A prostatitis-related marker and kit

Abstract

The present invention relates to a prostatitis-related marker. The marker is selected from at least one of DL-3-phenyllactic acid, pantothenic acid, and L-pyroglutamic acid. The inventors have found through research that, compared with healthy controls, the levels of DL-3-phenyllactic acid, pantothenic acid, and L-pyroglutamic acid in the serum of prostatitis patients are significantly increased. The ROC curve analysis results show that DL-3-phenyllactic acid, pantothenic acid, and L-pyroglutamic acid can be used alone as biomarkers for the diagnosis of prostatitis, with good specificity and sensitivity, especially when DL-3-phenyllactic acid and L-pyroglutamic acid are used in combination, the sensitivity and specificity can be as high as 95%. The present invention provides a new biomarker for the diagnosis of prostatitis, which has the advantages of convenient sampling, non-invasiveness, and rapid detection.

Description

Technical Field

[0001] The present invention relates to the technical field of biotechnology, and in particular to a prostatitis-related marker and a kit. Background Art

[0002] Prostatitis refers to the clinical manifestations of pelvic pain or discomfort, abnormal urination, sexual dysfunction, etc. caused by the infection of the prostate by pathogenic bacteria or stimulation by certain non-infectious factors. Prostatitis is common in men from adolescence to old age, and the incidence rate is higher in adult men under 50 years old. It is a common disease of the urinary system. According to data, about half of men will suffer from prostatitis.

[0003] Prostatitis can be divided into the following types: Type I prostatitis: acute prostatitis, which often occurs after fatigue, drinking, colds, and too frequent sexual life. Some patients are secondary to type II prostatitis. Indwelling urinary catheters, rectal or perineal prostate puncture, bacteria from infection foci in other parts of the body can also spread to the prostate through the bloodstream. Common pathogens are Gram-negative enteric bacilli, as well as staphylococci and streptococci, and occasionally anaerobic bacteria; when patients often hold urine or have stones or other conditions that cause urinary tract obstruction, retrograde infection through the urethra can occur; Type II prostatitis: chronic prostatitis, the main causative factor is pathogen infection, which is common in long-term and repeated lower urinary tract infections, repeated presence of pathogens, and the main pathogen is infection caused by urine reflux into the prostate. If type I prostatitis is not treated in time or is delayed, it can also develop into type II prostatitis; Type III and type IV prostatitis: The pathogenesis is currently unknown, and may be related to pathogen infection, abnormal immune response, urine reflux stimulation, psychological factors, neuroendocrine factors, etc. Acute prostatitis is often accompanied by acute cystitis, seminal vesiculitis, epididymitis, etc., and may cause frequent urination, urgent urination, painful urination, and difficulty urinating. For example, seminal vesiculitis may also cause hematospermia. Chronic prostatitis can cause mental and neurological symptoms, such as dizziness, head swelling, fatigue, exhaustion, and insomnia; it can also be accompanied by urinary system symptoms, such as urethral white discharge. Prostatitis can also lead to some complications, including acute seminal vesiculitis, epididymitis, spermatic cord lymphadenopathy, sexual dysfunction, and infertility.

[0004] The existing auxiliary diagnosis tests for prostatitis mainly include urine routine, prostatic fluid routine, rectal digital examination, lower urinary tract urodynamic examination and prostate B-ultrasound, etc. These examination methods have certain defects, such as the detection method is too cumbersome, lack of specificity and sensitivity, etc. At present, there is still a lack of an efficient and convenient biological marker for auxiliary diagnosis of prostatitis. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide a prostatitis-related marker, which has good sensitivity and specificity and can be used as a new biomarker for the detection of prostatitis, and has the advantages of convenient sampling, non-invasiveness and rapid detection.

[0006] The specific technical solution is:

[0007] A prostatitis-related marker, wherein the marker is selected from at least one of DL-3-phenyllactic acid, pantothenic acid, and L-pyroglutamic acid.

[0008] Furthermore, the marker is DL-3-phenyllactic acid.

[0009] Furthermore, the markers are DL-3-phenyllactic acid and L-pyroglutamic acid.

[0010] Furthermore, the markers are DL-3-phenyllactic acid, pantothenic acid and L-pyroglutamic acid.

[0011] The present invention also provides application of the marker in preparing a reagent for detecting prostatitis.

[0012] The present invention also provides a reagent for detecting prostatitis, and the reagent can detect the content of the marker.

[0013] Furthermore, the reagent is a reagent used for liquid chromatography-mass spectrometry detection.

[0014] Furthermore, the detection sample of the reagent is serum.

[0015] The invention also provides application of the reagent in preparing a prostatitis detection kit.

[0016] The present invention also provides a kit for detecting prostatitis, which comprises the reagent and a 25-35v / v% methanol aqueous solution containing 0.1-0.3w / v% formic acid.

[0017] Furthermore, the kit comprises the reagent, and a 30 v / v% methanol aqueous solution containing 0.1 w / v% formic acid.

[0018] Furthermore, the detection sample of the kit is serum.

[0019] The present invention provides a new biomarker for the diagnosis of prostatitis. The inventor has found through research that, compared with healthy controls, the contents of DL-3-phenyllactic acid, pantothenic acid and L-pyroglutamic acid in the serum of prostatitis patients are significantly increased. Further ROC curve analysis shows that when used to diagnose prostatitis, the AUC (area under the ROC curve) of DL-3-phenyllactic acid is 0.773, the sensitivity is 63.2%, and the specificity is 81.1%; the AUC of L-pyroglutamic acid is 0.725, the sensitivity is 78.9%, and the specificity is 56.8%; the AUC of pantothenic acid is 0.721, the sensitivity is 65.8%, and the specificity is 70.3%. It shows that DL-3-phenyllactic acid, pantothenic acid and L-pyroglutamic acid can be used as biomarkers for the diagnosis of prostatitis, and have good specificity and sensitivity. In particular, when DL-3-phenyllactic acid and L-pyroglutamic acid are used in combination, the sensitivity and specificity can be as high as 95%. The present invention provides a new biomarker for the diagnosis of prostatitis, which has the advantages of convenient sampling, non-invasiveness and rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The figure shows the comparison of DL-3-phenyllactic acid content between prostatitis patients and normal control group and the ROC curve analysis results for prostatitis diagnosis.

[0021] Figure 2 The figure shows the comparison of L-pyroglutamic acid content between prostatitis patients and normal control group and the ROC curve analysis results for prostatitis diagnosis.

[0022] Figure 3 The figure shows the comparison of pantothenic acid content between prostatitis patients and normal control group and the ROC curve analysis results for prostatitis diagnosis.

[0023] Figure 4 This is the ROC curve analysis result of the combined use of DL-3-phenyllactic acid and L-pyroglutamic acid in the diagnosis of prostatitis.

[0024] Figure 5 This is the ROC curve analysis result of DL-3-phenyllactic acid, pantothenic acid and L-pyroglutamic acid combined for the diagnosis of prostatitis.

[0025] Figure 6 This is the ROC curve analysis result of the combined use of DL-3-phenyllactic acid and pantothenic acid in the diagnosis of prostatitis. DETAILED DESCRIPTION

[0026] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0027] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0028] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0029] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0030] In the present invention, the term "multiple" refers to two or more than two. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent the following three situations: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0031] The structural formulas of DL-3-phenyllactic acid, pantothenic acid and L-pyroglutamic acid of the present invention are shown in formula (I), formula (II) and formula (III) respectively:

[0032]

[0033] Example 1

[0034] The contents of DL-3-phenyllactic acid, pantothenic acid and L-pyroglutamic acid in the serum of prostatitis patients and normal control group were detected by ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology. The specific method is as follows:

[0035] 1. Subject inclusion criteria

[0036] Inclusion criteria for normal controls (Control group): 1) normal digital rectal examination and prostate ultrasound examination; 2) no urinary system diseases, benign and malignant tumors, and chronic diseases;

[0037] Inclusion criteria for prostatitis patients (prostatitis group): 1) having typical clinical symptoms of urinary urgency, frequent urination, and dysuria; 2) abnormal results of routine prostatic fluid examination; 3) normal liver and kidney function, and no drug treatment or surgical resection.

[0038] All subjects signed the patient informed consent form, and this study was approved by the Ethics Committee of Guangzhou Panyu District Central Hospital.

[0039] The specific information and related laboratory index test results of the normal control group and prostatitis group are shown in Table 1:

[0040] Table 1 Specific information of normal people and patients and related laboratory examination indicators

[0041]

[0042] 2. Specimen collection:

[0043] All subjects had their fasting venous blood drawn in the morning before surgery (rectal examination, massage, puncture and other examination procedures were prohibited 1 week before blood collection) and 3 ml of serum was collected and stored at -80 degrees Celsius until analysis. Patient information and related laboratory test indicators were collected retrospectively from the patient's records.

[0044] 3. Ultra-high performance liquid chromatography-tandem mass spectrometry platform analysis

[0045] The extracts from serum extracted with methanol were analyzed based on an ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) platform, combined with UHPLC-triple quadrupole mass spectrometry targeted analysis. The raw data from the mass spectrometer were converted into mzXML file format using the MSConvert tool in the Proteowizard software package (v3.0.8789)4. The RXCMS software package was used to perform peak detection, peak filtering, and peak alignment to obtain a quantitative list of substances. The public databases HMDB, METLIN, massbank, LipidMaps, mzcloundi, KEGG and self-built substance libraries were used to identify the substances. The LOESs signal correction method based on the QC sample was used to achieve data correction and eliminate systematic errors. Substances with RSD>30% in the QC sample were filtered out during data quality control. The details are as follows:

[0046] (1) Curve configuration

[0047] Weigh appropriate amounts of DL-3-phenyllactic acid, pantothenic acid, and L-pyroglutamic acid standards and prepare single standard stock solutions with methanol or water. Take appropriate amounts of each stock solution to prepare mixed standards, dilute them one by one with 30% methanol aqueous solution (containing 0.1% formic acid) to appropriate concentrations to prepare working standard solutions. The information and concentration points of DL-3-phenyllactic acid, pantothenic acid, and L-pyroglutamic acid are shown in Tables 2 and 3, respectively. Both stock solutions and working standard solutions are stored at 0°C.

[0048] Table 2 DL-3-phenyllactic acid, pantothenic acid, L-pyroglutamic acid standard information

[0049] name English name CAS Number DL-3-phenyllactic acid Phenyllactic acid 828-01-3 L-Pyroglutamic acid Pyroglutamic acid 98-79-3 Pantothenic acid Pantothenic acid 79-83-4

[0050] Table 3 DL-3-phenyllactic acid, pantothenic acid, L-pyroglutamic acid at different concentrations

[0051]

[0052] Note: Concentration unit: ng / mL.

[0053] (2) Serum sample processing

[0054] Accurately pipette an appropriate amount of serum sample into a 2 mL EP tube, accurately add 400 μL of 30% methanol aqueous solution (containing 0.1% formic acid), vortex for 60 seconds, centrifuge at 12000 rpm and 4°C for 10 minutes, and take the supernatant and add it to the test bottle.

[0055] (3) Detection parameters

[0056] Chromatographic conditions: ACQUITY BEH C18 column, injection volume 5μL, column temperature 40℃, mobile phase A-water (containing 0.1% formic acid), B-methanol water (containing 0.1% formic acid). Gradient elution conditions are 0-6min, 28% B; 6-9min, 25-40% B; 9-10min, 40-50% B; 10-11min, 50% B; 11-12min, 28% B. Flow rate: 0.25mL / min.

[0057] Mass spectrometry conditions: electrospray ionization (ESI) source, negative ionization mode. Ion source temperature 500°C, ion source voltage -4500V, collision gas 6psi, nebulizer gas and auxiliary gas are 50psi. Multiple reaction monitoring (MRM) is used for scanning. The ion pairs used for quantitative analysis are shown in Table 4 below.

[0058] Table 4 Quantitative ion pairs used for analysis

[0059] Test substances Precursor ion Product ions DP EP CE CXP DL-3-phenyllactic acid 164.915 146.6 -60 -10 -16 -13 Pantothenic acid 218.033 88.1 -65 -10 -22 -3 L-Pyroglutamic acid 127.955 82 -45 -10 -20 -3

[0060] Each working standard solution and sample was tested separately. The concentration of the working standard solution was used as the horizontal axis and the peak area was used as the vertical axis. The linear range was investigated and the standard curve was drawn to obtain the regression equations, which are as follows:

[0061] DL-3-phenyllactic acid: Y = -1822.7 + 2156.3 * X, R = 0.9969;

[0062] L-pyroglutamate: Y = -414.7 + 21.005 * X, R = 0.9964;

[0063] Pantothenic acid: Y = -54.313 + 2.719*X.

[0064] The content of each substance in the sample was calculated based on the above regression equation and the peak area corresponding to each substance.

[0065] 4. Statistical Analysis

[0066] The R software package Ropls was used to perform principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA) dimensionality reduction analysis on the sample data. The P-value was calculated according to the T test, the variable projection importance (VIP) was calculated by the PLS-DA dimensionality reduction method, and the component difference fold change was calculated to measure the influence intensity and explanatory power of DL-3-phenyllactic acid, pantothenic acid, and L-pyroglutamate content on the classification and discrimination of samples. When the P-value value was <0.05 and the VIP value was >1, it was considered statistically significant. The area under the receiver operating characteristic curve (AUC) was used to evaluate the diagnostic accuracy of the model in identifying patients in each comparison. When AUC <0.5, it means that the diagnosis is meaningless; when AUC = 0.5-0.7, it means that the diagnostic accuracy is low; when AUC = 0.7-0.9, it means that the diagnostic accuracy is moderate; when AUC >0.9, it means that the diagnostic accuracy is high.

[0067] 5. Results Analysis

[0068] Comparison of DL-3-phenyllactic acid content between prostatitis patients and normal controls and ROC curve analysis results Figure 1 As shown, the content of DL-3-phenyllactic acid in the prostatitis patient group was significantly higher than that in the normal control group, with an AUC of 0.773, a sensitivity of 63.2%, and a specificity of 81.1%.

[0069] like Figure 2 As shown, the content of L-pyroglutamate in the prostatitis patient group was significantly higher than that in the normal control group. The ROC curve analysis results showed that the AUC was 0.725, the sensitivity was 78.9%, and the specificity was 56.8%.

[0070] like Figure 3 As shown, the content of pantothenic acid in the prostatitis patient group was significantly higher than that in the normal control group. The ROC curve analysis results showed that the AUC was 0.721, the sensitivity was 65.8%, and the specificity was 70.3%.

[0071] like Figure 4 As shown, the results of ROC curve analysis showed that when DL-3-phenyllactic acid and L-pyroglutamate were used together, the AUC was 0.988, the sensitivity was 95%, and the specificity was 95%, indicating that the combination of the two can effectively improve the diagnostic effect of prostatitis, and the sensitivity and specificity are greatly improved.

[0072] like Figure 5 As shown, the results of ROC curve analysis showed that when DL-3-phenyllactic acid, pantothenic acid and L-pyroglutamic acid were used together, the AUC was 0.985, the sensitivity was 95%, and the specificity was 95%, indicating that the combination of the three can effectively improve the diagnostic effect of prostatitis.

[0073] like Figure 6 As shown, the results of ROC curve analysis showed that when DL-3-phenyllactic acid and pantothenic acid were used together, the AUC was 0.548, the sensitivity was 55%, and the specificity was 60%, indicating that the combination of the two not only failed to improve the diagnostic effect of prostatitis, but also led to a decrease in diagnostic performance.

[0074] The above results indicate that DL-3-phenyllactic acid, pantothenic acid, and L-pyroglutamate can be used alone as biomarkers for the diagnosis of prostatitis, with good specificity and sensitivity; when DL-3-phenyllactic acid and L-pyroglutamate are used in combination, or when DL-3-phenyllactic acid, pantothenic acid, and L-pyroglutamate are used in combination, the diagnostic accuracy is greatly improved, with both sensitivity and specificity reaching up to 95%.

[0075] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. Use of a reagent for detecting prostatitis-related markers in the preparation of a reagent for detecting prostatitis, characterized in that: The marker is a combination of DL-3-phenyllactic acid, pantothenic acid and L-pyroglutamic acid; and the detection sample of the reagent is serum.

2. A reagent for detecting prostatitis, characterized in that: The reagent is used to detect the content of the prostatitis-related marker as described in claim 1; the detection sample of the reagent is serum.

3. The reagent according to claim 2, characterized in that The reagent is a reagent used for liquid chromatography-mass spectrometry detection.

4. Use of the reagent according to claim 2 or 3 in the preparation of a prostatitis detection kit; the detection sample of the reagent is serum.

5. A kit for detecting prostatitis, characterized in that: The kit comprises the reagent according to claim 2 or 3, and a 25-35v / v% methanol aqueous solution containing 0.1-0.3w / v% formic acid; the test sample of the kit is serum.

6. The kit according to claim 5, characterized in that The method comprises the reagent according to claim 2 or 3, and a 30 v / v % methanol aqueous solution containing 0.1 w / v % formic acid.

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