CN107245531B - Diarrhea pathogen multiple gene detection system and kit and application thereof - Google Patents
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Abstract
The invention relates to a diarrhea pathogen multiple gene detection system, a kit and application thereof. The detection system has 22 pairs of primers, wherein the primers comprise 19 pairs of diarrhea pathogens, 2 pairs of human genome internal references and 1 pair of system quality control internal reference primers. The diarrhea pathogens respectively aim at campylobacter jejuni, shigella, clostridium difficile, salmonella enteritidis, salmonella typhimurium, enterotoxigenic escherichia coli, escherichia coli O157, vibrio, enterocolitis yersinia, human astrovirus, norovirus II, human enterovirus, rotavirus and the like. The diarrhea pathogen multiple detection system and the kit thereof do not need to adopt steps such as conventional culture and the like, can directly carry out synchronous detection and analysis on a plurality of diarrhea pathogens on a fecal sample in the same reaction system, make up for the defects of low flux, long time consumption, low detection rate and the like of a conventional detection method, provide comprehensive, accurate and low-cost etiology diagnosis for clinic in the first time, and provide important references for individual medication and accurate medical treatment.
Description
Technical Field
The invention relates to a multiple gene detection product and a detection system used by the product, belonging to the technical field of biology.
Background
Infectious diarrhea is an intestinal infectious disease caused by various pathogens, and is one of the highest infectious diseases of the digestive system worldwide. The diarrhea is suffered by about 20 hundred million people every year in the world, and the disease is suffered by about 8 hundred million people every year in China. Infectious diarrhea has become the second largest infectious disease affecting human health, second only to respiratory infections.
The etiological diagnosis has important significance for preventing and treating infectious diarrhea. World gastroenterology organization Global guidelines-acute diarrhea in adults and children: global opinion (2012) and expert consensus on acute infectious diarrhea in adults (2013) clearly indicate that: "diagnosis of infectious diarrhea includes clinical diagnosis and etiological diagnosis, which not only provides basis for rational treatment, but also provides important clues for epidemiological investigation and prevention and control of the spread and prevalence of diarrhea diseases". At present, because the types of pathogens causing diarrhea are various and are dozens of pathogens, and the conventional detection method has large limitation, accurate etiological diagnosis is difficult to realize, so that clinical treatment is blindly performed, drug-resistant bacteria are generated, and infectious diarrhea cannot be controlled most timely and effectively.
However, conventional methods, procedures and clinical protocols for detecting infectious diarrhea pathogens have limitations.
The conventional method for detecting diarrhea-related pathogens at home and abroad currently comprises the following steps: bacterial culture: the species and serotype of the enteropathogenic bacteria are determined by separating, culturing and identifying the enteropathogenic bacteria and combining a serological typing method. The method has strong specificity, and can directly provide clear etiology diagnosis and in-vitro drug sensitivity test results for clinic. However, the pathogens causing infectious diarrhea are various, and the culture methods and culture conditions of intestinal pathogens of different species are different, so that the method cannot adopt a separation culture method (such as the type of culture medium and the culture environment) suitable for all the intestinal pathogens at the same time, and the pathogenic bacteria have low positive detection rate and high false negative rate, thereby causing high clinical missed diagnosis and misdiagnosis rate. Meanwhile, the method is long in time consumption and high in cost. ② an immunological method: etiological diagnosis is carried out by directly detecting specific antigens of pathogens in feces or specific antibodies in blood. This method is time consuming but has low sensitivity and specificity, especially the production of specific antibodies in the blood has a window (from infection to the production of detectable antibody components) leading to a high false negative rate. Meanwhile, the method cannot simultaneously detect and identify dozens of antigens or antibodies related to infectious diarrhea. ③ specific nucleotide detection method: diagnosis is carried out by specifically detecting gene segments of infectious diarrhea-associated pathogens. Its advantages are high specificity and sensitivity, but low cost and no need of simultaneously detecting and identifying multiple pathogens associated with infectious diarrhea.
The conventional detection process and steps of infectious diarrhea pathogens are as follows: firstly, collecting a feces sample of a diarrhea patient to culture intestinal pathogenic bacteria, namely directly inoculating an SS (suspended substance) plate, and separating and culturing salmonella/shigella; meanwhile, after the peptone water is inoculated for enrichment, a TCBS plate is inoculated for separating and culturing vibrio. ② culturing positive samples by conventional method, and performing biochemical identification, serological identification and in vitro drug sensitivity test. And (3) if the clinical symptoms of the negative person are in accordance with the intestinal bacterial infection symptoms, collecting the stool sample again to perform separation culture of pathogenic Escherichia coli, Yersinia enterocolitica, campylobacter jejuni or Clostridium difficile, respectively inoculating a Macconk plate, a campylobacter jejuni plate and a CCFA plate, and further performing biochemical identification, serological identification and in-vitro drug sensitivity test on the positive person. And thirdly, immunological or specific nucleic acid detection of enteroviruses is needed for patients with negative bacteria culture or other laboratory inspection indexes meeting the viral diarrhea. The detection process of infectious diarrhea pathogens in clinic has the disadvantages of multiple steps, high cost and long time. The comprehensive detection cost is at least 1000 yuan, and the time can be as long as 72 hours.
The methods of treatment and isolation of infectious diarrhea caused by different pathogens vary.
The clinical application guideline of antibacterial drugs in 2015 of national committee of health clearly indicates that: "the application of the antibacterial agent must be clearly applied after diagnosis according to the symptoms, signs and laboratory examination results of the patient". However, the conventional detection method at present has the defects of low detection rate, long time consumption, especially incapability of accurately identifying multiple pathogens at the same time and the like, and cannot provide timely, comprehensive and accurate pathogen diagnosis basis for clinic, so that the problems of low curative effect, incapability of timely controlling diarrhea, high incidence of drug-resistant strains, digestive flora disorder and the like caused by empirical treatment of broad-spectrum antibacterial drugs generally adopted in clinic are caused.
In conclusion, the current methods for detecting and diagnosing diarrhea pathogens cannot meet the clinical requirements, and the development of new technologies is urgently needed.
Disclosure of Invention
The invention aims to provide a diarrhea pathogen multiple gene detection system and a kit thereof, which can be used for quickly, comprehensively, accurately and at low cost, and application of the detection system in preparation of diagnostic products.
The invention provides a technical scheme for solving the technical problems, which comprises the following steps: a diarrhea pathogen multiple gene detection system comprises forward and reverse PCR amplification primers for respectively detecting campylobacter jejuni, Shigella, Clostridium difficile, salmonella enteritidis, salmonella typhimurium, enterotoxigenic Escherichia coli, enterohemorrhagic Escherichia coli, enteropathogenic Escherichia coli, intestinal adhesive Escherichia coli, intestinal invasive Escherichia coli, Escherichia coli O157, vibrio, Yersinia enterocolitica, human astrovirus, norovirus II, human enterovirus, rotavirus A, rotavirus B and rotavirus C.
The nucleotide sequence of the forward primer aiming at the campylobacter jejuni is shown as SEQ ID No.1, and the nucleotide sequence of the reverse primer aiming at the campylobacter jejuni is shown as SEQ ID No. 2;
the nucleotide sequence of the forward primer aiming at the shigella is shown as SEQ ID No.3, and the nucleotide sequence of the reverse primer aiming at the shigella is shown as SEQ ID No. 4;
the nucleotide sequence of the forward primer aiming at the clostridium difficile is shown as SEQ ID No.5, and the nucleotide sequence of the reverse primer aiming at the clostridium difficile is shown as SEQ ID No. 6;
the nucleotide sequence of the forward primer aiming at the salmonella enteritidis is shown as SEQ ID No.7, and the nucleotide sequence of the reverse primer aiming at the salmonella enteritidis is shown as SEQ ID No. 8;
the nucleotide sequence of the forward primer aiming at the salmonella typhimurium is shown as SEQ ID No.9, and the nucleotide sequence of the reverse primer aiming at the salmonella typhimurium is shown as SEQ ID No. 10;
the nucleotide sequence of the forward primer aiming at the enterotoxigenic escherichia coli is shown as SEQ ID No.11, and the nucleotide sequence of the reverse primer aiming at the enterotoxigenic escherichia coli is shown as SEQ ID No. 12;
the nucleotide sequence of the forward primer aiming at the enterohemorrhagic Escherichia coli is shown as SEQ ID No.13, and the nucleotide sequence of the reverse primer aiming at the enterohemorrhagic Escherichia coli is shown as SEQ ID No. 14;
the nucleotide sequence of the forward primer aiming at the enteropathogenic escherichia coli is shown as SEQ ID No.15, and the nucleotide sequence of the reverse primer aiming at the enteropathogenic escherichia coli is shown as SEQ ID No. 16;
the nucleotide sequence of the forward primer aiming at the intestinal adhesive escherichia coli is shown as SEQ ID No.17, and the nucleotide sequence of the reverse primer aiming at the intestinal adhesive escherichia coli is shown as SEQ ID No. 18;
the nucleotide sequence of the forward primer aiming at the intestinal invasive Escherichia coli is shown as SEQ ID No.19, and the nucleotide sequence of the reverse primer aiming at the intestinal invasive Escherichia coli is shown as SEQ ID No. 20;
the nucleotide sequence of the forward primer aiming at the vibrio is shown as SEQ ID No.21, and the nucleotide sequence of the reverse primer corresponding to the vibrio is shown as SEQ ID No. 22;
the nucleotide sequence of the forward primer aiming at the yersinia enterocolitica is shown as SEQ ID No.23, and the nucleotide sequence corresponding to the reverse primer of the yersinia enterocolitica is shown as SEQ ID No. 24;
the nucleotide sequence of the forward primer aiming at the Escherichia coli O157 is shown as SEQ ID No.25, and the nucleotide sequence of the reverse primer corresponding to the Escherichia coli O157 is shown as SEQ ID No. 26;
the nucleotide sequence of the forward primer aiming at the human astrovirus is shown as SEQ ID No.27, and the nucleotide sequence of the reverse primer corresponding to the human astrovirus is shown as SEQ ID No. 28;
the nucleotide sequence of the forward primer aiming at the norovirus II is shown as SEQ ID No.29, and the nucleotide sequence of the reverse primer aiming at the norovirus II is shown as SEQ ID No. 30;
the nucleotide sequence of the forward primer aiming at the human intestinal adenovirus is shown as SEQ ID No.31, and the nucleotide sequence of the reverse primer aiming at the human intestinal adenovirus is shown as SEQ ID No. 32;
the nucleotide sequence of the forward primer aiming at the rotavirus A is shown as SEQ ID No.33, and the nucleotide sequence of the reverse primer aiming at the rotavirus A is shown as SEQ ID No. 34;
the nucleotide sequence of the forward primer aiming at the rotavirus B is shown as SEQ ID No.35, and the nucleotide sequence of the reverse primer aiming at the rotavirus B is shown as SEQ ID No. 36;
the nucleotide sequence of the forward primer aiming at rotavirus C is shown as SEQ ID No.37, and the nucleotide sequence of the reverse primer aiming at rotavirus C is shown as SEQ ID No. 38.
The diarrhea pathogen multiple gene detection system also comprises forward and reverse PCR amplification primers for detecting human RNA internal reference, human DNA internal reference and system quality control internal reference;
the human RNA internal reference is B2M, the human DNA internal reference is RNaseP, and the system quality control internal reference is a plasmid containing a kanamycin resistance gene;
the nucleotide sequence of the forward primer for the human RNA internal reference is shown as SEQ ID No.39, and the nucleotide sequence of the reverse primer for the human RNA internal reference is shown as SEQ ID No. 40;
the nucleotide sequence of the forward primer aiming at the human DNA internal reference is shown as SEQ ID No.41, and the nucleotide sequence of the reverse primer aiming at the human DNA internal reference is shown as SEQ ID No. 42;
the nucleotide sequence of the forward primer aiming at the system quality control internal reference is shown as SEQ ID No.43, and the nucleotide sequence of the reverse primer aiming at the system quality control internal reference is shown as SEQ ID No. 44.
The final concentrations of the forward primers aiming at campylobacter jejuni, shigella, clostridium difficile, salmonella enteritidis, salmonella typhimurium, enterotoxigenic escherichia coli, enterohemorrhagic escherichia coli, enteropathogenic escherichia coli, intestinal adhesive escherichia coli, intestinal invasive escherichia coli, vibrio and rotavirus B in a detection system are all 200 nM; the final concentrations of the forward primers for yersinia enterocolitica, norovirus II and rotavirus C in the detection system are all 100nM, the final concentrations of the forward primers for escherichia coli O157 in the detection system are all 350nM, the final concentrations of the forward primers for human astrovirus in the detection system are all 300nM, the final concentrations of the forward primers for human enterovirus in the detection system are all 450nM, and the final concentrations of the forward primers for rotavirus A in the detection system are all 400 nM;
the final concentrations of the reverse primers aiming at campylobacter jejuni, shigella, clostridium difficile, salmonella enteritidis, salmonella typhimurium, enterotoxigenic escherichia coli, enterohemorrhagic escherichia coli, enteropathogenic escherichia coli, intestinal adhesive escherichia coli, escherichia coli O157, vibrio, human enterovirus, rotavirus B and rotavirus C in a detection system are all 100nM, the final concentrations of the reverse primers aiming at intestinal invasive escherichia coli in the detection system are all 200nM, the final concentrations of the reverse primers aiming at enterocolitis yersinia in the detection system are all 300nM, the final concentrations of the reverse primers aiming at human astrovirus and norovirus II in the detection system are all 400nM, and the final concentrations of the reverse primers aiming at rotavirus a in the detection system are all 450 nM;
the final concentration of forward and reverse primers in the detection system for human RNA internal reference, human DNA internal reference and system quality control internal reference is 1 μ M.
The diarrhea pathogen multiple gene detection system also comprises PCR buffer solution, MgCl2Solution, dNTPs, and a hot start DNA polymerase and reverse transcriptase mixture.
The above fluorescent label is CY5 or CY3 or FAM.
The diarrhea pathogen multiple gene detection system also comprises a positive control solution and a negative control substance; the positive control is a plasmid mixture comprising all target gene targets; the negative control solution was ultrapure water without nuclease.
The components used in the reaction system are 5 XPCR buffer 2 volume, 10. mu.M dNTPs 0.35 volume, 25mmol/L MgCl20.25 volume of the solution, 1 volume of the primer mixture, 0.4 volume of a mixture of 5U/. mu.L of the hot-start DNA polymerase and 5U/. mu.L of the reverse transcriptase, 1U/. mu.L
0.1 volume of UDG enzyme of L, 2.5 volumes of DNA template, and 2.5 volumes of pure water; the using amount of the gene template is 5-50 ng/system.
The invention provides another technical scheme for solving the technical problems, which comprises the following steps: a diarrhea pathogen multiple gene detection kit adopting the detection system.
The invention provides another technical scheme for solving the technical problems, which comprises the following steps: an application of the detection system in preparing a product for detecting and diagnosing diarrhea pathogens. The invention has the positive effects that:
(1) the diarrhea pathogen multiple gene detection system and the kit mix the copy numbers of plasmids of all target genes, and the peak heights of all target spots are equivalent by adjusting the primer concentration of all pathogens, so that the aim of equivalently amplifying all target genes is fulfilled.
(2) The diarrhea pathogen multiple gene detection system and the kit optimize a reaction system, add the UNG enzyme with pollution prevention, effectively eliminate the pollution of gene amplification fragments before gene amplification, and ensure the accuracy and reliability of results. Meanwhile, reverse transcriptase is added, so that reverse transcription and gene amplification are completed in one step, the detection process is simplified, the detection time is shortened, and the pollution caused by complicated operation is effectively reduced.
(3) The diarrhea pathogen multiple gene detection system and the kit are combined with capillary electrophoresis and fluorescence detection technologies, are different from the traditional gel electrophoresis analysis mode, can separate non-specific amplification products, primer dimers and specific amplification products, and reduce false positives to the maximum extent. The detection result of the kit has no miscellaneous peak and high specificity. Can detect pathogen with 10 copies and high sensitivity.
(4) The diarrhea pathogen multiple gene detection system and the diarrhea pathogen multiple gene detection kit do not need to adopt the steps of conventional culture of diarrhea pathogens and the like, the tissue sample is directly subjected to synchronous detection and analysis of multiple diarrhea pathogens in the same reaction system, all results are obtained by one-time detection, the defects of low flux, multiple steps, long time consumption, low detection rate and the like of a conventional detection method are overcome, the cost is low, the convenience is good, and comprehensive, accurate and low-cost etiology diagnosis is provided for clinic at the first time.
(5) The methods of treatment and isolation of infectious diarrhea caused by different pathogens vary. The clinical application guideline of antibacterial drugs in 2015 of national committee of health clearly indicates that: "the application of the antibacterial agent must be clearly applied after diagnosis according to the symptoms, signs and laboratory examination results of the patient". However, the conventional detection method at present has the defects of low detection rate, long time consumption, particularly incapability of accurately identifying multiple pathogens at the same time and the like, so that the problems of low curative effect, incapability of controlling diarrhea, high incidence of drug-resistant strains, disturbance of digestive flora and the like caused by empirical treatment by clinically and generally adopting broad-spectrum antibacterial drugs are caused. The diarrhea pathogen identification system with high flux, rapidness, accuracy and low cost is established, 19 common infectious diarrhea pathogens can be synchronously detected, the defects that a conventional detection method is low in detection rate and long in time consumption, multiple pathogens cannot be simultaneously identified and the like are effectively overcome, the types of the pathogens can be determined in the first time, so that a correct treatment scheme and isolation measures can be clinically adopted, infection diffusion is effectively prevented, and the generation of drug-resistant strains is reduced.
Drawings
FIG. 1 is a diagram of a reagent kit of the embodiment of the invention after performing a PCR reaction on a mixed positive control of diarrhea-associated pathogens and performing capillary electrophoresis analysis;
FIG. 2 is a diagram of a kit according to an embodiment of the present invention after serial dilution of diarrhea pathogens, PCR reaction, and capillary electrophoresis analysis;
FIG. 3 is a diagram of a sample 1 subjected to a PCR reaction and then to capillary electrophoresis analysis by the kit according to the embodiment of the present invention;
FIG. 4 is a diagram of a sample 2 after a PCR reaction and a capillary electrophoresis analysis by the kit according to the embodiment of the present invention;
FIG. 5 is a diagram of a sample 3 after a PCR reaction and a capillary electrophoresis analysis by the kit according to the embodiment of the present invention;
FIG. 6 is a diagram showing a sample 4 subjected to a PCR reaction and then to capillary electrophoresis analysis by the kit according to the embodiment of the present invention.
Detailed Description
The present invention is described in detail below by way of examples, it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make some insubstantial modifications and adaptations of the present invention based on the above-described disclosure. In the following examples, the reagents used were all analytical grade and were commercially available unless otherwise indicated. Experimental procedures not specifically identified herein are generally carried out under conventional conditions such as those described in the molecular cloning guidelines, published by scientific Press 2002, edited by J. SammBruk et al, or under 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 practice of the present invention.
Examples
Composition of kit
The diarrhea pathogen multiple gene detection kit of the embodiment comprises: primer mixture, PCR Buffer (5 XPCR Buffer), MgCl2Solution, dNTPs, hot start DNA polymerase and reverse transcriptase mixture, UDG enzyme, positive control and negative control.
The PCR buffer, hot start DNA polymerase and reverse transcriptase mixture were obtained from Qiagen (cat # 210212).
The hot-start DNA polymerase and the reverse transcriptase are mixed together to form a mixed solution, wherein the concentration of the hot-start DNA polymerase in the reaction system is 0.1U/. mu.L, and the concentration of the reverse transcriptase in the mixed solution is 0.1U/. mu.L.
The positive control solution is a plasmid mixture including all target gene targets.
The negative control solution was ultrapure water without nuclease.
The primer mixture comprises: a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for campylobacter jejuni; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for shigella; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for clostridium difficile; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for salmonella enteritidis; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for salmonella typhimurium; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for enterotoxigenic escherichia coli; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for enterohemorrhagic escherichia coli; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for enteropathogenic escherichia coli; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for Escherichia coli; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for Vibrio; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for enteroinvasive escherichia coli; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for yersinia enterocolitica; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for Escherichia coli O157; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for human astrovirus; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for norovirus II; the nucleotide sequence of a forward primer and the nucleotide sequence of a reverse primer aiming at the human intestinal adenovirus; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for rotavirus a; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for rotavirus B; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for rotavirus C; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for a human RNA internal reference; a nucleotide sequence of a forward primer and a nucleotide sequence of a reverse primer for a human DNA internal reference; the nucleotide sequence of the forward primer and the nucleotide sequence of the reverse primer aiming at the system quality control internal reference.
The human RNA internal reference is B2M, the human DNA internal reference is RNaseP, and the system quality control internal reference is a plasmid containing a kanamycin resistance gene.
The characteristics of each primer are shown in Table 1. The primers were synthesized by Shanghai Sangni Biotech Co., Ltd.
TABLE 1 diarrhea pathogen primer sequence characterization Table
Second, using method of kit
The specific detection steps of the diarrhea pathogen detection kit of the embodiment are as follows:
1. sample collection
And (3) immersing the fecal tissue specimen of a diarrhea patient into physiological saline, and extracting the total genome of the specimen by using a heel extraction kit, wherein the specific operation refers to the specification of an extraction kit product.
After obtaining the nucleic acid gene of the sample, the quality of the nucleic acid of the sample is controlled by measuring the concentration and the ratio of OD260/OD280 by an ultraviolet spectrophotometer. The preferred concentration of sample nucleic acid is 10 ng/. mu.L to 100 ng/. mu.L. The preferable range of the ratio of OD260/OD280 is 1.7-1.9.
PCR reaction
The reagents in the kit of this example were used to prepare PCR reaction systems with gene templates (sample nucleic acids, positive control solutions, or negative control solutions), respectively, the specific components of which are shown in table 2.
TABLE 2 PCR reaction System Components Table
The PCR reaction program was then performed on a PCR instrument (ABI Veriti 96 well), with the optimal reaction program as shown in Table 3.
TABLE 3 PCR reaction schedule
3. Capillary electrophoresis fragment analysis
To each well of the 96-well sample plate, 9. mu.L of formamide (AB. sciex, cat.: 608082) and 0.25. mu.L of an internal Standard (DNA Size Standard 500, AB. sciex, cat.: 608098) were added, and 1. mu.L of PCR product was added thereto.
The sample plate was placed into the machine and the Fragment separation procedure was run according to the operating manual for the AB3500DX capillary electrophoresis analyzer. And executing a default analysis method, and finally saving the data.
And (3) obtaining capillary electrophoresis peak images aiming at different sizes of PCR product fragments of all genes, wherein the abscissa represents the fragment length, and the ordinate represents the fluorescence intensity.
4. Analysis of results
The capillary electrophoresis analyzer automatically performs data analysis.
Thirdly, judging the detection result of the kit
1. Kit validity determination
The result judgment can be carried out when the following conditions are met:
1) negative control: only specific peaks of the internal reference of the system quality control are detected.
2) Positive control: one fluorescence signal was detected at each amplified fragment length and the fluorescence signal value was above 300.
2. And (3) judging the validity of the sample:
2) if the fluorescence signal value of the detected sample is at least one value higher than 32000, the sample is added in an excessive amount, and the capillary electrophoresis detection is recommended after the PCR product is properly diluted.
3) If the fluorescence signal values of the detected samples are all lower than 300, the sample addition amount is lower, and the PCR product addition amount or the PCR reaction cycle number can be properly increased; if the requirements are still not met, the sample is prepared again.
3. Criteria for determination of results
Identification of diarrhea pathogen infection
Corresponding peaks appear in target fragment regions of genes of the human DNA internal reference, the human RNA internal reference, the system quality control internal reference and the diarrhea pathogen, and fluorescence signal values are all higher than 300, so that the diarrhea pathogen can be judged to be infected.
4. Example of result judgment
The kit of this example was used to perform PCR reactions on individual positive controls and capillary electrophoresis analysis was used. The target fragment region of the gene shows corresponding peaks in 19 pathogens of campylobacter jejuni, shigella, clostridium difficile, salmonella enteritidis, salmonella typhimurium, enterotoxigenic escherichia coli, enterohemorrhagic escherichia coli, enteropathogenic escherichia coli, intestinal adhesive escherichia coli, intestinal invasive escherichia coli, escherichia coli O157, vibrio, yersinia enterocolitica, human astrovirus, norovirus II, human enteroadenovirus, rotavirus a, rotavirus B and rotavirus C. The results were very visual and the genes were all well amplified. Thus, each pair of primers can effectively amplify the corresponding target gene and has good specificity.
The spectrum of the mixture of all positive controls subjected to PCR reaction using the kit of this example after analysis by capillary electrophoresis is shown in FIG. 1. The target fragment region of the gene shows corresponding peaks in campylobacter jejuni, shigella, clostridium difficile, salmonella enteritidis, salmonella typhimurium, enterotoxigenic escherichia coli, enterohemorrhagic escherichia coli, enteropathogenic escherichia coli, enteroadhesive escherichia coli, enteroinvasive escherichia coli, escherichia coli O157, vibrio, yersinia enterocolitica, human astrovirus, norovirus II, human enteroadenovirus, rotavirus a, rotavirus B and rotavirus C19 pathogens. The results were very visual and the genes were all well amplified. Thus, the primers are not interfered with each other, and all target genes can be effectively amplified at the same time.
After the kit of the embodiment is used for carrying out PCR reaction on the negative control, capillary electrophoresis analysis is adopted, no target gene peak is generated, and only the non-specific background fluorescence signal exists at the position less than 100 nt. The detection system is proved to have very good specificity.
After serial dilution is carried out on a single diarrhea pathogen positive control by adopting the kit of the embodiment, the atlas obtained after PCR reaction and capillary electrophoresis analysis is shown in Table 4, and the atlas for detecting the clostridium difficile, the enterotoxigenic escherichia coli, the enteroadhesive escherichia coli, the salmonella enteritidis, the salmonella typhimurium, the vibrio, the yersinia enterocolitica and the rotavirus with the lowest sensitivity can reach 1000 copies; campylobacter jejuni, Escherichia coli enterohemorrhagic, Escherichia coli enteropathogenic, Escherichia coli invasive, and human astrovirus can reach 100 copies; shigella, norovirus type II and human enterovirus can reach 10 copies. The detection system has high sensitivity for detecting the diarrhea pathogen single infection.
TABLE 4 diarrhea pathogen detection sensitivity
The spectrum after mixing and serial dilution of all diarrhea pathogen positive controls by using the kit of the embodiment is analyzed by capillary electrophoresis after PCR reaction is carried out is shown in FIG. 2, 19 target pathogens can be detected at the dilution of 1000 copies/reaction, and the signal values are all higher than 300, so that the result is clear and easy to read. The detection system has high detection sensitivity to diarrhea pathogen multiple infection.
FIG. 3 shows a chromatogram obtained by analyzing a sample 1 by capillary electrophoresis after PCR reaction using the kit of this example. Human RNA, DNA and system quality control parameters are simultaneously appeared, the signal value is more than 300, corresponding peaks are appeared in the target fragment region of the Clostridium difficile (C.difficile) gene, and the signal value is more than 300. According to the result judgment standard, the patient is infected with clostridium difficile. The detection result is very intuitive.
FIG. 4 shows a chromatogram obtained by analyzing a sample 2 by capillary electrophoresis after PCR reaction using the kit of this example. Human RNA, human DNA and system quality control are simultaneously generated, the signal value is more than 300, and corresponding peaks are generated in the target fragment region of the salmonella typhimurium (S.typhimurium) gene, and the signal value is more than 300. According to the result judgment standard, the patient is infected with the salmonella typhimurium. The detection result is very intuitive.
FIG. 5 shows a chromatogram obtained by analyzing a sample 3 by capillary electrophoresis after PCR reaction using the kit of this example. Human RNA internal reference, human DNA internal reference and system quality control internal reference are simultaneously appeared, and the signal value is greater than 300, and the target fragment region of rotavirus (RoV) gene has the correspondent peaks, and its signal value is greater than 300. According to the result judgment standard, the patient is infected with rotavirus. The detection result is very intuitive.
FIG. 6 shows a spectrum obtained by analyzing a sample 4 by capillary electrophoresis after PCR reaction using the kit of this example. Human RNA, DNA and system quality control parameters are simultaneously generated, the signal value is more than 300, corresponding peaks are generated in target fragment regions of Clostridium difficile, Salmonella typhimurium and human intestinal adenovirus (S.typhimurium, C.difficile and HADV) genes, and the signal value is more than 300. According to the result judgment standard, the patient is infected with clostridium difficile, salmonella typhimurium and human intestinal adenovirus. The detection result is very intuitive.
It should be understood that the above examples are only for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. All embodiments need not be enumerated here, nor should they be enumerated. And such obvious variations or modifications which fall within the spirit of the invention are intended to be covered by the scope of the present invention.
SEQUENCE LISTING
<110> China east Hospital
<120> diarrhea pathogen multiple gene detection system, kit and application thereof
<130> do not
<160> 44
<170> PatentIn version 3.3
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Claims (8)
1. A diarrhea pathogen multiple gene detection product is characterized in that: comprises forward and reverse PCR amplification primers for respectively detecting campylobacter jejuni, shigella, clostridium difficile, salmonella enteritidis, salmonella typhimurium, enterotoxigenic escherichia coli, enterohemorrhagic escherichia coli, enteropathogenic escherichia coli, intestinal adhesive escherichia coli, intestinal invasive escherichia coli, escherichia coli O157, vibrio, yersinia enterocolitica, human astrovirus, norovirus II, human enteroadenovirus, rotavirus A, rotavirus B and rotavirus C;
the nucleotide sequence of the forward primer aiming at the campylobacter jejuni is shown as SEQ ID No.1, and the nucleotide sequence of the reverse primer aiming at the campylobacter jejuni is shown as SEQ ID No. 2;
the nucleotide sequence of the forward primer aiming at the shigella is shown as SEQ ID No.3, and the nucleotide sequence of the reverse primer aiming at the shigella is shown as SEQ ID No. 4;
the nucleotide sequence of the forward primer aiming at the clostridium difficile is shown as SEQ ID No.5, and the nucleotide sequence of the reverse primer aiming at the clostridium difficile is shown as SEQ ID No. 6;
the nucleotide sequence of the forward primer aiming at the salmonella enteritidis is shown as SEQ ID No.7, and the nucleotide sequence of the reverse primer aiming at the salmonella enteritidis is shown as SEQ ID No. 8;
the nucleotide sequence of the forward primer aiming at the salmonella typhimurium is shown as SEQ ID No.9, and the nucleotide sequence of the reverse primer aiming at the salmonella typhimurium is shown as SEQ ID No. 10;
the nucleotide sequence of the forward primer aiming at the enterotoxigenic escherichia coli is shown as SEQ ID No.11, and the nucleotide sequence of the reverse primer aiming at the enterotoxigenic escherichia coli is shown as SEQ ID No. 12;
the nucleotide sequence of the forward primer aiming at the enterohemorrhagic Escherichia coli is shown as SEQ ID No.13, and the nucleotide sequence of the reverse primer aiming at the enterohemorrhagic Escherichia coli is shown as SEQ ID No. 14;
the nucleotide sequence of the forward primer aiming at the enteropathogenic escherichia coli is shown as SEQ ID No.15, and the nucleotide sequence of the reverse primer aiming at the enteropathogenic escherichia coli is shown as SEQ ID No. 16;
the nucleotide sequence of the forward primer aiming at the intestinal adhesive escherichia coli is shown as SEQ ID No.17, and the nucleotide sequence of the reverse primer aiming at the intestinal adhesive escherichia coli is shown as SEQ ID No. 18;
the nucleotide sequence of the forward primer aiming at the intestinal invasive Escherichia coli is shown as SEQ ID No.19, and the nucleotide sequence of the reverse primer aiming at the intestinal invasive Escherichia coli is shown as SEQ ID No. 20;
the nucleotide sequence of the forward primer aiming at the vibrio is shown as SEQ ID No.21, and the nucleotide sequence of the reverse primer aiming at the vibrio is shown as SEQ ID No. 22;
the nucleotide sequence of the forward primer aiming at the yersinia enterocolitica is shown as SEQ ID No.23, and the nucleotide sequence of the reverse primer aiming at the yersinia enterocolitica is shown as SEQ ID No. 24;
the nucleotide sequence of the forward primer aiming at the Escherichia coli O157 is shown as SEQ ID No.25, and the nucleotide sequence of the reverse primer aiming at the Escherichia coli O157 is shown as SEQ ID No. 26;
the nucleotide sequence of the forward primer aiming at the human astrovirus is shown as SEQ ID No.27, and the nucleotide sequence of the reverse primer aiming at the human astrovirus is shown as SEQ ID No. 28;
the nucleotide sequence of the forward primer aiming at the norovirus II is shown as SEQ ID No.29, and the nucleotide sequence of the reverse primer aiming at the norovirus II is shown as SEQ ID No. 30;
the nucleotide sequence of the forward primer aiming at the human intestinal adenovirus is shown as SEQ ID No.31, and the nucleotide sequence of the reverse primer aiming at the human intestinal adenovirus is shown as SEQ ID No. 32;
the nucleotide sequence of the forward primer aiming at rotavirus A is shown as SEQ ID No.33, and the forward primer aiming at rotavirus A
The nucleotide sequence of the reverse primer of the rotavirus A is shown as SEQ ID No. 34;
the nucleotide sequence of the forward primer aiming at rotavirus B is shown as SEQ ID No.35, and the forward primer aiming at rotavirus B
The nucleotide sequence of the reverse primer of the rotavirus B is shown as SEQ ID No. 36;
the nucleotide sequence of the forward primer aiming at rotavirus C is shown as SEQ ID No.37, and the forward primer aiming at rotavirus C
The nucleotide sequence of the reverse primer of rotavirus C is shown as SEQ ID No. 38.
2. The diarrhea pathogen multiple gene detection product of claim 1, wherein: the kit also comprises forward and reverse PCR amplification primers for detecting human RNA internal reference, human DNA internal reference and system quality control internal reference; the human RNA internal reference is B2M, the human DNA internal reference is RNaseP, and the system quality control internal reference is a plasmid containing a kanamycin resistance gene;
the nucleotide sequence of the forward primer for the human RNA internal reference is shown as SEQ ID No.39, and the forward primer for the human RNA internal reference
The nucleotide sequence of the reverse primer of the human RNA internal reference is shown as SEQ ID No. 40;
the nucleotide sequence of the forward primer for the human DNA internal reference is shown as SEQ ID No.41, and
the nucleotide sequence of the reverse primer of the human DNA internal reference is shown as SEQ ID No. 42;
the nucleotide sequence of the forward primer aiming at the system quality control internal reference is shown as SEQ ID No.43, and the nucleotide sequence of the reverse primer aiming at the system quality control internal reference is shown as SEQ ID No. 44.
3. The diarrhea pathogen multiple gene detection product of claim 1, wherein: the final concentrations of the forward primers aiming at campylobacter jejuni, shigella, clostridium difficile, salmonella enteritidis, salmonella typhimurium, enterotoxigenic escherichia coli, enterohemorrhagic escherichia coli, enteropathogenic escherichia coli, intestinal adhesive escherichia coli, intestinal invasive escherichia coli, vibrio and rotavirus B in a detection system are all 200 nM; the final concentrations of the forward primers for yersinia enterocolitica, norovirus II and rotavirus C in the detection system are all 100nM, the final concentrations of the forward primers for escherichia coli O157 in the detection system are all 350nM, the final concentrations of the forward primers for human astrovirus in the detection system are all 300nM, the final concentrations of the forward primers for human enterovirus in the detection system are all 450nM, and the final concentrations of the forward primers for rotavirus A in the detection system are all 400 nM;
the final concentrations of the reverse primers aiming at campylobacter jejuni, shigella, clostridium difficile, salmonella enteritidis, salmonella typhimurium, enterotoxigenic escherichia coli, enterohemorrhagic escherichia coli, enteropathogenic escherichia coli, intestinal adhesive escherichia coli, escherichia coli O157, vibrio, human enteroadenovirus, rotavirus B and rotavirus C in a detection system are all 100nM, the final concentrations of the reverse primers aiming at intestinal invasive escherichia coli in the detection system are all 200nM, the final concentrations of the reverse primers aiming at enterocolitis yersinia in the detection system are all 300nM, the final concentrations of the reverse primers aiming at human astrovirus and norovirus II in the detection system are all 400nM, and the final concentrations of the reverse primers aiming at rotavirus a in the detection system are all 450 nM;
the final concentration of forward and reverse primers in the detection system for human RNA internal reference, human DNA internal reference and system quality control internal reference is 1 μ M.
4. The diarrhea pathogen multiple gene detection product of any one of claims 1 to 3, wherein: also included are PCR buffers, MgCl2Solution, dNTPs, and a hot start DNA polymerase and reverse transcriptase mixture.
5. The diarrhea pathogen multiple gene detection product of any one of claims 1 to 3, wherein: also included are fluorescent labels, which are CY5 or CY3 or FAM.
6. The diarrhea pathogen multiple gene detection product of any one of claims 1 to 3, wherein: positive and negative controls are also included; the positive control is a plasmid mixture comprising all target gene targets; the negative control was nuclease-free ultrapure water.
7. The diarrhea pathogen multiple gene detection product of claim 5, wherein: the components used in the reaction system are 5 XPCR buffer 2 volume, 10. mu.M dNTPs 0.35 volume, 25mmol/L MgCl20.25 volume of the solution, 1 volume of the primer mixture, 0.4 volume of a mixture of 5U/. mu.L of the hot-start DNA polymerase and 5U/. mu.L of the reverse transcriptase, 0.1 volume of 1U/. mu.L of the UDG enzyme, 2.5 volumes of the DNA template, and 2.5 volumes of pure water; the using amount of the gene template is 5-50 ng/system.
8. A diarrhea pathogen multi-gene detection kit using the detection product of claim 1.
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| CN108220464A (en) * | 2018-04-08 | 2018-06-29 | 陈思 | A kind of 16 kinds of food-borne pathogens detection kits |
| CN108546781A (en) * | 2018-04-26 | 2018-09-18 | 四川华汉三创生物科技有限公司 | It is a kind of detection lead to diarrhea pathogenic microorganism Nucleic acid combinations and its application |
| CN111826464B (en) * | 2020-07-16 | 2023-08-08 | 亚能生物技术（深圳）有限公司 | Primer probe for detecting various gastrointestinal viruses in one tube, screening method and kit |
| CN114277167A (en) * | 2021-12-31 | 2022-04-05 | 杭州千基生物科技有限公司 | Kit for detecting quantum dot nucleic acid of bacterial intestinal pathogens |
| CN114836581B (en) * | 2022-06-02 | 2024-03-12 | 昆明理工大学 | Primer combination for detecting pathogens of digestive tract infectious diseases |
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| Title |
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| Systematic application of multiplex PCR enhances the detection of bacteria, parasites, and viruses in stool samples;McAuliffe, Gary N.; Anderson, Trevor P.; Stevens, Mary; 等;《JOURNAL OF INFECTION》;20130418;第67卷(第2期);全文 * |
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