and customized treatment, making early risk assessment and intervention in early pregnancy possible, helping to prevent disease deterioration, reduce the risk of complications, and ensure the safety of mother and child. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] 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.

[0016] Figure 1 This is a graph showing the experimental results of adding different amounts of primers to PCR reagents; Figure 2 This is a performance comparison result diagram between different amplification programs of the kit; Figure 3 : The sensitivity amplification result diagram of the kit of the present invention; Figure 4 : Specific amplification result diagram of the kit of the present invention. DETAILED DESCRIPTION

[0017] 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.

[0018] Example 1: Primer set for detecting PPAP-A expression First, based on the gene sequence of PPAP-A 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: SEQ ID NO.1: 5'-CCCTCCCTGCCTTCTGAC-3' (PPAP-AF); SEQ ID NO.2: 5'-CCTGCCAGTTGGTGAAGC-3' (PPAP-AR); SEQ ID NO.3: 5'-VIC-AGCAGACTCCAGCCATTCCATTATCATTCA-BHQ1-3' (PPAP-AP); SEQ ID NO.4: 5'- GCCAGGTCATCACCATTGG-3' (β-actin-F); SEQ ID NO.5: 5'-CAGAGCCGGGAGACAGTC-3' (β-actin-R); SEQ ID NO.6: 5'-ROX-CCACTCACCCAGGAAGGAAGGCT-BHQ2-3' (β-actin-P); The primers and probe plasmids shown were commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0019] Example 2: Establishment of a diagnostic kit and diagnostic method for preeclampsia The primer composition provided in Example 1 was used to prepare the reagents.

[0020] PCR reagent components include: 10×Hifair® V Lyo-Enzyme Mix (Yishen Biotechnology (Shanghai) Co., Ltd.), 4×Hifair® V Lyo-Buffer (Yishen Biotechnology (Shanghai) Co., Ltd.), 1-5μM PPAP-A upstream primer, 1-5μM PPAP-A downstream primer, 1-5μM PPAP-A 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 nucleic acid extracted from the subject's venous blood is added.

[0021] All primers and plasmids were from Sangon Biotechnology (Shanghai) Co., Ltd.

[0022] The quality control reagents include positive quality control and negative quality control; the positive quality control has a concentration of 1×10 6 The artificial synthetic plasmid PPAP-A and the artificial synthetic plasmid β-actin were mixed in equal volumes; the negative quality control was TEBuffer.

[0023] 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.

[0024] Example 3: Optimization of the amount of primers added to the kit The PCR reagent preparation method is as follows: ① Preparation of PCR reagents for control group 1; 10×Hifair® V Lyo-Enzyme Mix (Yisheng Biotechnology (Shanghai) Co., Ltd.) (1X), 4×Hifair® V Lyo-Buffer (Yisheng Biotechnology (Shanghai) Co., Ltd.) (1X), 5μM PPAP-A upstream primer, 5μM PPAP-A downstream primer, 2.5μM PPAP-A 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.

[0025] ② Preparation of PCR reagents for control group 2; 10×Hifair® V Lyo-Enzyme Mix (Yisheng Biotechnology (Shanghai) Co., Ltd.) (1X), 4×Hifair® V Lyo-Buffer (Yisheng Biotechnology (Shanghai) Co., Ltd.) (1X), 5μM PPAP-A upstream primer, 5μM PPAP-A downstream primer, 5μM PPAP-A 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.

[0026] 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.

[0027] Figure 1 Experimental results of adding different amounts of primers to PCR reagents 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.

[0028] Example 4: Optimization of kit amplification procedure The kit amplification procedure is as follows: ①Control group 1: 37℃ for 5 min, 95℃ for 5 min, 95℃ for 15 s, 60℃ for 30 s, and cycle 45 times.

[0029] ②Control group 2: 37℃ for 5 min, 95℃ for 5 min, 95℃ for 15 s, 55℃ for 30 s, and cycle 45 times.

[0030] 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.

[0031] Figure 2 Figure 2 shows the performance comparison results between different amplification programs of the kit. 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.

[0032] Example 5: Establishment of reference range of PPAP-A expression Thirty patients with preeclampsia (early pregnancy: 11-14 weeks) were selected as the PE group, and another 30 healthy pregnant women were selected as the healthy control group. 3 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 was free of DNA influence. Real-time fluorescence quantitative PCR was used to detect the expression of PPAP-A mRNA. 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 .

[0033] As shown in the experimental data in Table 1, the expression level of PPAP-A in the blood of the PE group was significantly lower than that of the control group. According to the experimental data, the relative expression level of PPAP-A mRNA in PE patients ranged from 0.08 to 0.49. Pregnant women within this range have a high risk of preeclampsia. The expression level of PPAP-A mRNA in the blood of the subjects can be used to determine whether they are preeclampsia patients.

[0034] Table 1 is the relative expression data of PPAP-A mRNA in preeclampsia samples and healthy samples

[0035] Example 6: Determination of sensitivity of diagnostic kit for preeclampsia The sample No. 1 of the control group was selected for nucleic acid extraction and RNA quantification, with a concentration of 5*10 3cp / 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.

[0036] Figure 3 The sensitivity amplification result of the kit is shown in the figure The results show that 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 The kit has high sensitivity and can detect targets at lower concentrations, thus improving the accuracy of the test and detecting molecular weight changes at the early stages of the disease, which helps to achieve early intervention and treatment. This is very important for diagnosing diseases, evaluating conditions, and judging treatment effects.

[0037] Example 7: Diagnostic Kit Specific Test for Preeclampsia 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.

[0038] Figure 4 The results of the kit-specific amplification are shown in Figure 2. 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.

[0039] Example 8: Real sample testing of the diagnostic kit for preeclampsia 20 samples of venous blood from pregnant women were randomly selected for testing, and the test method described in Example 2 was used for testing. Samples 4, 11, and 17 were diagnosed as high risk for preeclampsia, and the remaining 17 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 4, 17, and 22 were diagnosed as preeclampsia by blood pressure and urine protein tests, and sample 11 did not suffer from preeclampsia. The clinical sensitivity calculation formula of this kit is: sensitivity = A / (A+C)×100%=3 / 4*100%=75% (where A is the number of samples that are positive for the kit and the gold standard is judged as positive, and C is the number of samples that are positive for the kit and the gold standard is judged as negative), and the clinical specificity is: clinical specificity = 1-A / (A+C)×100%=1-1 / 16*100%=93.8% (A is the number of patients who are negative for the kit and positive for the gold standard, and C is the number of patients who are negative for the kit and negative for the gold standard).

[0040] Table 2 is the data table of PPAP-A mRNA expression in 20 pregnant women's venous blood samples

[0041] Table 3 is the clinical sensitivity and clinical specificity data of the kit

[0042] The clinical sensitivity of the test kit is 3 / 4*100%=75% The clinical specificity of the test kit is 15 / 16*100%=93.8% 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 detecting preeclampsia by qPCR, characterized in that: The preeclampsia biomarker is PPAP-A, and a detection primer set is provided according to the gene sequence of PPAP-A and the sequence of the human housekeeping gene β-actin, wherein The forward primer of PPAP-A is shown in SEQ ID NO.1; the reverse primer of PPAP-A is shown in SEQ ID NO.2; the fluorescent probe of PPAP-A 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 detecting preeclampsia by qPCR according to claim 1, characterized in that: The fluorescent group VIC is added to the 5' end of the fluorescent probe of PPAP-A, and the quenching group BHQ1 is added to the 3' end of the fluorescent probe of PPAP-A; the fluorescent group ROX is added to the 5' end of the fluorescent probe of β-actin, and the quenching group BHQ2 is added to the 3' end of the fluorescent probe of β-actin.

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

4. A diagnostic kit for detecting preeclampsia by qPCR according to claim 3, characterized in that: The PCR reagents include 10×Hifair® V Lyo-Enzyme Mix (1X), 4×Hifair® V Lyo-Buffer (1X), 1-5μM PPAP-A upstream primer, 1-5μM PPAP-A downstream primer, 1-5μM PPAP-A fluorescent probe, 1-5μM β-actin upstream primer, 1-5μM β-actin downstream primer, 1-5μM β-actin fluorescent probe, DEPC water is made up to 20μL, and 5μL of template is added.

5. A diagnostic kit for detecting preeclampsia by qPCR according to claim 3, characterized in that: The quality control reagents include positive quality control and negative quality control. The positive quality control is obtained by mixing equal volumes of PPAP-A artificial synthetic plasmid and β-actin artificial synthetic plasmid at a concentration of 1×106 copies / mL; the negative quality control is TE Buffer.

6. A diagnostic method for detecting preeclampsia by qPCR, characterized in that: The diagnostic kit as described in any one of claims 1 to 5 is used, and the reaction program is constructed as follows: 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.

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