CN112852984B - Detection system for urinary system infection pathogen, kit and application thereof - Google Patents

Detection system for urinary system infection pathogen, kit and application thereof Download PDF

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CN112852984B
CN112852984B CN202110167340.3A CN202110167340A CN112852984B CN 112852984 B CN112852984 B CN 112852984B CN 202110167340 A CN202110167340 A CN 202110167340A CN 112852984 B CN112852984 B CN 112852984B
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nucleotide sequence
primer
reverse primer
forward primer
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CN112852984A (en
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张艳梅
赵虎
孙召洋
吴勇
南丽
余丁
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Ningbo Haier Shi Gene Technology Co ltd
Huadong Hospital
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Huadong Hospital
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Abstract

The invention relates to a detection system of urinary system infection pathogens, a kit and application thereof, wherein the detection system comprises 20 pairs of primers, including 18 pairs of detection primers, 1 pair of internal reference primers of human DNA, and 1 pair of internal reference primers of system quality control. The detection system of the urinary system infection pathogen drug resistance gene, the kit and the application thereof do not need to adopt conventional detection, can directly carry out synchronous identification and quantitative analysis of a plurality of pathogens on a urine sample in the same reaction system, have the advantages of strong specificity, high sensitivity, good repeatability, rapidness and accuracy, provide comprehensive, accurate and low-cost etiology diagnosis for clinic at the first time, and provide important references for personalized medication and accurate medical treatment.

Description

Detection system for urinary system infection pathogen, kit and application thereof
Technical Field
The invention relates to a multiplex gene detection product and a detection system used by the product, belonging to the technical field of biology.
Background
Urinary tract infection, also known as urinary tract infection (Urinary Tract Infections, UTI), is one of the most common infectious diseases in clinic. Multiple pathogens can invade the urinary system and grow and reproduce in large quantity, so that clinical symptoms such as frequent urination, urgent urination, painful urination, difficult urination, soreness of waist and legs and the like are caused, and serious diseases such as sepsis, acute and chronic kidney function damage and the like can be developed; the urologic sepsis and the septic shock progress very fast, and the death rate is up to 28.3% -41.1%. Over 50% of women experience UTI at least once throughout their lives. It has been found that a number of factors including women, older ages, long hospital stays, history of diabetes, invasive surgical procedures, long catheter residence time, and the type of antimicrobial drugs used are independent risk factors for inducing UTI in the hospital, which is also a major cause of increasing iatrogenic UTI in recent years. At the same time, the abuse of empirical and antibacterial drugs has led to an increasing resistance to UTI pathogens. Furthermore, in a substantial number of UTI patients, the infection recurs multiple times in a short period of time. In conclusion, the high incidence, high drug resistance and high recurrence rate of UTI bring great pain to patients, bring heavy treatment and economic burden to the social public health system, and even endanger the lives of the patients.
The pathogens causing UTI are various, and the pathogens commonly separated from urine samples are escherichia coli, enterococcus faecium, klebsiella pneumoniae, enterococcus faecalis, pseudomonas aeruginosa, candida albicans, proteus mirabilis, streptococcus agalactiae and the like in sequence. UTI infections are predominantly single species, and the complexity of UTI, which has been a mixed infection of two or more bacteria and a combined anaerobic, fungal or other non-classical microbial infection, has increased in recent years. Notably, a significant portion of UTI pathogens such as mycobacterium tuberculosis infection, gonococcus, chlamydia trachomatis, ureaplasma urealyticum, and the like are difficult to culture and identify. Taking tuberculosis of urinary system (urinary tuberculosis, UTB) as an example, the incidence rate of the tuberculosis accounts for about 4% of all tuberculosis, the proportion of the tuberculosis accounts for 30% -40% of the total tuberculosis outside the lung, and the tuberculosis is the most serious clinical type in the tuberculosis outside the lung, but the pathogen has extremely high omission rate and is easy to cause false negative and misdiagnosis. In summary, the great variety and complexity of UTB pathogens results in increased difficulty in diagnosis, delayed treatment, and even serious clinical complications. Therefore, rapid, accurate and comprehensive pathogen detection is critical for diagnosis and control of UTB.
UTI has high incidence rate, is easy to repeatedly infect, and has long duration, thereby seriously jeopardizing the health and life quality of patients. Early, rapid and accurate diagnosis of etiology is critical for the treatment of UTI. However, conventional methods for detecting related pathogens and drug resistance of UTI at home and abroad can not meet the requirements of clinical accurate diagnosis and treatment. The detection methods commonly used at present comprise: 1) Culturing and identifying: bacterial or fungal cultures are gold standards for UTI-related pathogen detection. The method has high specificity, and can directly provide definite etiology diagnosis and in-vitro drug sensitivity test results for clinic. However, culture identification suffers from a number of disadvantages: (1) time consuming: the common bacteria are cultured and incubated for 16-24 hours, and even longer time is needed for pathogens with slow growth and complex nutrition requirements; biochemical identification and drug sensitivity test are also needed after colony growth, at least 3 working days are needed; (2) incomplete coverage, low positive detection rate: as common culture is mostly aimed at single pathogens, but pathogenic bacteria such as mycobacterium tuberculosis, mycoplasma and the like which are difficult to culture but are extremely important are also included in UTI, false negatives are often caused. 2) Immunological methods: the specific pathogen in urine sample can be detected rapidly by colloidal gold, serology and other methods. However, the method has a narrow detection spectrum and cannot synchronously detect mixed infection. 3) Molecular biology method: in recent years, fluorescent quantitative PCR (reverse transcription-polymerase chain reaction) method (RT-PCR) is adopted in part of microbial laboratories at home and abroad, and the specificity gene fragments of common pathogens of UTI are detected to rapidly identify the pathogens, so that the positive detection rate can be greatly improved. However, RT-PCR can only detect one specific pathogen or conditional pathogen at a time, and cannot detect/identify multiple UTI-related pathogens simultaneously. Meanwhile, the method has high cost, the PCR detection charge of each pathogen is about 100 yuan, and the total detection cost of 20 most common clinical UTI pathogens at a time is at least 1200 yuan. In month 3 2017, the European urology institute (European Association of Urology, EAU) has updated guidelines for the management of urinary infections, indicating that "pathogens and sensitization to urinary infections should be clarified as early as possible in order to clinically select the optimal antimicrobial treatment regimen". Thus, there is a great need for a detection technique and method that can rapidly and simultaneously detect a variety of urinary system infectious pathogens.
Furthermore, the methods of treatment of urinary tract infections caused by different pathogens vary.
The national defense and accounting principal 2015 clinical application guidelines for antibacterial drugs clearly indicate that: the "antibacterial drug application" must be applicable after a clear diagnosis based on the patient's symptoms, signs and laboratory test results. However, the conventional detection method has the defects of low detection rate, long time consumption, incapability of accurately identifying various pathogens at the same time, and the like, and cannot provide timely, comprehensive and accurate pathogen diagnosis basis for clinic, so that broad-spectrum antibacterial drugs are commonly adopted for empirical treatment in clinic, and the problems of low curative effect, incapability of timely controlling the progress of urinary tract infection, high drug resistance rate of induced bacterial strains, increase of medical burden of patients and the like are caused.
The quantitative detection of pathogens causing urinary tract infection is helpful for knowing the pathogen load of patient infection and evaluating the severity of illness, and the qualitative and quantitative detection of pathogens can provide more comprehensive diagnosis information for clinic, and is helpful for clinicians to take more accurate medication schemes. For example, pathogens are distributed on the surface of normal human skin and belong to field bacteria. In determining whether or not the colonization bacteria is an infectious pathogenic bacteria, it is necessary to confirm whether or not the bacteria are dominant in number. At present, the clinical distinction between infection and pollution mainly refers to the degree of invasion of pathogens into the deep part of living tissues and whether the pathogen content of each gram of tissues or samples reaches a certain local value. The modern diagnosis and treatment guideline of urology indicates that the pathogen number of quantitative culture of middle-stage urine is more than 10 5 Infection with/mL < 10 3 The concentration of the catalyst in the solution/mL is usually 10 3 /mL～10 5 the/mL cannot exclude the possibility of infection, and re-sampling is required for review if necessary. Fungi, chlamydia, neisseria gonorrhoeae and salmonella typhiSpecific culture of Mycobacterium tuberculosis and anaerobic bacteria is required. Therefore, the type of pathogen infection should be quantified while being qualitative, thereby helping the clinician to ascertain whether urine culture pathogen positives are caused by urinary tract infections and thus determine whether antibiotic intervention should be taken. Pathogen infection is of great significance to the clinician in assessing the extent of infection in a patient and in formulating a reasonable treatment regimen.
In addition, when the quantitative detection finds that the content of the pathogen of urinary system infection is low, the patient is prompted to be possibly in an early stage of infection, so that a clinician is warned to take therapeutic intervention in time, and the exacerbation of the patient is avoided.
In view of the above, in order to determine the kind of the pathogen of urinary system infection as early as possible and to quantitatively detect the pathogen, development of a new technology is urgently required.
Disclosure of Invention
The invention aims to solve the technical problem of providing a urinary system infection pathogen detection system with the advantages of rapidness, comprehensiveness, accuracy and low cost, a kit and application of the detection system in preparing diagnostic products.
The invention provides a technical scheme for solving the technical problems, which is as follows: a pathogen detection system for urinary system infection comprises forward and reverse primers for respectively detecting escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae, enterobacter cloacae, acinetobacter baumannii, proteus mirabilis, streptococcus agalactiae, enterococcus faecium, enterococcus faecalis, staphylococcus aureus, candida albicans, candida tropicalis, candida glabrata, mycobacterium tuberculosis, chlamydia trachomatis, mycoplasma hominis, ureaplasma urealyticum and neisseria gonorrhoeae, and a detection sample is urine.
The nucleotide sequence of the forward primer for the escherichia coli is shown as SEQ ID No.1, and the nucleotide sequence of the reverse primer for the escherichia coli is shown as SEQ ID No. 2;
the nucleotide sequence of the forward primer for pseudomonas aeruginosa is shown as SEQ ID No.3, and the nucleotide sequence of the reverse primer for pseudomonas aeruginosa is shown as SEQ ID No. 4;
the nucleotide sequence of the forward primer for klebsiella pneumoniae is shown as SEQ ID No.5, and the nucleotide sequence of the reverse primer for klebsiella pneumoniae is shown as SEQ ID No. 6;
the nucleotide sequence of the forward primer for enterobacter cloacae is shown as SEQ ID No.7, and the nucleotide sequence of the reverse primer for enterobacter cloacae is shown as SEQ ID No. 8;
the nucleotide sequence of the forward primer for Acinetobacter baumannii is shown as SEQ ID No.9, and the nucleotide sequence of the reverse primer for Acinetobacter baumannii is shown as SEQ ID No. 10;
the nucleotide sequence of the forward primer for the Proteus mirabilis is shown as SEQ ID No.11, and the nucleotide sequence of the reverse primer for the Proteus mirabilis is shown as SEQ ID No. 12;
the nucleotide sequence of the forward primer for streptococcus agalactiae is shown as SEQ ID No.13, and the nucleotide sequence of the reverse primer for streptococcus agalactiae is shown as SEQ ID No. 14;
the nucleotide sequence of the forward primer for enterococcus faecium is shown as SEQ ID No.15, and the nucleotide sequence of the reverse primer for enterococcus faecium is shown as SEQ ID No. 16;
the nucleotide sequence of the forward primer for enterococcus faecalis is shown as SEQ ID No.17, and the nucleotide sequence of the reverse primer for enterococcus faecalis is shown as SEQ ID No. 18;
the nucleotide sequence of the forward primer for staphylococcus aureus is shown as SEQ ID No.19, and the nucleotide sequence of the reverse primer for staphylococcus aureus is shown as SEQ ID No. 20;
the nucleotide sequence of the forward primer for candida albicans is shown as SEQ ID No.21, and the nucleotide sequence of the reverse primer for candida albicans is shown as SEQ ID No. 22;
the nucleotide sequence of the forward primer for candida tropicalis is shown as SEQ ID No.23, and the nucleotide sequence of the reverse primer for candida tropicalis is shown as SEQ ID No. 24;
the nucleotide sequence of the forward primer for candida glabrata is shown as SEQ ID No.25, and the nucleotide sequence of the reverse primer for candida glabrata is shown as SEQ ID No. 26;
the nucleotide sequence of the forward primer for the mycobacterium tuberculosis is shown as SEQ ID No.27, and the nucleotide sequence of the reverse primer for the mycobacterium tuberculosis is shown as SEQ ID No. 28;
the nucleotide sequence of the forward primer for the chlamydia trachomatis is shown as SEQ ID No.29, and the nucleotide sequence of the reverse primer for the chlamydia trachomatis is shown as SEQ ID No. 30;
the nucleotide sequence of the forward primer for the mycoplasma hominis is shown as SEQ ID No.35, and the nucleotide sequence of the reverse primer for the mycoplasma hominis is shown as SEQ ID No. 36;
the nucleotide sequence of the forward primer for ureaplasma urealyticum is shown as SEQ ID No.33, and the nucleotide sequence of the reverse primer for ureaplasma urealyticum is shown as SEQ ID No. 34;
the nucleotide sequence of the forward primer for neisseria gonorrhoeae is shown in SEQ ID No.31 and the nucleotide sequence of the reverse primer for neisseria gonorrhoeae is shown in SEQ ID No. 32.
The urinary system infection pathogen detection system also comprises forward and reverse primers for detecting the internal control of human DNA and forward and reverse primers for detecting the internal control of system quality; the nucleotide sequence of the forward primer for the human DNA reference is shown as SEQ ID No.37, and the nucleotide sequence of the reverse primer for the human DNA reference is shown as SEQ ID No. 38; the nucleotide sequence of the forward primer for the system quality control internal reference is shown as SEQ ID No.39, and the nucleotide sequence of the reverse primer for the system quality control internal reference is shown as SEQ ID No. 40.
The systemic quality control reference may be beta-globin gene.
The final concentration of forward primers in a detection system for escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae, enterobacter cloacae, acinetobacter baumannii, proteus mirabilis, streptococcus agalactiae, enterococcus faecium, enterococcus faecalis, chlamydia trachomatis, mycoplasma hominis, ureaplasma urealyticum neisseria gonorrhoeae, human DNA internal reference and system quality control internal reference is 200nM; the final concentration of the reverse primer in a detection system for escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae, enterobacter cloacae, acinetobacter baumannii, proteus mirabilis, streptococcus agalactiae, enterococcus faecium, enterococcus faecalis, chlamydia trachomatis, mycoplasma hominis, ureaplasma urealyticum neisseria gonorrhoeae, human DNA internal reference and system quality control internal reference is 200nM; the final concentration of forward primers for staphylococcus aureus, candida albicans, candida tropicalis, candida glabrata and mycobacterium tuberculosis in a detection system is 400nM; the final concentration of the reverse primers against Staphylococcus aureus, candida albicans, candida tropicalis, candida glabrata and Mycobacterium tuberculosis in the detection system was 400nM.
The urinary system infection pathogen detection system also comprises multiple PCR premix, multiple PCR enzyme solution and nuclease-free pure water; the multiplex PCR premix is formed by mixing 10 times of PCR buffer solution, mgCl2 and dNTPs; the multiplex PCR enzyme solution is formed by mixing a hot start DNA polymerase and a UNG enzyme.
All forward primers were provided with fluorescent markers, CY5 or CY3 or FAM.
The urinary system infection pathogen detection system also comprises a positive control substance and a negative control substance; the positive control is a plasmid mixture comprising all target gene targets; the negative control is nuclease-free ultrapure water.
The component amount in the reaction system is 1 volume of 10 XPCR buffer, 10 mu M dNTPs are 0.2 volume altogether, 25mmol/L MgCl2 solution is 0.8 volume, primer mixture is 1 volume, 5U/mu L hot start DNA polymerase is 0.4 volume, 1U/mu L UNG enzyme is 0.5 volume, DNA template is 5 volume, and nuclease-free pure water is 1.1 volume; the usage amount of the DNA template is 5-50 ng/system.
The invention provides another technical scheme for solving the technical problems as follows: a urinary system infection pathogen detection kit comprising the detection system.
The invention provides another technical scheme for solving the technical problems as follows: an application of the detection system in preparing the products for detecting and diagnosing the pathogen of urinary system infection.
The invention has the positive effects that:
(1) The detection product of the urinary system infection pathogen can identify a plurality of urinary system infection pathogens through urine, the copy numbers of plasmids and the like of all target genes are mixed together, and the peak heights of all target points are equivalent by adjusting the primer concentration of each pathogen, so that the purpose of equivalently amplifying all target genes is achieved. Pathogens that can be quantitatively detected by two methods: standard curve method and pathogen peak area to known copy number IC ratio method. The method can detect urine samples of patients with urinary system infection or suspected patients, provide etiology diagnosis information about urinary system infection pathogens, help clinicians to timely determine pathogen types and adopt effective treatment schemes, reduce the use of empirical antibiotics, and reduce medical cost.
(2) The anti-pollution UNG enzyme is added in the detection product of the urinary system infection pathogen, so that the pollution of the gene amplification fragments is effectively eliminated before the gene amplification, and the accuracy and the reliability of the result are ensured.
(3) The detection product of the urinary system infection pathogen is different from the traditional gel electrophoresis analysis mode, and can separate nonspecific amplification products, primer dimers and specific amplification products, so that the detection result has no impurity peak, and the detection specificity and sensitivity are ensured. Through tests, the method has fewer miscellaneous peaks and shows high specificity; and can detect pathogens as low as 1000cfu/mL or 10 copies/mu L, and has high sensitivity.
(4) The detection product of the urinary system infection pathogen adds the human DNA internal reference and the IC internal reference, thereby ensuring the detection accuracy. The internal reference of the human DNA can monitor the extraction quality of the nucleic acid of the sample, and the appearance of the characteristic peak of the internal reference of the human DNA indicates the successful extraction of the nucleic acid, so that the false negative caused by the failure of the extraction of the nucleic acid can be effectively avoided; the internal reference characteristic peak of the human DNA does not appear to indicate the failure of nucleic acid extraction, and false positive caused by miscellaneous peaks can be effectively avoided. The IC can monitor the reaction process of PCR and capillary electrophoresis, and the characteristic peak of the IC does not show the failure of reaction, so that false negative can be effectively avoided. The IC is added while the nucleic acid extraction is carried out on the sample, and the IC is used as a system quality control internal reference for monitoring the whole detection process, so that the relative quantification of pathogens can be carried out.
(5) The dosage regimen and treatment of urinary system infections caused by different pathogens vary. The national defense and accounting principal 2015 clinical application guidelines for antibacterial drugs clearly indicate that: the "antibacterial drug application" must be applicable after a clear diagnosis based on the patient's symptoms, signs and laboratory test results. However, the conventional detection method has the defects of low detection rate, long time consumption, incapability of accurately identifying various pathogens at the same time, and the like, so that the problems of low curative effect, incapability of timely controlling urinary system infection, high incidence of drug-resistant strains, waste of national medical resources, increase of medical cost of patients and the like caused by the fact that broad-spectrum antibacterial drugs are generally adopted for empirical treatment in clinic. The invention establishes a high-flux, rapid, accurate and low-cost urinary system infection pathogen identification system, can synchronously detect 18 common pathogens of urinary system infection, effectively overcomes the defects of low detection rate, long time consumption, incapability of simultaneously identifying multiple pathogens and the like of the conventional detection method, and can clearly determine the types of the pathogens within 2.5 hours so as to clinically take correct treatment schemes and isolation measures, effectively prevent infection diffusion and reduce the generation of drug-resistant strains.
Drawings
FIG. 1 is a chart of the kit of example 1 of the present invention after performing a PCR reaction on a mixed positive control and then performing capillary electrophoresis analysis;
FIG. 2 is a chart showing the analysis of capillary electrophoresis after PCR reaction of negative control in the kit of example 1 of the present invention;
FIG. 3 is a chart showing the analysis of capillary electrophoresis after PCR reaction of sample 1 in the kit of example 1 of the present invention;
FIG. 4 is a chart showing the analysis of capillary electrophoresis after PCR reaction of sample 2 in the kit of example 1 of the present invention;
FIG. 5 is a chart showing the analysis of capillary electrophoresis after PCR reaction of sample 3 in the kit of example 1 of the present invention;
FIG. 6 is a quantitative standard curve of the gradient concentration simulated urine for the detection of Streptococcus agalactiae by the kit of example 1 of the present invention;
FIG. 7 is a quantitative standard curve of the test sample for Escherichia coli in the test sample of the kit of example 1. The method comprises the steps of carrying out a first treatment on the surface of the
FIG. 8 is a quantitative standard curve of the test kit of example 1 of the present invention for detecting the gradient concentration of Acinetobacter baumannii in simulated urine;
FIG. 9 is a quantitative standard curve of the gradient concentration simulated urine for detecting Enterobacter cloacae by the kit of example 1 of the present invention;
FIG. 10 is a quantitative standard curve of the gradient concentration simulated urine for the detection of enterococcus faecium by the kit of example 1 of the present invention;
FIG. 11 is a standard curve of the gradient concentration simulated urine for the detection of Staphylococcus aureus by the kit of example 1 of the present invention;
FIG. 12 is a standard curve of the gradient concentration simulated urine for detection of Mycoplasma hominus by the kit of example 1 of the present invention.
Detailed Description
The present invention is described in detail below by way of examples, which are necessary to be pointed out herein for further illustration of the invention and are not to be construed as limiting the scope of the invention, since numerous insubstantial modifications and adaptations of the invention will be to those skilled in the art in light of the foregoing disclosure. In the examples which follow, reagents used were all analytically pure and were all available from commercial sources unless specifically indicated. The experimental method, in which specific conditions are not specified, is generally carried out according to conventional conditions such as those described in the "molecular cloning Experimental guidelines" published in 2002 by J.Sam Brookfield et al, or according to the conditions recommended by the manufacturer. 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. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention.
Example 1
1. Composition of the kit
The urinary system infection pathogen detection kit of this embodiment includes: multiplex PCR premix, multiplex PCR enzyme solution, primer mix, positive control, negative control, system quality control internal reference (IC), and nuclease-free purified water. The multiplex PCR premix is prepared by mixing 10 XPCR buffer, mgCl2 and dNTPs. The multiplex PCR enzyme solution is prepared by mixing a hot start DNA polymerase and a UNG enzyme.
The multiplex PCR premix and the multiplex PCR enzyme solution were both from Roche Inc. (cat# 210212).
The positive control is a plasmid mixture that includes all the target gene of interest.
The negative control was nuclease-free ultrapure water.
The primer mixture comprises primer pairs for respectively detecting 18 pathogen target genes, primer pairs for detecting human DNA internal references and primer pairs for detecting system quality control internal references (ICs), the characteristics of each primer sequence are shown in table 1, and the primers are synthesized by Shanghai Biotechnology Co.
TABLE 1 characterization of primer sequences
2. Method for using kit
The specific detection steps of the urinary system infection pathogen detection kit of the embodiment are as follows:
(1) Collecting a urine sample of a patient: clean mid-stage urine is collected from patients suspected or diagnosed with urinary system infections.
(2) Sample nucleic acid extraction: nucleic acid extraction was performed on 300 μl of clean mid-stage urine samples, 80 μl of each of positive and negative controls was extracted, and 3 μl of IC was added to each sample involved in the extraction for co-extraction.
(3) Preparing a reaction system: according to the specification, a reaction system was prepared in a proportion of 2. Mu.L of a multiplex PCR premix, 0.9. Mu.L of a multiplex PCR enzyme solution, 1. Mu.L of a primer mixture and 1.1. Mu.L of purified water without nuclease for each reaction, and after vortexing, the mixture was centrifuged with a centrifuge and then split-packed in PCR reaction tubes.
(4) Adding a nucleic acid template: the extracted nucleic acid was added to a PCR reaction tube containing the prepared reaction system, and 5. Mu.L of nucleic acid was added per one person.
(5) Multiplex PCR amplification; the PCR amplification reaction conditions of the kit are shown in Table 2.
TABLE 2 multiplex PCR amplification conditions
(6) And carrying out capillary electrophoresis analysis on the amplified products, and judging the results according to the peak pattern diagram.
Taking 3500Dx genetic analyzer matched highly deionized formamide (HiDi) 8.75 mu L, SIZE-500 Plus 0.25 mu L, mixing, adding 1 mu L of PCR product, and performing capillary electrophoresis to separate samples. And judging the pathogen type and the pathogen quantity according to the peak position of the peak pattern.
3. Determination of detection results of a kit
1. Kit validity determination
The following conditions are satisfied at the same time, so that the result judgment can be performed:
1) Negative control: only the reference specific peak of the system quality control is detected.
2) Positive control: one fluorescent signal was detected at each amplified fragment length and the fluorescent signal value was higher than 500.
2. Sample validity determination:
1) If at least one of the fluorescence signal values of the detected sample is higher than 32000, the sample is excessively added, and the PCR product is recommended to be properly diluted and then subjected to capillary electrophoresis detection.
2) If the fluorescence signal values of the detected samples are lower than 500, the sample addition amount is lower, and the PCR product addition amount can be properly increased or the PCR reaction cycle number can be properly increased; if still unsatisfactory, the sample is prepared again.
3. Result criterion
Identification of urinary system infection pathogens: the target fragment areas of the human DNA internal reference, the system quality control internal reference and the pathogen gene show corresponding peaks, and the fluorescence signal values are higher than 500, so that the infection of related pathogens can be judged.
The quantitative method comprises the following steps: the method comprises the steps of simulating urine or gradient concentration plasmids by detecting gradient concentration (cfu/mL) of related urinary system infectious pathogens, determining correlation between different gradient concentrations and corresponding detection peak areas, making a standard curve, and further relatively quantifying pathogen concentration according to the peak areas of detection samples, and evaluating the pathogen load infected in the samples. The specific method is that the gradient concentration of each pathogen is taken as an abscissa, the detection peak area of each gradient concentration is taken as an ordinate, a quantitative standard curve is drawn, and the correlation between different concentrations and corresponding detection peak areas is determined. The data analysis software used was Origin 2018, the type of function selected was MichaeliMenten, R 2 The closer the value is to 1, the stronger the correlation between the gradient concentration of each pathogen and the detection peak area.
In addition, the relative quantification of pathogens can be performed based on the peak area ratio of pathogens to ICs in the test sample.
4. Result judgment example
The use method of the kit of the embodiment is adopted to carry out PCR reaction on the mixed solution of all positive controls, and capillary electrophoresis analysis is adopted to analyze the map as shown in figure 1. The target fragment region of the gene has 20 detection targets of escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae, enterobacter cloacae, acinetobacter baumannii, proteus mirabilis, streptococcus agalactiae, enterococcus faecium, staphylococcus aureus, candida albicans, candida tropicalis, candida glabrata, mycobacterium tuberculosis, chlamydia trachomatis, mycoplasma hominis, ureaplasma urealyticum, neisseria gonorrhoeae, and human DNA internal control and system quality control internal control. The result is very visual, and the gene amplification is good. It is shown that the primers have no interference, and can effectively amplify all target genes at the same time.
The map after PCR reaction of the negative control by the kit using method of the embodiment and analysis by capillary electrophoresis is shown in FIG. 2, only characteristic peaks of human DNA reference (hum_DNA) and system quality control reference (IC) appear, no characteristic peak of any pathogen appears, and only a non-specific background fluorescence signal is present at a position less than 100 nt. The specificity of the detection system is good.
The kit of the embodiment is used for serially diluting positive controls of single urinary system infectious pathogens, and capillary electrophoresis analysis is adopted after PCR reaction for evaluating the sensitivity of the method for detecting 18 pathogens. As shown in Table 3, the minimum sensitivity of the method for detection of 13 pathogens is 1000cfu/mL, and the minimum sensitivity for detection of 5 pathogens is 10 copies/. Mu.L. The sensitivity of the detection system to urinary system infection pathogen single infection detection is higher.
TABLE 3 sensitivity of detection of urinary system infectious pathogens
The map of sample 1 after PCR reaction and capillary electrophoresis analysis by the method of using the kit of this example is shown in FIG. 3. The human DNA internal reference and the system quality control internal reference are simultaneously appeared, the signal value is more than 500, and the target fragment region of the Neisseria Gonorrhoeae (NG) gene is respectively provided with a corresponding peak, and the signal value is more than 500. Based on the outcome criteria, the patient was shown to be infected with neisseria gonorrhoeae. The signal value of Neisseria Gonorrhoeae (NG) gene is 32167, the peak area is 293179, and the detection result is very visual.
The map of sample 2 after PCR reaction and capillary electrophoresis analysis by the method of using the kit of this example is shown in FIG. 4. The human DNA internal reference and the system quality control internal reference are simultaneously appeared and the signal value is more than 500, and the target fragment areas of enterococcus faecium (Efm) and escherichia coli (Eco) genes are respectively provided with corresponding peaks and the signal value is more than 500. According to the result judgment standard, the patient is infected with enterococcus faecium and Escherichia coli. The signal value of the enterococcus faecium (Efm) gene is 31268, the peak area is 280581, the signal value of the escherichia coli (Eco) gene is 31384, the peak area is 179914, and the detection result is very visual.
The map of sample 3 after PCR reaction and capillary electrophoresis analysis by the method of using the kit of this example is shown in FIG. 5. The human DNA internal reference and the system quality control internal reference are simultaneously appeared, the signal value is more than 500, and the target fragment areas of the Chlamydia Trachomatis (CT) and staphylococcus aureus (Sau) genes are respectively provided with corresponding peaks, and the signal value is more than 500. Based on the results criteria, the patient was shown to be infected with both Chlamydia trachomatis and Staphylococcus aureus. The signal value of the Chlamydia Trachomatis (CT) gene is 31952, the peak area is 236416, the signal value of the staphylococcus aureus (Sau) gene is 3462, the peak area is 20089, and the detection result is very visual.
The map of the gradient concentration simulated urine of streptococcus agalactiae after PCR reaction and capillary electrophoresis analysis by the use of the kit of the embodiment is shown in figure 6. Serial diluted gradient concentration simulated urine (10) 3 cfu/mL～10 6 cfu/mL) increases with increasing concentration of simulated urine, and the correlation coefficient R between the concentration of simulated urine for Streptococcus agalactiae infection (cfu/mL) and the detection peak area (rfu) 2 ＞0.99。
The map of the gradient concentration simulated urine of Escherichia coli after PCR reaction and capillary electrophoresis analysis by the kit of this example is shown in FIG. 7. Serial diluted gradient concentration simulated urine (10) 3 cfu/mL～10 6 cfu/mL) increases with increasing concentration of the simulated urine, and a correlation coefficient R between the concentration of the simulated urine (cfu/mL) and the detection peak area (rfu) of Escherichia coli infection 2 ＞0.99。
The map of the gradient concentration simulated urine of Acinetobacter baumannii subjected to PCR reaction by using the kit using method of the embodiment and capillary electrophoresis analysis is shown in FIG. 8. Serial diluted gradient concentration simulated urine (10) 3 cfu/mL～10 6 cfu/mL) with simulated urineThe concentration is increased, the concentration of the acinetobacter baumanii infection simulated urine (cfu/mL) is increased, and the correlation coefficient R between the concentration and the detection peak area (rfu) is increased 2 ＞0.99。
The map of the gradient concentration simulated urine of enterobacter cloacae subjected to PCR reaction by using the kit using method of the embodiment and then analyzed by capillary electrophoresis is shown in FIG. 9. Serial diluted gradient concentration simulated urine (10) 3 cfu/mL～10 6 cfu/mL) increases with increasing concentration of simulated urine, and the correlation coefficient R between the concentration of simulated urine (cfu/mL) and the detection peak area (rfu) for infection by Enterobacter cloacae 2 ＞0.99。
The map of the gradient concentration simulated urine of enterococcus faecium subjected to PCR reaction and capillary electrophoresis analysis by the kit using method of the embodiment is shown in FIG. 10. Serial diluted gradient concentration simulated urine (10) 3 cfu/mL～10 6 cfu/mL) increases with increasing concentration of simulated urine, and the correlation coefficient R between the concentration of simulated urine (cfu/mL) and the detection peak area (rfu) of enterococcus faecium infection 2 ＞0.99。
The map of the gradient concentration simulated urine of staphylococcus aureus after PCR reaction and capillary electrophoresis analysis by the kit using method of the embodiment is shown in figure 11. Serial diluted gradient concentration simulated urine (10) 3 cfu/mL～10 6 cfu/mL) increases with increasing concentration of simulated urine, and a correlation coefficient R between the concentration of simulated urine (cfu/mL) and the detection peak area (rfu) for Staphylococcus aureus infection 2 ＞0.99。
The map of the gradient concentration simulated urine of mycoplasma hominis after PCR reaction and capillary electrophoresis analysis by the use of the kit of the embodiment is shown in FIG. 12. Serial diluted gradient concentration simulated urine (10 copies/microliter-10) 4 Copy/microliter) of the detected peak area increases with increasing concentration of the simulated urine, and the correlation coefficient between the concentration of human mycoplasma infection simulated urine (copy/microliter) and the detected peak area (rfu)R 2 ＞0.99。
It is apparent that the above examples are merely illustrative of the present invention and are not limiting of the embodiments of the present invention. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While remaining within the scope of the invention, obvious variations or modifications are incorporated by reference herein.
Sequence listing
<110> Huadong Hospital
Ningbo Health Gene Technologies Co.,Ltd.
<120> detection system for urinary system infection pathogen, kit and application thereof
<130> none of
<141> 2021-02-07
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<212> DNA
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gtatgcccat atacgcgtcg atgccactcg gcc 33
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<213> Artificial sequence (none)
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<210> 9
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<213> Artificial sequence (none)
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<212> DNA
<213> Artificial sequence (none)
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<212> DNA
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<212> DNA
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<212> DNA
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<212> DNA
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<211> 35
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<212> DNA
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<211> 27
<212> DNA
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tgatctccga gatgtcatag cccttta 27
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<211> 27
<212> DNA
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gtagactcgg ctcctgttta actgaat 27
<210> 27
<211> 26
<212> DNA
<213> Artificial sequence (none)
<400> 27
tgaatgcgag ctcgagggat accagt 26
<210> 28
<211> 25
<212> DNA
<213> Artificial sequence (none)
<400> 28
gtccagtaga ggacgccaag agggt 25
<210> 29
<211> 28
<212> DNA
<213> Artificial sequence (none)
<400> 29
agggcttcct tacccataaa cttacgca 28
<210> 30
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<212> DNA
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<400> 30
atgctgtgac ttgttgtagt gtgtgaa 27
<210> 31
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<212> DNA
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<400> 31
tatcgatgcg gacacccaat acctgc 26
<210> 32
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<400> 32
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<212> DNA
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gttggtgtac cattccaata ccagtt 26
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<212> DNA
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Claims (6)

1. A urinary system infection pathogen detection system, characterized by: the kit comprises forward and reverse primers for respectively detecting escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae, enterobacter cloacae, acinetobacter baumannii, proteus mirabilis, streptococcus agalactiae, enterococcus faecium, enterococcus faecalis, staphylococcus aureus, candida albicans, candida tropicalis, candida glabrata, mycobacterium tuberculosis, chlamydia trachomatis, human-type mycoplasma, ureaplasma urealyticum and neisseria gonorrhoeae, wherein a detection sample is urine;
the nucleotide sequence of the forward primer for the escherichia coli is shown as SEQ ID No.1, and the nucleotide sequence of the reverse primer for the escherichia coli is shown as SEQ ID No. 2;
the nucleotide sequence of the forward primer for pseudomonas aeruginosa is shown as SEQ ID No.3, and the nucleotide sequence of the reverse primer for pseudomonas aeruginosa is shown as SEQ ID No. 4;
the nucleotide sequence of the forward primer for klebsiella pneumoniae is shown as SEQ ID No.5, and the nucleotide sequence of the reverse primer for klebsiella pneumoniae is shown as SEQ ID No. 6;
the nucleotide sequence of the forward primer for enterobacter cloacae is shown as SEQ ID No.7, and the nucleotide sequence of the reverse primer for enterobacter cloacae is shown as SEQ ID No. 8;
the nucleotide sequence of the forward primer for Acinetobacter baumannii is shown as SEQ ID No.9, and the nucleotide sequence of the reverse primer for Acinetobacter baumannii is shown as SEQ ID No. 10;
the nucleotide sequence of the forward primer for the Proteus mirabilis is shown as SEQ ID No.11, and the nucleotide sequence of the reverse primer for the Proteus mirabilis is shown as SEQ ID No. 12;
the nucleotide sequence of the forward primer for streptococcus agalactiae is shown as SEQ ID No.13, and the nucleotide sequence of the reverse primer for streptococcus agalactiae is shown as SEQ ID No. 14;
the nucleotide sequence of the forward primer for enterococcus faecium is shown as SEQ ID No.15, and the nucleotide sequence of the reverse primer for enterococcus faecium is shown as SEQ ID No. 16;
the nucleotide sequence of the forward primer for enterococcus faecalis is shown as SEQ ID No.17, and the nucleotide sequence of the reverse primer for enterococcus faecalis is shown as SEQ ID No. 18;
the nucleotide sequence of the forward primer for staphylococcus aureus is shown as SEQ ID No.19, and the nucleotide sequence of the reverse primer for staphylococcus aureus is shown as SEQ ID No. 20;
the nucleotide sequence of the forward primer for candida albicans is shown as SEQ ID No.21, and the nucleotide sequence of the reverse primer for candida albicans is shown as SEQ ID No. 22;
the nucleotide sequence of the forward primer for candida tropicalis is shown as SEQ ID No.23, and the nucleotide sequence of the reverse primer for candida tropicalis is shown as SEQ ID No. 24;
the nucleotide sequence of the forward primer for candida glabrata is shown as SEQ ID No.25, and the nucleotide sequence of the reverse primer for candida glabrata is shown as SEQ ID No. 26;
the nucleotide sequence of the forward primer for the mycobacterium tuberculosis is shown as SEQ ID No.27, and the nucleotide sequence of the reverse primer for the mycobacterium tuberculosis is shown as SEQ ID No. 28;
the nucleotide sequence of the forward primer for the chlamydia trachomatis is shown as SEQ ID No.29, and the nucleotide sequence of the reverse primer for the chlamydia trachomatis is shown as SEQ ID No. 30;
the nucleotide sequence of the forward primer for the mycoplasma hominis is shown as SEQ ID No.35, and the nucleotide sequence of the reverse primer for the mycoplasma hominis is shown as SEQ ID No. 36;
the nucleotide sequence of the forward primer for ureaplasma urealyticum is shown as SEQ ID No.33, and the nucleotide sequence of the reverse primer for ureaplasma urealyticum is shown as SEQ ID No. 34;
the nucleotide sequence of the forward primer for neisseria gonorrhoeae is shown as SEQ ID No.31, and the nucleotide sequence of the reverse primer for neisseria gonorrhoeae is shown as SEQ ID No. 32;
all the forward primers are provided with fluorescent markers, and the fluorescent markers are CY5 or CY3 or FAM;
the urinary system infection pathogen detection system also comprises a positive control and a negative control; the positive control is a plasmid mixture comprising all target gene targets; the negative control is nuclease-free ultrapure water.
2. The urinary system infection pathogen detection system according to claim 1, wherein: the kit also comprises forward and reverse primers for detecting the internal reference of the human DNA and forward and reverse primers for detecting the internal reference of the system quality control; the nucleotide sequence of the forward primer for the human DNA reference is shown as SEQ ID No.37, and the nucleotide sequence of the reverse primer for the human DNA reference is shown as SEQ ID No. 38; the nucleotide sequence of the forward primer for the system quality control internal reference is shown as SEQ ID No.39, and the nucleotide sequence of the reverse primer for the system quality control internal reference is shown as SEQ ID No. 40.
3. The urinary system infection pathogen detection system according to claim 2, wherein: the final concentration of forward primers in a detection system for escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae, enterobacter cloacae, acinetobacter baumannii, proteus mirabilis, streptococcus agalactiae, enterococcus faecium, enterococcus faecalis, chlamydia trachomatis, mycoplasma hominis, ureaplasma urealyticum, neisseria gonorrhoeae, human DNA internal reference and system quality control internal reference is 200nM; the final concentration of the reverse primer in a detection system for escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae, enterobacter cloacae, acinetobacter baumannii, proteus mirabilis, streptococcus agalactiae, enterococcus faecium, enterococcus faecalis, chlamydia trachomatis, mycoplasma hominis, ureaplasma urealyticum, neisseria gonorrhoeae, human DNA internal reference and system quality control internal reference is 200nM; the final concentration of forward primers for staphylococcus aureus, candida albicans, candida tropicalis, candida glabrata and mycobacterium tuberculosis in a detection system is 400nM; the final concentration of the reverse primers against Staphylococcus aureus, candida albicans, candida tropicalis, candida glabrata and Mycobacterium tuberculosis in the detection system was 400nM.
4. A urinary system infection pathogen detection system according to any one of claims 1 to 3, wherein: the kit also comprises multiplex PCR premix, multiplex PCR enzyme solution and nuclease-free pure water; the multiplex PCR premix consists of 10 XPCR buffer solution and MgCl 2 Mixing with dNTPs; the multiplex PCR enzyme solution is formed by mixing a hot start DNA polymerase and a UNG enzyme.
5. The urinary system infection pathogen detection system according to claim 4, wherein: the component amount in the reaction system is 1 volume of 10 XPCR buffer, 10 mu M dNTPs are 0.2 volume, 25mmol/L MgCl 2 0.8 volume of solution, 1 volume of primer mixture, 0.4 volume of 5U/. Mu.L of hot-start DNA polymerase, 0.5 volume of 1U/. Mu.L of UNG enzyme, DNA template5 volumes of pure water without nuclease 1.1 volumes; the usage amount of the DNA template is 5-50 ng/system.
6. A urinary system infection pathogen detection kit comprising the detection system of claim 1.
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