Cervical cancer auxiliary diagnosis detection kit and application thereof

Abstract

The invention discloses a cervical cancer auxiliary diagnosis detection kit and application thereof, and belongs to the technical field of molecular diagnosis, the kit comprises a substance for detecting methylation modification of CNTNAP2 and PEX5L genes, GRCh38 is taken as a reference genome, the detection area of the CNTNAP2 gene is located between 145813214-145813565 of a chromosome 7, and the detection area of the PEX5L gene is located between 179754514-179754949 of a chromosome 3. The two gene targets are combined for cervical cancer methylation detection for the first time, and the scheme has the characteristics of high sensitivity, high specificity, simplicity, convenience and rapidness in operation and the like, and provides a new choice for clinical auxiliary diagnosis, early screening and clinical shunt of cervical cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular diagnosis, and in particular to a cervical cancer auxiliary diagnosis detection kit developed based on methylation gene targets CNTNAP2 and PEX5L and an application thereof. Background Art

[0002] Gynecological tumors are the primary disease that threatens women's health, and cervical cancer is the most common gynecological tumor, ranking first among malignant tumors of the female reproductive system. It has a high morbidity and mortality rate, and the incidence rate is increasing year by year. At present, common detection methods for cervical cancer include hrHPV detection and cytology (TCT) examination. These detection methods are widely used in clinical practice, but they all have some limitations. For example, a negative result of hrHPV detection can indicate a low possibility of cancer in the short term, but since many women will recover on their own after infection, the significance of a positive result is not clear. At the same time, there is also excessive diagnosis and treatment, which adds a burden to patients and the medical system. Cytology collects exfoliated cells from the cervix for observation and analysis under a microscope to determine whether there are diseases such as cervical inflammation, precancerous lesions or cervical cancer. The test results are affected by the technical level of laboratory personnel, have a certain degree of subjectivity, and the clinical sensitivity is relatively low, and there may be missed diagnosis. Therefore, the early diagnosis of cervical cancer still faces great challenges. Therefore, it is still urgent to find an efficient, accurate and simple early diagnosis method.

[0003] In recent years, methylation detection, as a new molecular diagnostic technology, has shown potential clinical application value, and has attracted much attention in the clinical diagnosis and auxiliary diversion of cervical cancer. Several studies have shown that the detection of methylation of related genes in samples such as human tissues or exfoliated cells has a high clinical application value for screening cervical cancer and precancerous lesions, which can effectively improve the accuracy of detection and reduce clinical overdiagnosis and treatment.

[0004] Monitoring the methylation level of paired box family gene 1 (PAX1) in cervical cancer shows a high correlation and can inhibit the malignant phenotype of cervical cancer cell lines, indicating the importance of gene methylation in clinical screening and treatment. Junctional Adhesion Molecule 3 (JAM3) was found to be highly methylated in cervical cancer and intraepithelial neoplasia, and the methylation level can be used as a prognostic marker for high-grade intraepithelial neoplasia (CIN II, CIN III). Currently, multiple cervical cancer-related gene methylation detection kits are on the market, involving methylation gene targets including PAX1, JAM3, ASTN1, DLX1, etc. Summary of the invention

[0005] During the long-term practical research process, the inventors discovered a new gene target combination that can be used for cervical cancer methylation detection, and based on this gene target combination, provided a detection kit and detection method for auxiliary diagnosis of cervical cancer, providing a new approach for early auxiliary diagnosis of cervical cancer.

[0006] In a first aspect, the present invention provides the use of a substance for detecting methylation modification of the CNTNAP2 gene (Contactin Associated Protein 2) and the PEX5L gene (Peroxisomal Biogenesis Factor 5 Like) in the preparation of a product for auxiliary diagnosis of cervical cancer.

[0007] Furthermore, a large number of validation experiments showed that, with GRCh38 as the reference genome, the methylation detection CpG site region of the CNTNAP2 gene is preferably located between 145813214 and 145813565 on chromosome 7, and the methylation detection CpG site region of the PEX5L gene is preferably located between 179754514 and 179754949 on chromosome 3.

[0008] Preferably, in the above application, the substances for detecting methylation modification of CNTNAP2 gene and PEX5L gene are primers and fluorescent probes for specifically detecting methylation modification of CNTNAP2 gene and PEX5L gene.

[0009] In a second aspect, the present invention provides a cervical cancer auxiliary diagnosis detection kit, specifically, the kit comprises: Primers for specifically detecting methylation modification of CNTNAP2 gene, the nucleotide sequences of which are shown in SEQ ID NO.1-2; A fluorescent probe for specifically detecting methylation modification of CNTNAP2 gene, the nucleotide sequence of which is shown in SEQ ID NO.3; Primers for specifically detecting methylation modification of the PEX5L gene, the nucleotide sequences of which are shown in SEQ ID NO.4-5; A fluorescent probe for specifically detecting methylation modification of the PEX5L gene, whose nucleotide sequence is shown in SEQ ID NO.6.

[0010] Preferably, the above-mentioned auxiliary diagnosis and detection kit for cervical cancer also includes primers and fluorescent probes for detecting internal reference genes. In some embodiments of the present invention, the ACTB gene is used as the internal reference gene, and the nucleotide sequences of the primers for specifically detecting the ACTB gene are shown in SEQ ID NO.7-8, and the nucleotide sequence of the fluorescent probe for specifically detecting the ACTB gene is shown in SEQ ID NO.9.

[0011] It can be understood that in the above-mentioned cervical cancer auxiliary diagnosis detection kit, the 5' end of the fluorescent probe nucleotide sequence is labeled with a fluorescent group and the 3' end is labeled with a quenching group, wherein the fluorescent group can be selected from FAM, VIC, HEX, ROX, CY3, CY5, etc., and the quenching group can be selected from TAMRA, BHQ1, BHQ2, MGBNFQ, QSY, etc.

[0012] In the above-mentioned cervical cancer auxiliary diagnosis detection kit, the primers and fluorescent probes for specific detection of CNTNAP2 gene, PEX5L gene and ACTB gene can be prepared in proportion into a PCR reaction solution and stored. The PCR reaction solution can also include PCR buffer, dNTPs, DNA polymerase and nuclease-free water.

[0013] Preferably, the above-mentioned cervical cancer auxiliary diagnosis detection kit also includes positive quality control products and negative quality control products. Specifically, the positive quality control products include cell line DNA in which both CNTNAP2 gene and PEX5L gene are methylated, and the negative quality control products include cell line DNA in which CNTNAP2 gene and PEX5L gene are unmethylated.

[0014] Preferably, the above-mentioned cervical cancer auxiliary diagnosis detection kit also includes a nucleic acid extraction reagent and / or a sulfite conversion reagent.

[0015] In a third aspect, the present invention provides the use of the above-mentioned cervical cancer auxiliary diagnosis detection kit in detecting the methylation status of CNTNAP2 and PEX5L genes in the samples to be tested, wherein the samples to be tested include but are not limited to cervical exfoliated cells, vaginal secretions, urine, biopsy tissue, blood, blood free DNA, etc.

[0016] Furthermore, in the above detection application, the following steps are included: S1. Obtain DNA of the sample to be tested; S2. treating the DNA obtained in step S1 with sulfite to obtain converted DNA; S3. Construct a reaction system, perform fluorescence quantitative PCR detection on the DNA obtained in step S2, obtain a Ct value, and calculate a ΔCt value based on the difference between the Ct value of the CNTNAP2 and PEX5L genes and the Ct value of the internal reference gene; S4. Determine the positive judgment threshold and judge the methylation status of each gene according to the ΔCt value.

[0017] Preferably, in the reaction system described in step S3, each 25 μL includes 5 μL DNA and PCR buffer, 0.5-1.0 μL dNTPs (10 mM each), 0.5-1.2 μL primers (10 μM) shown in SEQ ID NO.1, 0.5-1.2 μL primers (10 μM) shown in SEQ ID NO.2, 0.25-0.6 μL fluorescent probe (10 μM) shown in SEQ ID NO.3, 0.5-1.2 μL primers (10 μM) shown in SEQ ID NO.4, 0.5-1.2 μL primers (10 μM) shown in SEQ ID NO.5, 0.25-0.6 μL fluorescent probe (10 μM) shown in SEQ ID NO.6, 0.1-0.6 μL primers (10 μM) shown in SEQ ID NO.7, 0.1-0.6 μL primers (10 μM) shown in SEQ ID NO.8, 0.1-0.6 μL primers (10 μM) shown in SEQ ID NO.9, 0.25-0.6 μL primers (10 μM) shown in SEQ ID NO.10 0.1-0.6μL of fluorescent probe (10μM) shown in NO.9, 0.2-0.8μL of DNA polymerase (5U / μL), and the rest is nuclease-free water.

[0018] Fourthly, based on the judgment of the methylation status of CNTNAP2 and PEX5L genes in the sample to be tested, the cervical cancer auxiliary diagnosis detection kit of the present invention can achieve auxiliary diagnosis or early screening of cervical cancer by detecting clinically collected samples.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The present invention is the first to use the two targets, CNTNAP2 gene and PEX5L gene, to jointly perform cervical cancer methylation detection. Clinical sample testing has verified that the two target detection effects of the present invention are effectively complementary, and the dual-target combination detection system has high sensitivity and specificity, and is simple and fast to operate. It is helpful for early detection and auxiliary diagnosis of cervical cancer, and provides a new and reliable choice for clinical screening of cervical cancer, diversion of colposcopy referral population, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solution of the present application, the following is a brief introduction to the drawings used in the present application. Obviously, the drawings described below are only some embodiments of the present application, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0021] Figure 1 This is a fluorescence PCR amplification curve of a cervical exfoliated cell sample from a cervical cancer positive patient in Example 4 of the present application. DETAILED DESCRIPTION

[0022] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the terms "including" and "having" in the specification and claims of the present invention and the above-mentioned figure descriptions and any variations thereof are intended to cover non-exclusive inclusions.

[0024] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0025] Example 1 This example provides a methylation gene target combination that can be used for cervical cancer detection, specifically composed of the CNTNAP2 gene and the PEX5L gene. When constructing a product for auxiliary diagnosis of cervical cancer based on the methylation modification of the CNTNAP2 and PEX5L genes, the corresponding CpG site region position detected is specifically between the chromosome corresponding site coordinates shown in Table 1.

[0026] Table 1 CpG site region information for methylation detection

[0027] Example 2 Based on the gene target combination in Example 1 and the CpG site region corresponding to each gene detection, this example provides a fluorescent PCR kit that can realize auxiliary diagnosis of cervical cancer, and the kit includes a PCR reaction solution. Specifically, the PCR reaction solution includes primers and fluorescent probes for specific detection of CNTNAP2 gene and PEX5L gene, and primers and fluorescent probes for specific detection of internal reference gene ACTB, and the sequences of the above primers and probes are shown in Table 2.

[0028] Table 2 Sequence information of primers and probes used in the kit

[0029] In the PCR reaction solution of this example, in addition to the above primers and probes, it also includes PCR buffer, dNTPs, DNA polymerase, nuclease-free water, etc. Specifically, the composition of each 20 μL PCR reaction solution is shown in Table 3.

[0030] Table 3 Composition of PCR reaction solution in the kit

[0031] The kit of this example also includes a positive quality control and a negative quality control. The positive quality control is a cell line DNA in which both the CNTNAP2 gene and the PEX5L gene are methylated, and the negative quality control is a cell line DNA in which both the CNTNAP2 gene and the PEX5L gene are unmethylated.

[0032] Example 3 Based on the fluorescent PCR kit in Example 2, this example provides a fluorescent PCR detection method, comprising the following steps: (1) DNA extraction.

[0033] Collect samples, process them with nucleic acid extraction reagents, and obtain sample DNA.

[0034] (2) Sulfite conversion of DNA.

[0035] The sample DNA, negative quality control products and positive quality control products were treated with sulfite to obtain converted DNA.

[0036] (3) Fluorescence PCR detection.

[0037] The corresponding fluorescent groups and quenching groups were labeled on the fluorescent probes shown in Table 2, and the PCR reaction solution was prepared according to Table 3 and the converted DNA was subjected to fluorescent PCR detection, wherein the DNA loading amount was 5 μL and the total reaction volume was 25 μL.

[0038] The detected Ct value was obtained, and the ΔCt value was calculated based on the difference between the Ct value of the CNTNAP2 and PEX5L genes and the Ct value of the internal reference ACTB gene.

[0039] (4) Interpretation of results.

[0040] The positive judgment value interval was set and established, and the positive and negative status of each gene was determined according to the ΔCt value. If any one of the CNTNAP2 and PEX5L gene targets was positive, the sample was determined to be positive, and if both targets were negative, the sample was determined to be negative.

[0041] Example 4 Based on the method in Example 3, this example tested the cervical exfoliated cell samples collected clinically, including the following: (1) Extraction of DNA from cervical exfoliated cells.

[0042] The nucleic acid extraction kit used in this example was purchased from the commercially available QIAGEN company (Cat. No. 51104), and the extraction was performed by the following operation: ① Collect the cells by centrifugation (300g for 5 min), discard the supernatant, and resuspend them in 200μL PBS (you can resuspend the remaining cells in the cell preservation solution after centrifuging the sample and discarding the supernatant, or use PBS according to the instructions).

[0043] ②Add 20μL QIAGEN protease.

[0044] ③ Add 200 μL AL Buffer to the centrifuge tube and pulse vortex for 15 seconds.

[0045] ④After incubation at 56 degrees for 10 minutes, briefly centrifuge to collect the droplets on the tube cover.

[0046] ⑤ Add 200 μL of anhydrous ethanol, pulse vortex for 15 seconds, and briefly centrifuge to collect the droplets on the tube cap.

[0047] ⑥ Add the mixed solution to the centrifuge column, centrifuge at 8000rpm for 1min, and discard the filtrate.

[0048] ⑦ Add 500 μL AW1 buffer, centrifuge at 8000 rpm for 1 min, and discard the filtrate.

[0049] ⑧Add 500 μL AW2 buffer, centrifuge at 14000 rpm for 3 min, and discard the filtrate.

[0050] ⑨Idle at 14000rpm for 1min.

[0051] ⑩ Place the centrifuge column in a new 1.5 mL centrifuge tube, add 200 μL Buffer AE, place at room temperature for 1 min, and centrifuge at 12,000 rpm for 1 min.

[0052] (2) Sulfite conversion of DNA.

[0053] The sulfite conversion kit used in this example was purchased from the commercially available ZYMO Research Company (Cat. No. 5030 / 5031), and the conversion was performed as follows: ① Prepare the transformation system according to Table 4.

[0054] Table 4 Sulfite conversion system

[0055] Note: If the DNA volume is less than 20 μL, make up with nuclease-free water.

[0056] ② Place the prepared transformation system into a PCR instrument for incubation, where the program is: 98°C, 8 min; 54°C, 60 min; 4°C for up to 20 hours.

[0057] ③Put the purification column on the collection tube and add 600μL of M-Binding Buffer to the purification column.

[0058] ④ Add all the samples incubated in step ② to the purification column to which M-Binding Buffer has been added in step ③, close the tube cap, and invert several times to mix.

[0059] ⑤ Centrifuge at full speed (>10000×g) for 30 seconds and discard the waste liquid in the collection tube.

[0060] ⑥ Add 100 μL of M-Wash Buffer to the purification column and centrifuge at full speed for 30 seconds.

[0061] ⑦ Add 200 μL of L-Desulphonation Buffer to the purification column and incubate at room temperature (20-30°C) for 15 minutes. After incubation, centrifuge at full speed for 30 seconds and discard the waste liquid in the collection tube.

[0062] ⑧ Add 200 μL of M-Wash Buffer to the purification column and centrifuge at full speed for 30 seconds; repeat this step once.

[0063] ⑨ Remove the purification column from the collection tube and put it into a new clean 1.5mL centrifuge tube. Add 10-20μL of M-Elution Buffer to the purification column, let it stand for 1min, and centrifuge at full speed for 30s to elute the converted DNA.

[0064] (3) Fluorescence PCR detection.

[0065] The detection conditions used in this example are: the fluorescence PCR instrument is ABI7500, the FAM fluorescence channel is set to detect CNTNAP2, the VIC fluorescence channel is set to detect PEX5L, the CY5 fluorescence channel is set to detect ACTB, and the Passive Reference is set to none. The amplification reaction conditions are: 95℃ for 5 minutes; 95℃ for 15 seconds, 60℃ for 60 seconds (collecting fluorescence), and 45 cycles.

[0066] Each experiment should simultaneously test positive quality control products and negative quality control products as positive and negative quality control products. After the PCR reaction is completed, the baseline and fluorescence threshold are appropriately adjusted according to the amplification curve to obtain the Ct value of the sample. The negative control FAM and VIC channels of each reaction should have no obvious amplification curve, no Ct value or Ct value>39, and the CY5 channel should have an obvious amplification curve, and the Ct value is ≤37. The positive control FAM and VIC channels should have obvious amplification curves, Ct values ​​≤39, and the CY5 channel should have obvious amplification curves, Ct values ​​≤37. On the premise that the positive and negative quality control is qualified, the CY5 channel of the sample internal standard ACTB has an obvious amplification curve, and the Ct value is ≤37, otherwise it indicates that the sample concentration is too low and resampling is required for the detection process.

[0067] If all the above conditions are met, calculate the ΔCt value; ΔCt value = gene Ct value - internal reference Ct value. If the sample gene has no amplification curve showing NoCt, the Ct value is defined as 45.

[0068] (4) Interpretation of results.

[0069] According to the ΔCt value, the results were interpreted according to Table 5.

[0070] Table 5 Determination of positive and negative properties of the samples to be tested

[0071] According to the above detection steps, the test results of 30 (P1-30) clinically collected exfoliated cell samples from cervical cancer positive patients (the pathological status of cervical cancer patients has been confirmed by clinical pathology) and 20 (N1-20) clinically collected exfoliated cell samples provided by normal healthy volunteers are shown in Table 6, where Figure 1 This is a fluorescence PCR amplification curve of a cervical exfoliated cell sample from a patient with positive cervical cancer.

[0072] Table 6 Statistics of fluorescent PCR test results of clinical samples

[0073] In Table 6, ΔCt value 1 = CNTNAP2 gene Ct value - internal reference Ct value, ΔCt value 2 = PEX5L gene Ct value - internal reference Ct value, if the sample gene has no amplification curve showing NoCt, the Ct value is defined as 45. The results in Table 6 are summarized and statistically calculated in the form of a four-table table in Table 7.

[0074] Table 7 Paired statistics of kit test results and sample clinical diagnosis results

[0075] After summarizing in Table 7, the detection sensitivity, specificity and overall compliance rate of the kit were calculated according to the following formulas: Sensitivity = a / M1 × 100%; Specificity = d / N1 × 100%; Overall compliance rate = (a+d) / N × 100%.

[0076] According to Table 7, the detection performance of the two genes CNTNAP2 and PEX5L for the above clinical samples by single target and combined dual target was calculated, and the results are shown in Table 8; when the dual targets were detected jointly, if any one target was positive, the sample was judged to be positive, and if both targets were negative, the sample was judged to be negative.

[0077] Table 8 Statistics of clinical diagnostic performance of dual targets

[0078] In summary, the methylation gene targets and detection kit provided by the present invention have the ability to be used for auxiliary diagnosis and early screening of cervical cancer, and have high sensitivity and specificity, and have application value in clinical screening and clinical triage of cervical cancer.

[0079] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, without departing from the scope of the main purpose of the present invention, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present invention.

Claims

1. Application of substances for detecting methylation modification of CNTNAP2 and PEX5L genes in the preparation of products for auxiliary diagnosis of cervical cancer.

2. The use according to claim 1, characterized in that: Taking GRCh38 as the reference genome, the detection region of the CNTNAP2 gene is located between 145813214 and 145813565 of chromosome 7, and the detection region of the PEX5L gene is located between 179754514 and 179754949 of chromosome 3.

3. The use according to claim 2, characterized in that: The substances are primers and fluorescent probes for specifically detecting methylation modification of CNTNAP2 gene and PEX5L gene.

4. A cervical cancer auxiliary diagnosis detection kit, characterized in that: It includes primers and fluorescent probes for specifically detecting CNTNAP2 gene and PEX5L gene; the nucleotide sequence of the primers for detecting CNTNAP2 gene is shown in SEQ ID NO.1-2, and the nucleotide sequence of the fluorescent probe is shown in SEQ ID NO.3; The nucleotide sequences of the primers used to detect the PEX5L gene are shown in SEQ ID NO.4-5, and the nucleotide sequence of the fluorescent probe is shown in SEQ ID NO.

6.

5. The auxiliary diagnosis and detection kit for cervical cancer according to claim 4, characterized in that: The detection kit includes primers and fluorescent probes for detecting an internal reference gene; the internal reference gene is preferably ACTB, the nucleotide sequences of the primers for detecting the ACTB gene are shown in SEQ ID NO.7-8, and the nucleotide sequence of the fluorescent probe is shown in SEQ ID NO.

9.

6. The auxiliary diagnosis and detection kit for cervical cancer according to claim 4, characterized in that: The detection kit comprises a positive quality control product and a negative quality control product, wherein the positive quality control product comprises a cell line DNA in which both the CNTNAP2 gene and the PEX5L gene are methylated, and the negative quality control product comprises a cell line DNA in which the CNTNAP2 gene and the PEX5L gene are not methylated.

7. Use of the auxiliary diagnostic test kit for cervical cancer as described in any one of claims 4 to 6 in detecting the methylation status of CNTNAP2 and PEX5L genes in a sample to be tested, wherein the sample to be tested is a mixture of any one or more of cervical exfoliated cells, vaginal secretions, urine, biopsy tissue, blood, blood free DNA, etc.

8. The use according to claim 7, characterized in that: The following steps are involved: S1. Obtain DNA of the sample to be tested; S2. treating the DNA obtained in step S1 with sulfite to obtain converted DNA; S3. Construct a reaction system, perform fluorescence quantitative PCR detection on the DNA obtained in step S2, obtain a Ct value, and calculate a ΔCt value based on the difference between the Ct value of the CNTNAP2 and PEX5L genes and the Ct value of the internal reference gene; S4. Determine the positive judgment threshold and judge the methylation status of each gene according to the ΔCt value.

9. The use according to claim 8, characterized in that In the reaction system described in step S3, each 25 μL includes 5 μL DNA and PCR buffer, dNTPs (10 mM each) 0.5-1.0 μL, primers 10 μM 0.5-1.2 μL as shown in SEQ ID NO.1, primers 10 μM 0.5-1.2 μL as shown in SEQ ID NO.2, fluorescent probes 10 μM 0.25-0.6 μL as shown in SEQ ID NO.3, primers 10 μM 0.5-1.2 μL as shown in SEQ ID NO.4, primers 10 μM 0.5-1.2 μL as shown in SEQ ID NO.5, fluorescent probes 10 μM 0.25-0.6 μL as shown in SEQ ID NO.6, primers 10 μM 0.1-0.6 μL as shown in SEQ ID NO.7, primers 10 μM 0.1-0.6 μL as shown in SEQ ID NO.8, fluorescent probes 10 μM as shown in SEQ ID NO.9 0.1-0.6μL, DNA polymerase 5U / μL 0.2-0.8μL, and the rest is nuclease-free water.

10. Use of the cervical cancer auxiliary diagnosis detection kit according to any one of claims 4 to 6 in auxiliary diagnosis or screening of cervical cancer, wherein the use is not for the purpose of diagnosis and treatment of the disease.

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