CN114277167A

CN114277167A - Kit for detecting quantum dot nucleic acid of bacterial intestinal pathogens

Info

Publication number: CN114277167A
Application number: CN202111670676.8A
Authority: CN
Inventors: 尹华立; 裘惠良; 刘娜娜
Original assignee: Hangzhou Qianji Biotechnology Co ltd
Current assignee: Hangzhou Qianji Biotechnology Co ltd
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2022-04-05

Abstract

The invention relates to the technical field of biomedicine, in particular to a kit for detecting quantum dot nucleic acid of bacterial enteric pathogens. The kit comprises a detection membrane strip, fluorescence detection liquid and reaction liquid, wherein the detection membrane strip comprises a nylon membrane and a capture probe fixed on the nylon membrane; the fluorescence detection solution comprises quantum dots which are used for marking the surface of the capture probe and are coupled with streptavidin; the reaction solution comprises: reaction liquid I, reaction liquid II and reaction liquid III. The invention has the beneficial effects that: the kit for detecting the quantum dot nucleic acid of the bacterial enteric pathogens with high flux, high sensitivity and high specificity is provided, the steps are fewer than those of the existing chromogenic gene chip, the detection time is obviously shortened, the equipment cost is lower than that of an organic fluorescent gene chip, and the clinical popularization is facilitated.

Description

Kit for detecting quantum dot nucleic acid of bacterial intestinal pathogens

Technical Field

The invention relates to the technical field of biomedicine, in particular to a kit for detecting quantum dot nucleic acid of bacterial enteric pathogens.

Background

At present, the incidence of Infectious Diarrhea (ID) in China is high, and the infectious diarrhea is one of important public health problems affecting human health. Infectious Diarrhea (ID), also known as acute gastroenteritis, is a group of common intestinal Infectious diseases with diarrhea and abdominal pain as main clinical manifestations, and common pathogens of Infectious Diarrhea (ID) include viruses, bacteria, fungi, parasites, etc., and is characterized by increased frequency of defecation, increased stool volume, and thin stool quality. The clinical manifestations include nausea, vomiting, fever, abdominal pain, tenesmus, weakness, etc., and severe cases may be accompanied by electrolyte disturbance, dehydration, renal insufficiency, multiple organ failure, etc.

The pathogenic bacteria of the bacterial intestinal tract are mainly: vibrio parahaemolyticus, Vibrio cholerae, Clostridium difficile, Staphylococcus aureus, Campylobacter jejuni, Aeromonas hydrophila, Salmonella, Yersinia enterocolitica. Shigella, diarrheagenic Escherichia coli (enteroinvasive Escherichia coli, enteroaggregative Escherichia coli, enteropathogenic Escherichia coli, enterotoxigenic Escherichia coli, Shiga toxin-producing Escherichia coli, enterohemorrhagic Escherichia coli), and the like.

Quantum Dots (QD), also known as semiconductor nanocrystals, are approximately spherical, have three-dimensional sizes in the range of 2-10nm, and have significant Quantum effects. The quantum dots are generally made of semiconductor materials of II-VI group elements (such as CdS, CdSe, CdTe, ZnSe, ZnS and the like) or III-V group elements (cadmium-free quantum dots, such as InP, InAs and the like), and a core/shell structure (such as common CdSe/ZnS core/shell structure quantum dots and the like) can also be made of two or more semiconductor materials. The physical, optical and electrical characteristics of the quantum dots are far superior to those of the existing organic fluorescent dye, and the quantum dots have the advantages of high sensitivity, good stability, long shelf life and the like, and are the best choice for a new generation of fluorescent labeled probe.

The quantum dot as a marking probe is particularly suitable for the application fields of high sensitivity, multi-index simultaneous detection and the like, and has the following advantages:

1) the quantum fluorescence efficiency is high, the molar extinction coefficient is large, the fluorescence intensity is more than 20 times stronger than the light intensity of the strongest organic fluorescent material, the quantum fluorescence detection device is suitable for high-sensitivity detection, and single quantum dot tracing can be realized by combining a high-resolution fluorescence microscope;

2) the optical-stability and photobleaching resistance are good, and the optical-bleaching resistant optical-film is suitable for long-time stable excitation dynamic observation and result archiving;

3) the fluorescence lifetime is long, the background fluorescence lifetime of the organic fluorescent dye or the biological sample is generally only 1-10 nanoseconds, the fluorescence lifetime of the quantum dots can last for 10-100 nanoseconds, and the background interference can be reduced and the sensitivity can be improved through the time resolution characteristic;

4) the emission wavelength is different due to the composition and the particle size, so that the quantum dots with similar characteristics but different emission wavelengths after surface modification are easy to prepare;

5) a broad and continuous absorption spectrum, realizing single light source multi-color excitation;

6) the emission spectrum is narrow and symmetrical, and the interference among different quantum dots in the multicolor excitation process can be reduced;

7) the quantum dots have larger Stokes shift and are easily distinguished from organic fluorescent dyes with smaller Stokes shift and background fluorescent light, and the background can be eliminated by adjusting the wavelength of the excitation light or using an optical filter, so that the sensitivity is improved.

8) The surface modified product has better biocompatibility, is coupled with various biomolecules, and has no non-specific adsorption.

Quantum dot materials were synthesized in glass matrix by Alexey I.Ekimov and in colloidal solution by Louis E.Brus in the 80 s of the 20 th century, and then the chemical modification technology of quantum dot surface ligands was gradually improved. As quantum dots have a plurality of advantages compared with the traditional fluorescent dyes, in 1998 Alivisatos and Nie, the quantum dots are applied to the biomolecular markers, and bioactive molecules such as antibodies or antigens are connected to active groups of the quantum dot surface modification ligands, so that the quantum dot bioluminescence dyeing is realized, and the application research of quantum dot biomarker materials is initiated.

The gene chip technology can simultaneously fix a large number of probes on a support to form a microarray, so that a large number of sequences in a clinical sample can be detected and analyzed at one time, and the defects of complicated operation, low automation degree, small number of operation sequences, low detection efficiency and the like of the traditional nucleic acid Blotting hybridization (Southern Blotting, Northern Blotting and the like) technology are overcome. The gene chip technology has the detection characteristics of high flux, high speed and high efficiency, so that a powerful detection tool is provided for the detection and identification of the pathogenic bacteria with high flux. However, the existing gene chip mainly uses organic fluorescent dye, BCIP/NBT and DAB as detection methods, and the organic fluorescent dye has a plurality of defects: the fluorescence intensity is not high, the stability is poor, the fluorescence is easy to be bleached by light, the emission peak width is large, the Stokes displacement is small, and the BCIP/NBT and DAB modes have the technical defects of complicated operation steps, low detection sensitivity, poor repeatability and the like.

At present, the detection method of bacterial enteric pathogen infection is mainly a culture method, enteric pathogens are identified through separation culture, serological typing methods are combined to determine the species and serotype of the pathogens, but the pathogens causing enteric infection are various, the culture methods and culture conditions of the pathogens of different species are different, the method cannot adopt a culture method suitable for all the pathogens at the same time, the positive detection rate is low, false negative is high, missed diagnosis and misdiagnosis are caused, and meanwhile, the steps are complicated, the time consumption is long, and the cost is high.

The prior related patents are as follows:

the Chinese patent publication No. CN103911443A discloses a gene chip for detecting 11 common infectious diarrhea pathogens and application thereof, including Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio cholerae, Vibrio alginolyticus, Vibrio freniscus, Shigella, Escherichia coli, Aeromonas, Salmonella, Campylobacter, and Proteus, wherein the substrate is a glass slide (the probe length is more than 35bp), the detection regions are 16S, gyrB, wzy and ipaH genes, Klenow enzyme labeling is performed by random primers after amplification, and the result interpretation is performed by laser excitation scanning after hybridization, so that 11 infectious diarrhea pathogens can be detected simultaneously.

The Chinese patent publication No. CN107083446A discloses a diarrhea pathogen multiple gene detection system and a kit and application thereof, wherein 15 pairs of primers, 1 pair of primers for human genome internal reference (RnaseP gene) and 1 pair of system quality control internal reference (kalamycin resistance gene) are adopted, the primers select specific genes to design, and meanwhile, a CY5 or FAM or CY3 mark is adopted to detect 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 and yersinia enterocolitica by adopting a capillary electrophoresis method.

The Chinese patent publication No. CN105441539A discloses a kit for simultaneously detecting 12 diarrhea pathogenic bacteria and its application, which adopts 15 pairs of primers and a pair of internal reference primers, enteropathogenic Escherichia coli, enterotoxigenic Escherichia coli, enterohemorrhagic Escherichia coli, enteroaggregative Escherichia coli, enteroinvasive Escherichia coli, salmonella, campylobacter jejuni, campylobacter coli, vibrio cholerae, vibrio parahaemolyticus, and yersinia enterocolitica, and the size and presence of amplified products are judged by electrophoresis after PCR amplification. Meanwhile, the design genes of the primers are the bfPA gene and the eaeA gene of the Escherichia coli EPEC, the elt gene, the stp gene and the sth gene of the Escherichia coli ETEC, the stx1 and stx2 genes of the Escherichia coli EHEC, the aggR gene of the Escherichia coli EAEC, the virA gene of the Escherichia coli EIEC, the virA gene of the Shigella, the ompC gene of the salmonella, the hipO gene of the campylobacter jejuni, the ceuE gene of the campylobacter coli, the ompW gene of the vibrio cholerae, the toxR gene of the vibrio parahaemolyticus and the ail gene of the yersinia enterocolitica, and the detection sensitivity is as follows: for target genes of campylobacter jejuni ceu E, campylobacter coli hipO, vibrio parahaemolyticus toxR and stx2 of EHEC, the sensitivity is 5000 copies/reaction; the sensitivity to other target genes was 500 copies/response.

As can be seen from the above patent documents and the prior art, no nucleic acid detection kit for detecting 14 bacterial enteric pathogens with high throughput, high sensitivity and high specificity by utilizing the optical characteristics of quantum dot materials and the characteristics of gene chips is established at present.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: provides a high-flux, high-sensitivity and high-specificity kit for detecting the quantum dot nucleic acid of the bacterial enteric pathogens.

In order to solve the technical problems, the invention adopts the technical scheme that: providing a kit for detecting quantum dot nucleic acid of bacterial enteric pathogens, wherein the kit comprises a detection membrane strip, fluorescent detection liquid and reaction liquid, and the detection membrane strip comprises a nylon membrane and a capture probe fixed on the nylon membrane; the fluorescence detection solution comprises quantum dots which are used for marking the surface of the capture probe and are coupled with streptavidin; the reaction solution comprises: reaction liquid I, reaction liquid II and reaction liquid III;

the reaction solution I comprises the following detection primers:

primer ID 1: AGAGGCGATGAAGGACGTAC (SEQ No. 1);

primer ID 2: AGGAGCCGATGAAGGACGTG (SEQ No. 2);

primer ID 3: GAGGCGATGAAAGACGTAG (SEQ No. 3);

primer ID 4: AGAGGCGATGAAGGGCGTGC (SEQ No. 4);

primer ID 5: AAAGGCGATGAAGGACGTAC (SEQ No. 5);

primer ID 6: AGAGGCGATGAAGGGCGTGC (SEQ No. 6);

primer ID 7: AGCCGATGAAGGACGTTAC (SEQ No. 7);

primer ID 8: CTGCTTGTACGTACACGGTTTC (SEQ No. 8);

primer ID 9: CCCTCCTGCTTCCCACAGG (SEQ No. 9);

primer ID 10: CATATTCAGACAGGATATC (SEQ No. 10);

primer ID 11: ACGTACACGGTTTCAGGTTCTT (SEQ No. 11);

primer ID 12: CCGGATTCAGACAGGATTC (SEQ No. 12);

primer ID 13: TGGTCCTCCCAGATTCCGACGG (SEQ No. 13);

primer ID 14: TATGGTTGGGATAAGGCTGG (SEQ No. 14);

primer ID 15: CGAGCTTAGTGATACTTGTG (SEQ No. 15);

the reaction solution II comprises the following detection primers:

primer ID 16: CATATCTTAGAAATGAACTC (SEQ No. 16);

primer ID17: TCTGAAGTAATTCTTGAATC (SEQ No. 17);

primer ID18: AGCTTCAGTCGCGATCT (SEQ No. 18);

primer ID19: TCGGAATCATAGAACGGT (SEQ No. 19);

primer ID20: CAATAGGTACTCCATTAC (SEQ No. 20);

primer ID21: CCTGTGCCACTATCAATCAT (SEQ No. 21);

primer ID22: CGCCGATATATTATTAAAG (SEQ No. 22);

primer ID23: ACCACTCTGCAACGTGTC (SEQ No. 23);

primer ID24: ATCAGGATATTCACTTACT (SEQ No. 24);

primer ID25: TAATACCGGTCTCTATATTC (SEQ No. 25);

primer ID26: ATGTTCGTGGATGCCATGTC (SEQ No. 26);

primer ID27: TCGATTTATTCAACAAAGCA (SEQ No. 27);

primer ID28: TTACAATCACTCTCGGC (SEQ No. 28);

primer ID29: GGTAATGAAGTTAGTAGGTT (SEQ No. 29);

primer ID 14: TATGGTTGGGATAAGGCTGG (SEQ No. 14);

primer ID 15: CGAGCTTAGTGATACTTGTG (SEQ No. 15);

the reaction solution III is Hotstart Taq DNA polymerase.

Preferably, in the above-mentioned kit for detecting a quantum dot nucleic acid of a bacterial enteric pathogen, the capture probe comprises:

probe IDP 1: TAGCATATCAGAAGGCACACC (SEQ No. 30);

probe IDP 2: CTGGAAAGCTTGGCGATACAG (SEQ No. 31);

probe IDP 3: CATACCACATATAAGAGGTC (SEQ No. 32);

probe IDP 4: AAGGCGCGCGATACAGGGTGA (SEQ No. 33);

probe IDP 5: GTAGGACTGCAATGTGCAAG (SEQ No. 34);

probe IDP 6: GGAAAGGCCGGCGATACAG (SEQ No. 35);

probe IDP 7: GGAAAGTCGCACGGTACAG (SEQ No. 36);

probe IDP 8: TACACGCGTTAGACGAACG (SEQ No. 37);

probe IDP 9: CCATTTATCGCAATCAGAT (SEQ No. 38);

probe IDP 10: CATTTGGTCAGGTCGGAGC (SEQ No. 39);

probe IDP 11: GACTATTTCATCAGGAGG (SEQ No. 40);

probe IDP 12: GAACTCCATTAACGCCAG (SEQ No. 41);

probe IDP 13: GGAATACCATATTCTCAGAT (SEQ No. 42);

probe IDP 14: GCTAAACCAGTAGAGTC (SEQ No. 43);

probe IDP 15: GAATTGCCTCATAGACTAC (SEQ No. 44);

preferably, in the above kit for detecting a quantum dot nucleic acid of a bacterial enteric pathogen, the detection membrane strip further comprises an internal control probe for monitoring nucleic acid extraction and amplification of a sample: ICP1: TTTGCTAATCATGTTCATACC (SEQ No. 45).

Preferably, in the kit for detecting a quantum dot nucleic acid of a bacterial enteric pathogen, the capture probe is an oligonucleotide single-stranded DNA, an amino group is labeled at the 3 'end or the 5' end of the oligonucleotide single-stranded DNA, an inter-arm is connected between the oligonucleotide single-stranded DNA and the amino group, the inter-arm is one or a combination of two of a fatty acid Cn chain and an oligo dTn, n in the fatty acid Cn chain is an integer of 1 to 12, and n in the oligo dTn is an integer of 1 to 30.

Preferably, in the kit for detecting the quantum dot nucleic acid of the bacterial enteric pathogen, 5' ends of primers SEQ No. 8-SEQ No.13, SEQ No.15, SEQ No.17, SEQ No.19, SEQ No.21, SEQ No.23, SEQ No.25, SEQ No.27 and SEQ No.29 in the detection primers are modified with biotin labels, a spacer arm is connected between the detection primers and biotin, the spacer arm is one or a combination of two of a fatty acid Cn chain and oligo dTn, n in the fatty acid Cn chain is an integer from 1 to 12, and n in the oligo dTn is an integer from 1 to 30.

Preferably, in the above-mentioned kit for detecting a quantum dot nucleic acid of a bacterial enteric pathogen, the excitation wavelength of the quantum dot is 200-500nm, the emission wavelength of the quantum dot is 400-700nm, and the size of the quantum dot is 1-200 nm.

Preferably, in the kit for detecting a quantum dot nucleic acid of a bacterial enteric pathogen, the quantum dot is CdSe/ZnS.

The invention has the beneficial effects that: the invention provides a high-throughput, high-sensitivity and high-specificity kit for detecting quantum dot nucleic acid of bacterial enteric pathogens, and solves the problems of long time (more than 3 days), high cost, low positive rate and the like of the conventional detection culture method. The quantum dot nucleic acid detection method for establishing the bacterial enteric pathogens for the first time has the following advantages:

1) compared with the existing organic fluorescence method or color method gene chip, the kit has less detection steps, obviously shortens the detection time, has lower equipment cost (low light source requirement) than the organic fluorescence gene chip, has the existing carrier of glass, has complex preparation process and complex and fussy detection process, needs a laser scanner with high cost for detection instruments, and is not beneficial to clinical popularization.

2) The invention adopts a multiple PCR method, can simultaneously detect and identify 9 types of 14 bacterial intestinal pathogens in one detection, has shorter time than a culture method, and solves the problem that some difficultly-cultured bacteria can not be cultured. The invention can also guide the use of antibiotics, avoid drug resistance caused by abuse of antibiotics, and provide basis for early diagnosis of patients with mixed infection or unobvious early clinical manifestations.

3) The kit for detecting the quantum dot nucleic acid of the bacterial enteric pathogen has high sensitivity, and all detection targets can reach 10³cfu/ml。

4) The kit for detecting the quantum dot nucleic acid of the bacterial intestinal pathogens has high specificity. The invention designs primers and probes separately according to specific genes of different pathogenic bacteria to ensure the high specificity of each pathogenic bacteria, and the specific related genes are 23SrRNA, ipaH, eaeA, elt, est, stx1, stx2, aggR and the like, which is different from the type identification effect which can be achieved only by aiming at a single or multiple targets disclosed in the existing literature.

5) The kit for detecting the quantum dot nucleic acid of the bacterial enteric pathogen is additionally provided with an internal control target for detection, the internal control target is endogenous nucleic acid (human genome manager gene) of a sample, each sample can obtain an effective signal during detection, the whole process of nucleic acid extraction of each sample to PCR amplification and quantum dot nucleic acid detection is guaranteed, and false negative is effectively avoided.

6) The kit for detecting the quantum dot nucleic acid of the bacterial enteric pathogen can use a simple ultraviolet imaging instrument to carry out fluorescence-signal detection or an automatic hybridization detector to carry out detection, and errors caused by artificial interpretation are avoided to the greatest extent.

Drawings

FIG. 1 is a diagram showing the results of the sensitivity of the kit for detecting various pathogens according to the embodiment of the present invention (Vibrio parahaemolyticus, Vibrio cholerae), Clostridium difficile, Staphylococcus aureus, Aeromonas hydrophila, Campylobacter jejuni, Yersinia enterocolitica, Salmonella, Escherichia coli diarrheal (enteroaggregative Escherichia coli, pathogenic Escherichia coli, enterotoxigenic Escherichia coli, Shiga toxin-producing Escherichia coli), Shigella/enteroinvasive Escherichia coli 10⁴/10³cfu/ml)；

FIG. 2 is a diagram showing the results of detecting the specificity of each genome with the kit according to the embodiment of the present invention (Enterobacter cloacae, Escherichia coli, Candida albicans, Staphylococcus epidermidis, Lactobacillus bulgaricus, Enterovirus 71, Coxsackie virus 16, norovirus, astrovirus, adenovirus, Saxavirus, rotavirus);

FIG. 3 is a schematic diagram of the detection accuracy of 10 clinical samples detected by the kit according to the embodiment of the present invention.

Detailed Description

In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.

The most key concept of the invention is as follows: a detection kit for detecting bacterial enteric pathogens by using quantum dot nucleic acid with high flux, high sensitivity and high specificity is established by using the optical characteristics and gene chip characteristics of quantum dot materials, and can be used for simultaneously detecting 9 common types of 14 intestinal bacterial pathogens.

The detection spectrum of the kit for detecting the quantum dot nucleic acid of the bacterial enteric pathogen is as follows: vibrio (vibrio parahaemolyticus, vibrio cholerae), clostridium difficile, staphylococcus aureus, aeromonas hydrophila, campylobacter jejuni, enterocolitis yersinia, salmonella, diarrheagenic escherichia coli (enteroaggregative escherichia coli, pathogenic escherichia coli, enterotoxigenic escherichia coli, shiga toxin-producing escherichia coli), shigella/enteroinvasive escherichia coli.

The invention designs the detection primers and the capture probes capable of identifying corresponding pathogenic bacteria according to different pathogen genome information, screens the primers and the probes according to the genome detection sensitivity, combines and optimizes the primers meeting the requirements from single weight to multiple weight, determines the optimal primer combination and different reaction systems, and reduces the related influence among the primers. And finally determining the primer combination form of each reaction system, and dividing the reaction system into 2 tubes.

Example 1

Preparation and application of quantum dot nucleic acid detection kit for bacterial enteric pathogens

Firstly, a quantum dot nucleic acid detection principle:

and performing molecular hybridization on the nucleic acid amplification product with the biotin label and a probe on a detection membrane strip, combining the biotin and a quantum dot coupled with streptavidin, and observing whether each site has a fluorescent signal by the detection membrane strip through a fluorescence detector to judge whether the probe is hybridized with the nucleic acid product, thereby determining whether the sample contains related target nucleic acid.

The capture probe is characterized in that amino is marked at the 3 'end or the 5' end of oligonucleotide single-stranded DNA, a spacer arm is arranged between the amino and the oligonucleotide single-stranded DNA, the spacer arm is one or the combination of two of a fatty acid Cn chain and oligo dTn, n in the fatty acid Cn chain is an integer from 1 to 12, and n in the oligo dTn is an integer from 1 to 30.

The detection membrane strip is made of a nylon membrane, and capture probes (1-50uM) with certain concentration are dotted on the activated nylon membrane and distributed on the nylon membrane in a microarray mode.

The quantum dots are quantum dots (CdSe/ZnS) with a plurality of coupled streptavidin on the surface, and the number of the specifically coupled streptavidin is more than or equal to 1. The excitation wavelength of the quantum dot is 200-500nm, and the emission wavelength of the quantum dot is 400-700 nm. The size of the quantum dots is 1-200 nm.

The 5 'end of the nucleic acid amplification product is provided with a biotin label, specifically, the 5' end of one primer of the nucleic acid amplification is modified with the biotin label, the primer is connected with biotin to form a spacer arm, the spacer arm is one or a combination of two of a Cn chain and oligo dTN, n in the fatty acid Cn chain is an integer of 1-12, and n in the oligo dTN is an integer of 1-30.

The nucleic acid amplification method comprises polymerase chain reaction (such as PCR) and isothermal amplification (such as TMA/RPA/LAMP).

Quantum dot nucleic acid detection process:

1) firstly, carrying out nucleic acid amplification by using a plurality of pairs of primers, wherein biotin is modified at the 5' end of one primer in one pair of primers for gene amplification, the primer is connected with biotin to form a spacer arm, the spacer arm is one or the combination of two of a fatty acid Cn chain and oligo dTN, n in the fatty acid Cn chain is an integer from 1 to 12, and n in the oligo dTN is an integer from 1 to 30.

2) After the nucleic acid amplification, the product is subjected to a nucleic acid denaturation treatment by high-temperature heat denaturation or alkali denaturation. The high-temperature heating denaturation is more than 95 ℃, and the alkali denaturation is a monovalent metal alkaline substance.

3) And adding the denatured product and the detection membrane strip into a hybridization solution preheated to a certain temperature (40-55 ℃) in advance for hybridization, wherein the hybridization time is 30min-2 h. The hybridization solution was 2 × SSC with 0.1% SDS.

4) After hybridization, transferring the detection membrane strip into a washing solution preheated to a certain temperature (40-55 ℃) in advance for washing for 5-15 min. The wash was 0.5 SSC with 0.1% SDS.

5) After washing, removing the washing solution, adding the washing solution into an incubation solution at a certain temperature for incubation for 5-30min, wherein the temperature is 20-37 ℃, and the incubation solution is formed by adding SA-QD quantum dots (the excitation wavelength is 200-500nM and the emission wavelength is 400-700nM) at a concentration of 0.01nM-5nM into 2 XSSC and 0.1% SDS. The size of the quantum dots is 1-200 nm.

6) After the incubation is finished, removing the incubation liquid, and adding a certain amount of washing liquid for washing for 5-15 min. The wash was 0.5 SSC with 0.1% SDS.

7) And after washing, placing the detection membrane strip in a fluorescence instrument for fluorescence detection.

Second, design and screening of primers

The genes of 23SrRNA, ipaH, eaeA, elt, est, stx1, stx2, aggR and the like of each pathogenic bacterium are inquired and downloaded in an NCBI database of a bioinformatics website, the regions with the highest homology of each target are found out through BLAST comparison, and amplification primers are designed. Primers with sensitivity meeting the requirements are screened by a large number of experimental tests (single amplification and multiple combined amplification). The specific detection primer sequences and sequence numbers are as follows:

primer ID 1: AGAGGCGATGAAGGACGTAC (SEQ No. 1);

primer ID 2: AGGAGCCGATGAAGGACGTG (SEQ No. 2);

primer ID 3: GAGGCGATGAAAGACGTAG (SEQ No. 3);

primer ID 4: AGAGGCGATGAAGGGCGTGC (SEQ No. 4);

primer ID 5: AAAGGCGATGAAGGACGTAC (SEQ No. 5);

primer ID 6: AGAGGCGATGAAGGGCGTGC (SEQ No. 6);

primer ID 7: AGCCGATGAAGGACGTTAC (SEQ No. 7);

primer ID 8: CTGCTTGTACGTACACGGTTTC (SEQ No. 8);

primer ID 9: CCCTCCTGCTTCCCACAGG (SEQ No. 9);

primer ID 10: CATATTCAGACAGGATATC (SEQ No. 10);

primer ID 11: ACGTACACGGTTTCAGGTTCTT (SEQ No. 11);

primer ID 12: CCGGATTCAGACAGGATTC (SEQ No. 12);

primer ID 13: TGGTCCTCCCAGATTCCGACGG (SEQ No. 13);

primer ID 14: TATGGTTGGGATAAGGCTGG (SEQ No. 14);

primer ID 15: CGAGCTTAGTGATACTTGTG (SEQ No. 15);

primer ID 16: CATATCTTAGAAATGAACTC (SEQ No. 16);

primer ID17: TCTGAAGTAATTCTTGAATC (SEQ No. 17);

primer ID18: AGCTTCAGTCGCGATCT (SEQ No. 18);

primer ID19: TCGGAATCATAGAACGGT (SEQ No. 19);

primer ID20: CAATAGGTACTCCATTAC (SEQ No. 20);

primer ID21: CCTGTGCCACTATCAATCAT (SEQ No. 21);

primer ID22: CGCCGATATATTATTAAAG (SEQ No. 22);

primer ID23: ACCACTCTGCAACGTGTC (SEQ No. 23);

primer ID24: ATCAGGATATTCACTTACT (SEQ No. 24);

primer ID25: TAATACCGGTCTCTATATTC (SEQ No. 25);

primer ID26: ATGTTCGTGGATGCCATGTC (SEQ No. 26);

primer ID27: TCGATTTATTCAACAAAGCA (SEQ No. 27);

primer ID28: TTACAATCACTCTCGGC (SEQ No. 28);

primer ID29: GGTAATGAAGTTAGTAGGTT (SEQ No. 29);

primers with sequence numbers of SEQ No. 8-SEQ No.13, SEQ No.15, SEQ No.17, SEQ No.19, SEQ No.21, SEQ No.23, SEQ No.25, SEQ No.27 and SEQ No.29 are modified with biotin labels.

Third, confirmation of amplification reaction liquid System

Determining the composition of each reaction liquid system through a large number of multiple combination tests and system optimization tests, wherein the specific conditions are as follows:

the reaction system (1 part by weight) of the reaction solution I is shown in Table 1;

TABLE 1

The reaction system (1 part by weight) of the reaction solution II is shown in Table 2;

TABLE 2

The detection of each sample needs to be carried out by amplifying 2 reaction systems at the same time, each reaction system has 21ul, the extracted nucleic acid template has 4ul, and the total volume is 25 ul.

Fourthly, determination of PCR reaction program

Through a large number of test tests, the amplification program can effectively amplify the primers in each reaction system to the maximum extent, and the detection sensitivity of each pathogen reaches 10³cfu/ml. The specific procedure is as follows in table 4 (touchdown PCR procedure was used);

TABLE 4

Design of capture probe

The method comprises the following steps of inquiring and downloading genes such as 23SrRNA, ipaH, eaeA, elt, est, stx1, stx2 and aggR of each pathogenic bacterium in an NCBI database of a bioinformatics website, finding out a region with the highest target specificity through BLAST comparison, designing probes through hybridization tests of each capture probe at the same hybridization temperature, and determining probe sequences of each pathogenic bacterium and drug-resistant gene through a large number of sensitivity test tests and specificity tests, wherein the specific sequences and the serial numbers are as follows:

probe IDP 1: TAGCATATCAGAAGGCACACC (SEQ No. 30);

probe IDP 2: CTGGAAAGCTTGGCGATACAG (SEQ No. 31);

probe IDP 3: CATACCACATATAAGAGGTC (SEQ No. 32);

probe IDP 4: AAGGCGCGCGATACAGGGTGA (SEQ No. 33);

probe IDP 5: GTAGGACTGCAATGTGCAAG (SEQ No. 34);

probe IDP 6: GGAAAGGCCGGCGATACAG (SEQ No. 35);

probe IDP 7: GGAAAGTCGCACGGTACAG (SEQ No. 36);

probe IDP 8: TACACGCGTTAGACGAACG (SEQ No. 37);

probe IDP 9: CCATTTATCGCAATCAGAT (SEQ No. 38);

probe IDP 10: CATTTGGTCAGGTCGGAGC (SEQ No. 39);

probe IDP 11: GACTATTTCATCAGGAGG (SEQ No. 40);

probe IDP 12: GAACTCCATTAACGCCAG (SEQ No. 41);

probe IDP 13: GGAATACCATATTCTCAGAT (SEQ No. 42);

probe IDP 14: GCTAAACCAGTAGAGTC (SEQ No. 43);

probe IDP 15: GAATTGCCTCATAGACTAC (SEQ No. 44);

probe ICP1: TTTGCTAATCATGTTCATACC (SEQ No. 45).

Wherein the ICP1 probe is an internal control probe and is used for monitoring false negative in the process of extracting and amplifying the sample nucleic acid.

The 5' end of each probe was labeled with an amino group, and an oligo dT10 was located between the amino group and the oligonucleotide chain.

Sixth, preparation of detection membrane strip

Each capture probe is synthesized by a primer synthesis unit, then diluted by diluent to the required concentration, and then fixed on a nylon membrane through the condensation reaction of amino and carboxyl to prepare the detection membrane strip.

One layout of the test strips is shown in Table 5 below;

TABLE 5

The pathogens corresponding to the upper points of the membrane strip are shown in table 6 below;

TABLE 6

Seventh, determination of hybridization conditions

After PCR amplification is finished, 2-tube amplification products are mixed and subjected to denaturation treatment at 95 ℃ for 10min, and then hybridization, washing, incubation, washing and fluorescence detection are carried out. In the hybridization step, the hybridization temperature has a great influence on the interpretation of the result, the hybridization temperature is too low, non-specific capture can occur to cause false positive, the hybridization temperature is too high, the binding rate of the target product and the capture probe can be reduced, and finally the sensitivity is reduced when the time comes to cause false negative. The subsequent washing temperature, the length of the incubation time and the concentration of SA-QD in the incubation solution also have great influence on the result.

And (3) hybridization:

and adding the denatured PCR product and the detection membrane strip into 1ml of hybridization solution which is pre-incubated to 48 ℃, and carrying out hybridization for 1.5h by gentle shaking at 48 ℃. While preheating 1ml of the wash liquor to 48 ℃.

The hybridization solution is 2 SSC and 0.1% SDS. The wash was 0.5 SSC with 0.1% SDS.

Washing:

and taking out the detection membrane strip, transferring the detection membrane strip into a washing solution preheated to 48 ℃, and washing for 5min by shaking.

And (3) incubation:

1uM SA-QD was added to 1ml of the hybridization solution to prepare an incubation solution. Transferring the detection membrane strip into an incubation solution, incubating at room temperature, and shaking gently for 30 min.

Washing:

and taking out the detection membrane strip, transferring the detection membrane strip into a washing solution, and washing the detection membrane strip for 5min by gentle shaking at room temperature.

In summary, the present embodiment provides a kit for detecting a quantum dot nucleic acid of a bacterial intestinal pathogen, the kit includes the above-mentioned detection membrane strip, a fluorescence detection solution and a reaction solution, the detection membrane strip includes a nylon membrane and a capture probe fixed on the nylon membrane; the fluorescence detection solution comprises quantum dots which are used for marking the surface of the capture probe and are coupled with streptavidin; the reaction solution comprises: reaction liquid I, reaction liquid II and reaction liquid III.

The application flow of the kit for detecting the quantum dot nucleic acid of the bacterial enteric pathogen in the embodiment is as follows:

1. and mixing the reaction solution I, the reaction solution II and the reaction solution III according to a ratio of 20.75 ul: mixing at a ratio of 0.25 ul.

2. 4ul of sample nucleic acid was added to the mixed reaction solution, and the mixture was placed in a PCR apparatus for PCR amplification. The amplification procedure is as in table 4;

3. after PCR amplification, the products are mixed and then denatured, and the nucleic acid denaturation method includes high-temperature heating denaturation. The high-temperature heating denaturation is more than 95 ℃.

4. And adding the denatured product and the detection membrane strip into a hybridization solution preheated to 48 ℃ in advance for hybridization, wherein the hybridization time is 30min-2 h. The hybridization solution was 2 × SSC with 0.1% SDS.

5. And after hybridization, transferring the detection membrane strip into a washing solution preheated to 48 ℃ in advance for washing for 5-15 min. The wash was 0.5 SSC with 0.1% SDS.

6. After washing, removing the washing solution, adding the washing solution into an incubation solution at room temperature for incubation for 5-30min, wherein the incubation solution is formed by adding SA-QD quantum dots (the excitation wavelength is 200-500nM, and the emission wavelength is 400-700nM) at the concentration of 0.01-5 nM into 2-SSC and 0.1% SDS. The size of the quantum dots is 1-200 nm.

7. After the incubation is finished, removing the incubation liquid, and adding a certain amount of washing liquid for washing for 5-15 min. The wash was 0.5 SSC with 0.1% SDS.

8. And after washing, placing the detection membrane strip in a fluorescence instrument for fluorescence detection.

Example 2

The effect verification analysis of the kit for detecting the quantum dot nucleic acid of the bacterial enteric pathogen

1. Sensitivity detection

The reaction systems were prepared as in example 1 and dispensed in 21ul, 4ul being added to each reaction system to extract pathogen nucleic acid at different concentrations.

PCR amplification procedure: PCR amplification was performed according to the procedure described in example 1.

The assay was tested according to the kit protocol in example 1. The detection results are shown in FIG. 1. The detection result shows that the sensitivity of each detection target can reach 10³cfu/ml。

2. Specificity detection

Reaction systems were prepared according to example 1 and split-charged at 21ul, and 4ul of genome was added to each reaction system, and detected genomes were enterobacter cloacae, escherichia coli, candida albicans, staphylococcus epidermidis, lactobacillus bulgaricus, enterovirus type 71, coxsackievirus type 16, norovirus, astrovirus, adenovirus, zakhstan virus, rotavirus, respectively.

Hybridization assays were performed according to the kit protocol of example 1. The results of the measurements are shown in FIG. 2, respectively. The result shows that all detection point positions have no fluorescent signals, which indicates that the specificity of the detection probe meets the requirement.

3. Clinical sample testing

10 clinical excrement samples are taken, genome extraction is firstly carried out, and the specific extraction reagent adopts an excrement nucleic acid extraction kit (magnetic bead method) produced by Hangzhou Qianji biotechnology limited to carry out the following extraction process:

1) adding a proper amount of normal saline into a fecal sample tube, uniformly mixing, and taking 50ul for nucleic acid extraction;

2) adding 250ul of lysate I for 10 min;

3) then 20ul of lysis solution II, 5ul of magnetic bead solution and 600ul of binding solution are added for 5-10 min;

4) placing the EP tube on a magnetic separator, separating and discarding supernatant;

6) adding 0.5ml of washing solution into the EP tube, and fully washing;

7) placing the EP tube on a magnetic separator, separating and discarding supernatant;

8) adding 0.5ml of washing solution into the EP tube, and fully washing;

7) placing the EP tube on a magnetic separator, separating and discarding supernatant, and air drying for 2-5 min;

8) adding 60ul of eluent into an EP tube, resuspending the magnetic beads, and heating at 56 deg.C for 5 min;

9) the EP tube was placed on a magnetic separator, the supernatant was separated and transferred to a clean EP tube and stored at-20 ℃ until use.

A reaction system was prepared in accordance with example 1, and then reaction solutions were dispensed in an amount of 21 ul/reaction, 4ul of the extracted nucleic acid was added to each reaction solution, PCR amplification was performed in accordance with example 1, and hybridization test was performed on the amplified product in accordance with example 1. The results are shown in FIG. 3. The detection result is consistent with the Sanger sequencing identification result.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.

Sequence listing

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Claims

1. A kit for detecting quantum dot nucleic acid of bacterial enteric pathogens is characterized by comprising a detection membrane strip, fluorescent detection liquid and reaction liquid, wherein the detection membrane strip comprises a nylon membrane and a capture probe fixed on the nylon membrane; the fluorescence detection solution comprises quantum dots which are used for marking the surface of the capture probe and are coupled with streptavidin; the reaction solution comprises: reaction liquid I, reaction liquid II and reaction liquid III;

the reaction solution I comprises the following detection primers:

primer ID 1: AGAGGCGATGAAGGACGTAC (SEQ No. 1);

primer ID 2: AGGAGCCGATGAAGGACGTG (SEQ No. 2);

primer ID 3: GAGGCGATGAAAGACGTAG (SEQ No. 3);

primer ID 4: AGAGGCGATGAAGGGCGTGC (SEQ No. 4);

primer ID 5: AAAGGCGATGAAGGACGTAC (SEQ No. 5);

primer ID 6: AGAGGCGATGAAGGGCGTGC (SEQ No. 6);

primer ID 7: AGCCGATGAAGGACGTTAC (SEQ No. 7);

primer ID 8: CTGCTTGTACGTACACGGTTTC (SEQ No. 8);

primer ID 9: CCCTCCTGCTTCCCACAGG (SEQ No. 9);

primer ID 10: CATATTCAGACAGGATATC (SEQ No. 10);

primer ID 11: ACGTACACGGTTTCAGGTTCTT (SEQ No. 11);

primer ID 12: CCGGATTCAGACAGGATTC (SEQ No. 12);

primer ID 13: TGGTCCTCCCAGATTCCGACGG (SEQ No. 13);

primer ID 14: TATGGTTGGGATAAGGCTGG (SEQ No. 14);

primer ID 15: CGAGCTTAGTGATACTTGTG (SEQ No. 15);

the reaction solution II comprises the following detection primers:

primer ID 16: CATATCTTAGAAATGAACTC (SEQ No. 16);

primer ID17: TCTGAAGTAATTCTTGAATC (SEQ No. 17);

primer ID18: AGCTTCAGTCGCGATCT (SEQ No. 18);

primer ID19: TCGGAATCATAGAACGGT (SEQ No. 19);

primer ID20: CAATAGGTACTCCATTAC (SEQ No. 20);

primer ID21: CCTGTGCCACTATCAATCAT (SEQ No. 21);

primer ID22: CGCCGATATATTATTAAAG (SEQ No. 22);

primer ID23: ACCACTCTGCAACGTGTC (SEQ No. 23);

primer ID24: ATCAGGATATTCACTTACT (SEQ No. 24);

primer ID25: TAATACCGGTCTCTATATTC (SEQ No. 25);

primer ID26: ATGTTCGTGGATGCCATGTC (SEQ No. 26);

primer ID27: TCGATTTATTCAACAAAGCA (SEQ No. 27);

primer ID28: TTACAATCACTCTCGGC (SEQ No. 28);

primer ID29: GGTAATGAAGTTAGTAGGTT (SEQ No. 29);

primer ID 14: TATGGTTGGGATAAGGCTGG (SEQ No. 14);

primer ID 15: CGAGCTTAGTGATACTTGTG (SEQ No. 15);

the reaction solution III is Hotstart Taq DNA polymerase.

2. The kit of claim 1 for quantum dot nucleic acid detection of bacterial enteric pathogens, wherein the capture probe comprises:

probe IDP 1: TAGCATATCAGAAGGCACACC (SEQ No. 30);

probe IDP 2: CTGGAAAGCTTGGCGATACAG (SEQ No. 31);

probe IDP 3: CATACCACATATAAGAGGTC (SEQ No. 32);

probe IDP 4: AAGGCGCGCGATACAGGGTGA (SEQ No. 33);

probe IDP 5: GTAGGACTGCAATGTGCAAG (SEQ No. 34);

probe IDP 6: GGAAAGGCCGGCGATACAG (SEQ No. 35);

probe IDP 7: GGAAAGTCGCACGGTACAG (SEQ No. 36);

probe IDP 8: TACACGCGTTAGACGAACG (SEQ No. 37);

probe IDP 9: CCATTTATCGCAATCAGAT (SEQ No. 38);

probe IDP 10: CATTTGGTCAGGTCGGAGC (SEQ No. 39);

probe IDP 11: GACTATTTCATCAGGAGG (SEQ No. 40);

probe IDP 12: GAACTCCATTAACGCCAG (SEQ No. 41);

probe IDP 13: GGAATACCATATTCTCAGAT (SEQ No. 42);

probe IDP 14: GCTAAACCAGTAGAGTC (SEQ No. 43);

probe IDP 15: GAATTGCCTCATAGACTAC (SEQ No. 44).

3. The kit for quantum dot nucleic acid detection of bacterial enteric pathogens of claim 2, wherein said detection membrane strip further comprises an internal control probe for monitoring sample nucleic acid extraction and amplification: ICP1: TTTGCTAATCATGTTCATACC (SEQ No. 45).

4. The kit for detecting the quantum dot nucleic acid of the bacterial enteric pathogen according to claim 2, wherein the capture probe is a single-stranded oligonucleotide DNA, the 3 'end or the 5' end of the single-stranded oligonucleotide DNA is labeled with an amino group, an intermediate arm is connected between the single-stranded oligonucleotide DNA and the amino group, the intermediate arm is one or a combination of two of a fatty acid Cn chain and an oligo dTN, n in the fatty acid Cn chain is an integer from 1 to 12, and n in the oligo dTN is an integer from 1 to 30.

5. The kit for detecting the quantum dot nucleic acid of the bacterial enteric pathogen according to claim 1, wherein 5' ends of primers SEQ No. 8-SEQ No.13, SEQ No.15, SEQ No.17, SEQ No.19, SEQ No.21, SEQ No.23, SEQ No.25, SEQ No.27 and SEQ No.29 in the detection primers are modified with biotin labels, an intermediate arm is connected between the detection primers and biotin, the intermediate arm is one or a combination of two of a fatty acid Cn chain and oligo dTN, n in the fatty acid Cn chain is an integer from 1 to 12, and n in the oligo dTN is an integer from 1 to 30.

6. The kit for detecting the quantum dot nucleic acid of the bacterial enteric pathogen according to claim 1, wherein the excitation wavelength of the quantum dot is 200-500nm, the emission wavelength of the quantum dot is 400-700nm, and the size of the quantum dot is 1-200 nm.

7. The kit for the quantum dot nucleic acid detection of bacterial enteric pathogens of claim 1, wherein the quantum dot is CdSe/ZnS.