CN114200121A - Prostatitis related marker and kit - Google Patents
Prostatitis related marker and kit Download PDFInfo
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- CN114200121A CN114200121A CN202111537179.0A CN202111537179A CN114200121A CN 114200121 A CN114200121 A CN 114200121A CN 202111537179 A CN202111537179 A CN 202111537179A CN 114200121 A CN114200121 A CN 114200121A
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Abstract
The invention relates to a prostatitis-related marker. The marker is at least one selected from DL-3-phenyllactic acid, pantothenic acid and L-pyroglutamic acid. The inventor finds that compared with healthy controls, the content of DL-3-phenyllactic acid, pantothenic acid and L-pyroglutamic acid in the serum of the prostatitis patients is obviously increased. The ROC curve analysis result shows that DL-3-phenyllactic acid, pantothenic acid and L-pyroglutamic acid can be independently used as biomarkers for diagnosing prostatitis, and have better specificity and sensitivity, and particularly, when DL-3-phenyllactic acid and pantothenic acid are combined, the sensitivity and the specificity can reach 95 percent. The invention provides a new biomarker for diagnosing prostatitis, and has the advantages of convenient sampling, no wound, quick detection and the like.
Description
Technical Field
The invention relates to the technical field of biotechnology, in particular to a prostatitis related marker and a kit.
Background
Prostatitis refers to the clinical manifestations of pelvic region pain or discomfort, abnormal urination, sexual dysfunction and the like caused by the fact that the prostate is infected by pathogenic bacteria or stimulated by some non-infectious factors. Prostatitis is better in males from puberty to old, and the prevalence rate of adult males under 50 years old is higher, and the prostatitis is a common disease of the urinary system. Data show that about half of men suffer from prostatitis.
Prostatitis can be divided into the following categories: type i prostatitis: acute prostatitis, which is usually caused by fatigue, alcohol drinking, cold and frequent sexual life, is secondary to type II prostatitis in some patients. The bacteria of the infection focus of other parts of the body can also spread to the prostate through the blood stream when the urinary catheter is left and the prostate is punctured through rectum or perineum, and the common pathogenic bacteria are gram-negative enteric bacilli, staphylococcus and streptococcus, and are occasionally anaerobic bacteria; when the patient frequently holds urine or has stones or other conditions to cause the urinary tract to be blocked, the patient can be infected in a retrograde way through the urethra; type II prostatitis: the pathogenic factor of the chronic prostatitis is mainly pathogen infection which is commonly seen in long-term repeated lower urinary tract infection, the pathogen exists repeatedly, the pathogen mainly enters the prostate through urine counter-current to cause the infection, the type I prostatitis is not treated in time or is not cured for a long time, and the type II prostatitis can also be developed; prostatitis type iii and prostatitis type iv: the pathogenesis is unknown at present, and can be related to pathogen infection, abnormal immune response, urine reflux stimulation, psychopsychological factors, neuroendocrine factors and the like. Acute prostatitis often accompanies acute cystitis, seminal vesiculitis, epididymitis, etc., and there may be frequent micturition, urgent micturition, odynuria, dysuria, for example, seminal vesiculitis may also present with hemospermia. While chronic prostatitis can cause mental and neurological symptoms such as dizziness, fullness in head, fatigue, insomnia; it is also associated with urinary symptoms such as urinary dripping. Prostatitis also causes some complications, including acute seminal vesiculitis, epididymitis, swollen spermatic cord lymph nodes, sexual dysfunction and infertility.
The existing examination for the auxiliary diagnosis of prostatitis mainly comprises urine routine, prostatic fluid routine, rectal digital examination, lower urinary tract urodynamic examination, prostate B-ultrasound and the like, and all the examination methods have certain defects, such as excessively complicated detection methods, lack of specificity and sensitivity and the like. At present, an efficient and convenient biological marker for auxiliary diagnosis of prostatitis is not available.
Disclosure of Invention
Based on this, the invention aims to provide a prostatitis-related marker which has better sensitivity and specificity, can be used as a novel biomarker for prostatitis detection, and has the advantages of convenient sampling, no wound, quick detection and the like.
The specific technical scheme is as follows:
a marker associated with prostatitis, the marker being selected from at least one of DL-3-phenyllactic acid, pantothenic acid, L-pyroglutamic acid.
Further, the marker is DL-3-phenyllactic acid.
Further, the markers are DL-3-phenyllactic acid and pantothenic acid.
Further, the markers are DL-3-phenyllactic acid, pantothenic acid and L-pyroglutamic acid.
The invention also provides application of the marker in preparation of a reagent for detecting prostatitis.
The invention also provides a reagent for detecting prostatitis, and the reagent can detect the content of the marker.
Further, the reagent is used for detection by a liquid chromatography-mass spectrometry combined technology.
Further, the detection sample of the reagent is serum.
The invention also provides application of the reagent in preparation of a prostatitis detection kit.
The invention also provides a kit for detecting prostatitis, which comprises the reagent and 25-35 v/v% methanol aqueous solution containing 0.1-0.3 w/v% formic acid.
Further, the kit comprises the reagent, and a 30 v/v% aqueous methanol solution containing 0.1 w/v% formic acid.
Further, the detection sample of the kit is serum.
The invention provides a new biomarker for diagnosing prostatitis, and the inventor finds that the content of DL-3-phenyllactic acid, pantothenic acid and L-pyroglutamic acid in the serum of a prostatitis patient is obviously increased compared with that of a healthy control patient through research. Further by ROC curve analysis, when used for diagnosing prostatitis, the AUC (area under ROC curve) of DL-3-phenyllactic acid is 0.773, the sensitivity is 63.2%, and the specificity is 81.1%; AUC for pantothenate is 0.725, sensitivity is 78.9%, specificity is 56.8%; pyroglutamic acid has an AUC of 0.721, a sensitivity of 65.8% and a specificity of 70.3%. The results show that DL-3-phenyllactic acid, pantothenic acid and L-pyroglutamic acid can be used as biomarkers for diagnosing prostatitis, and have good specificity and sensitivity. Especially when DL-3-phenyllactic acid and pantothenic acid are combined, the sensitivity and specificity can reach 95%. The invention provides a new biomarker for diagnosing prostatitis, and has the advantages of convenient sampling, no wound, quick detection and the like.
Drawings
FIG. 1 is a graph showing the results of ROC curve analysis of the content of DL-3-phenyllactic acid in the group of patients with prostatitis as compared with the group of normal controls and used for diagnosis of prostatitis.
FIG. 2 is a graph showing the results of ROC curve analysis for comparison of pantothenic acid content in prostatitis patients and normal controls and diagnosis of prostatitis.
FIG. 3 is a graph showing the results of ROC curve analysis of L-pyroglutamic acid content in the group of patients with prostatitis as compared with the group of normal controls.
FIG. 4 is a graph showing the results of ROC curve analysis of the combination of DL-3-phenyllactic acid and pantothenic acid for the diagnosis of prostatitis.
FIG. 5 is a graph showing the results of ROC curve analysis of the combination of DL-3-phenyllactic acid, pantothenic acid and L-pyroglutamic acid for the diagnosis of prostatitis.
FIG. 6 is a graph showing the results of ROC curve analysis of the combination of DL-3-phenyllactic acid and L-pyroglutamic acid for diagnosis of prostatitis.
Detailed Description
Experimental procedures according to the invention, in which no particular conditions are specified in the following examples, are generally carried out under conventional conditions, or under conditions recommended by the manufacturer. The various chemicals used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to only those steps or modules listed, but may alternatively include other steps not listed or inherent to such process, method, article, or device.
The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to only those steps or modules listed, but may alternatively include other steps not listed or inherent to such process, method, article, or device.
The "plurality" referred to in the present invention means two or more. "and/or" describes the association relationship of the associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.
The structural formulas of the DL-3-phenyllactic acid, the pantothenic acid and the L-pyroglutamic acid are respectively shown as a formula (I), a formula (II) and a formula (III):
example 1
Detecting the contents of DL-3-phenyllactic acid, pantothenic acid and L-pyroglutamic acid in serum of a prostatitis patient and a normal control group by using an ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology, wherein the specific method comprises the following steps:
1. subject inclusion criteria
Normal human controls (Control group) were included as standard: 1) the patients are normal through digital rectal examination and B-ultrasonic prostate examination; 2) eliminating urinary system diseases, various benign and malignant tumors, and various chronic diseases;
prostatitis patients (prostatis group) were included in the criteria: 1) has typical clinical symptoms of urgent micturition, frequent micturition and odynuria; 2) abnormal results of routine examination of prostatic fluid; 3) liver and kidney function and the like are not abnormal, and the patients do not receive treatment of medicines or surgical excision.
All subjects signed patient informed consent, which was approved by the ethical committee of the central hospital in the district of Panyu, Guangzhou city.
The specific information and the relevant laboratory index detection results of the normal control group and the prostatitis group are shown in table 1:
TABLE 1 specific information of normal and patient and related laboratory examination indexes
2. Collecting a specimen:
all subjects sampled 3ml of early morning fasting venous blood (examination operations such as rectal digital examination, massage, puncture and the like are prohibited 1 week before blood sampling) before surgery and stored at-80 ℃ until analysis. Patient information and related laboratory test indices are collected retrospectively from the patient's records.
3. Ultra-high performance liquid chromatography-tandem mass spectrometry platform analysis
The extracts in serum extracted with methanol were analyzed on a platform based on ultra performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with UHPLC-triple quadrupole mass spectrometry targeting analysis. The raw data of the mass spectrometer is converted into an mzXML file format by using the mscovert tool in the Proteowizard software package (v3.0.8789) 4. And (5) performing peak detection, peak filtration and peak alignment by adopting RXCMS software to obtain a substance quantitative list. Public databases HMDB, METLIN, massbank, LipidMaps, mzcloundi, KEGG and self-established substance libraries were used for substance identification. And the LOESs signal correction method based on the QC samples realizes data correction and eliminates system errors. And filtering out substances with RSD > 30% in QC samples in data quality control. The method comprises the following specific steps:
(1) arrangement of standard music
Weighing appropriate amount of DL-3-phenyllactic acid, pantothenic acid and L-pyroglutamic acid standard substance, and preparing single-standard mother liquor with methanol or water. Appropriate amount of each mother liquor is measured to prepare a mixed standard product, and the mixed standard product is diluted one by one to appropriate concentration by 30% methanol aqueous solution (containing 0.1% formic acid) to prepare working standard solution. 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 the mother liquor and the working standard solution were stored at 0 ℃.
TABLE 2 information on DL-3-phenyllactic acid, pantothenic acid, L-pyroglutamic acid standards
| Name (R) | English name | CAS number |
| DL-3-phenylLactic acid | Phenyllactic acid | 828-01-3 |
| Pantothenic acid | Pyroglutamic acid | 98-79-3 |
| L-pyroglutamic acid | Pantothenic acid | 79-83-4 |
TABLE 3 respective concentration points of DL-3-phenyllactic acid, pantothenic acid, L-pyroglutamic acid
Note: the concentration unit is ng/mL.
(2) Serum sample processing
Accurately sucking a proper amount of serum sample into a 2mL EP tube, accurately adding 400 μ L of 30% methanol aqueous solution (containing 0.1% formic acid), vortexing and shaking for 60s, centrifuging at 12000rpm at 4 ℃ for 10min, and taking supernatant to add into a detection bottle.
(3) Detecting parameters
Chromatographic conditions are as follows: by using ACQUITY
BEH C18 chromatographic column, sample size 5 μ L, column temperature 40 deg.C, mobile phase A-water (containing 0.1% formic acid), B-methanol water (containing 0.1% formic acid). Gradient elution conditions are 0-6 min, and 28% of B; for 6-9 min, 25-40% of B; 9-10 min, 40-50% B; 10-11 min, 50% B; 11-12 min, 28% B. Flow rate: 0.25 mL/min.
Mass spectrum conditions: electrospray ionization (ESI) source, negative ion ionization mode. The ion source temperature was 500 ℃, and the ion source was charged. 4500V, 6psi of impinging gas, 50psi of atomizing and assisting gas. Scanning was performed using Multiple Reaction Monitoring (MRM). The ion pairs used for the quantitative analysis are shown in table 4 below.
TABLE 4 quantitative ion pairs for analysis
| Detecting substance | Parent ion | Daughter 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 |
And respectively detecting each working standard solution and each sample. Taking the concentration of the working standard solution as a horizontal coordinate and the peak area as a vertical coordinate, investigating a linear range and drawing a standard curve to obtain a regression equation, wherein the regression equation is as follows:
DL-3-phenyllactic acid: y ═ 1822.7+ 2156.3X, R ═ 0.9969;
pantothenic acid: y-414.7 + 21.005X, R-0.9964;
l-pyroglutamic acid: y-54.313 + 2.719X.
And calculating the content of each substance in the sample according to the regression equation and the peak area corresponding to each substance.
4. Statistical analysis
And respectively performing Principal Component Analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA) dimension reduction analysis on the sample data by adopting an R software package Ropls. P-value is calculated according to T test, variable projection importance (VIP) is calculated by a PLS-DA dimension reduction method, fold change calculation module difference multiple is calculated, and influence strength and interpretability of the content of DL-3-phenyllactic acid, pantothenic acid and L-pyroglutamic acid on sample classification discrimination are measured. Statistical significance was considered when P-value <0.05 and VIP value > 1. The area under the subject's working characteristic curve (AUC) was used to assess the diagnostic accuracy of the model in identifying patients in each comparison. AUC <0.5, indicating no diagnostic significance; when AUC is 0.5-0.7, the diagnosis accuracy is low; when AUC is 0.7-0.9, the diagnosis accuracy is moderate; AUC >0.9, indicating high diagnosis accuracy.
5. Analysis of results
The results of comparing the content of DL-3-phenyllactic acid in the patients with prostatitis with that of the normal control and ROC curve analysis are shown in FIG. 1, the content of DL-3-phenyllactic acid in the group of patients with prostatitis is significantly higher than that in the group of normal control, AUC is 0.773, sensitivity is 63.2%, and specificity is 81.1%.
As shown in FIG. 2, the content of pantothenic acid in the group of patients with prostatitis was significantly higher than that in the group of normal controls, and ROC curve analysis showed that AUC was 0.725, sensitivity was 78.9% and specificity was 56.8%.
As shown in FIG. 3, the content of L-pyroglutamic acid in the group of patients with prostatitis is significantly higher than that in the group of normal controls, and ROC curve analysis results show that AUC is 0.721, sensitivity is 65.8%, and specificity is 70.3%.
As shown in FIG. 4, the results of ROC curve analysis show that when DL-3-phenyllactic acid and pantothenic acid are used in combination, AUC is 0.988, sensitivity is 95%, and specificity is 95%, which indicates that the combination of the two can effectively improve the diagnosis effect on prostatitis, and greatly improve the sensitivity and specificity.
As shown in FIG. 5, the results of ROC curve analysis show that when DL-3-phenyllactic acid, pantothenic acid and L-pyroglutamic acid are used in combination, AUC is 0.985, sensitivity is 95% and specificity is 95%, which indicates that the combination of the three can effectively improve the diagnosis effect on prostatitis.
As shown in FIG. 6, the results of ROC curve analysis show that when DL-3-phenyllactic acid and L-pyroglutamic acid are used in combination, AUC is 0.548, sensitivity is 55% and specificity is 60%, which indicates that the combination of the two can not improve the diagnosis effect on prostatitis, but also can cause the reduction of diagnosis performance.
The results show that DL-3-phenyllactic acid, pantothenic acid and L-pyroglutamic acid can be independently used as biomarkers for diagnosing prostatitis, and have better specificity and sensitivity; when DL-3-phenyllactic acid and pantothenic acid are used together or DL-3-phenyllactic acid, pantothenic acid and L-pyroglutamic acid are used together, the diagnosis accuracy is greatly improved, and the sensitivity and the specificity can reach 95 percent.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Claims (10)
1. A prostatitis-related marker, wherein the marker is at least one selected from the group consisting of DL-3-phenyllactic acid, pantothenic acid, and L-pyroglutamic acid.
2. The prostatitis-related marker of claim 1 wherein the marker is DL-3-phenyllactic acid and pantothenic acid.
3. The prostatitis-related marker of claim 1 wherein the marker is DL-3-phenyllactic acid, pantothenic acid, and L-pyroglutamic acid.
4. Use of the prostatitis-related marker of any one of claims 1 to 3 in the preparation of a reagent for detecting prostatitis.
5. A reagent for detecting prostatitis, wherein the reagent can detect the amount of the prostatitis-related marker according to any one of claims 1 to 3.
6. The reagent of claim 5, wherein the reagent is a reagent for detection by a liquid chromatography-mass spectrometry technique.
7. Use of the reagent of claim 5 or 6 in the preparation of a prostatitis detection kit.
8. A kit for the detection of prostatitis comprising the reagent according to claim 5 or 6 and a 25 to 35 v/v% aqueous methanol solution containing 0.1 to 0.3 w/v% formic acid.
9. The kit according to claim 8, comprising the reagent according to claim 5 or 6, and a 30 v/v% aqueous methanol solution containing 0.1 w/v% formic acid.
10. The reagent or the kit according to any one of claims 5 to 9, wherein the detection sample of the reagent or the kit is serum.
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