CN111349708A - Quantum dot nucleic acid detection kit and method for simultaneously detecting 11 lower respiratory tract pathogenic bacteria - Google Patents
Quantum dot nucleic acid detection kit and method for simultaneously detecting 11 lower respiratory tract pathogenic bacteria Download PDFInfo
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Abstract
The invention relates to the technical field of biological medicines, in particular to a detection kit and a method for simultaneously detecting 11 types of lower respiratory tract pathogenic bacteria quantum dots. The kit comprises reaction liquid, a positive reference substance, a negative reference substance, fluorescence detection liquid, a denaturant, a neutral agent and a membrane strip. The reaction solution comprises a respiratory tract pathogenic bacterium detection primer, dN (U) TP, UDG, a PCR buffer system and DNA polymerase, the positive control substance comprises Klebsiella pneumoniae recombinant plasmid, the negative control substance comprises human genome DNA, the fluorescence detection solution comprises streptavidin coupled quantum dots, the denaturant comprises alkali liquor, the neutral agent comprises acid liquor, and the membrane strip comprises a nylon membrane coupled with a specific detection probe. The kit and the method can simultaneously detect 11 common pathogenic bacteria of lower respiratory tract infection rapidly, sensitively and characteristically, and the set endogenous internal standard can evaluate whether the sample is qualified or not and effectively monitor the occurrence of false negative, thereby assisting clinical realization of early diagnosis and early treatment.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a nucleic acid detection kit and a method for simultaneously detecting 11 lower respiratory tract pathogenic bacteria quantum dots.
Background
Respiratory Tract Infection (RTI) is the most common disease in humans, can occur in any sex, age and territory, and is one of the leading causes of morbidity and mortality in the population worldwide. The clinical symptoms and physical signs caused by respiratory tract infection are similar, the clinical manifestations mainly comprise symptoms such as rhinitis, pharyngitis, laryngitis, tonsillitis and the like, and severe symptoms can cause tracheitis, bronchitis, pneumonia and the like, but the treatment method, the curative effect and the course of the infection caused by different pathogens are different. It has now been demonstrated that most respiratory diseases are caused by pathogens other than bacteria, with respiratory viruses being the most common.
Respiratory tract infections are divided into upper respiratory tract infections and lower respiratory tract infections. The upper respiratory tract infections are classified as viral (70-80%) and bacterial (20-25%). Acute upper respiratory infection: common cold, acute sinusitis, tonsillitis, laryngitis, pharyngitis, epiglottitis, etc. mainly manifested by acute nasopharyngitis. Acute lower respiratory tract infection: acute tracheobronchitis, bronchiolitis, pneumonia, among which pneumonia is the leading cause of death in children under 5 years of age. Acute lower respiratory tract infection is acute infection of trachea, bronchus and lung tissues, comprises acute trachea, bronchitis, pneumonia and the like, is frequently caused in cold seasons, and is often caused by downward spread of viruses or bacteria such as upper respiratory tract infection, rhinitis, influenza (flu) and the like. It is well developed for hospitalized patients, children and patients with low immunity.
At present, the clinical pathogen detection mainly depends on the traditional methods for detecting related antibodies by bacterial culture and serology, but the operation method is complicated, the obtained result period is long, only one pathogen can be identified each time, and the positive detection rate is low and is only 30-40%, so that an efficient, sensitive, rapid and accurate pathogen detection method is established, and the method plays a vital role in clinical early diagnosis and accurate treatment.
The invention provides a bacterial quantum dot gene chip capable of simultaneously detecting 11 common lower respiratory tract infections, which has the characteristics of high flux, high sensitivity, high specificity and the like.
Disclosure of Invention
The invention aims to develop a quantum dot nucleic acid detection kit and a method capable of simultaneously detecting 11 respiratory tract pathogenic bacteria aiming at tracheitis, bronchitis and pneumonia caused by common lower respiratory tract bacterial infection, so as to provide laboratory basis for clinical diagnosis and prevent infection in communities or hospitals.
In order to solve the problems, the invention adopts the following technical scheme:
a quantum dot nucleic acid detection kit for simultaneously detecting 11 lower respiratory tract pathogenic bacteria comprises a reaction solution, a positive reference substance, a negative reference substance, a fluorescence detection solution, a denaturant, a neutral agent and a membrane strip; wherein, the membrane strip comprises a nylon membrane and a specific capture probe coupled 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 denaturant comprises 0.01N-1.5N sodium hydroxide solution; the neutralizers comprise 0.01N-1.5N hydrochloric acid solution; the positive control substance comprises a Klebsiella pneumoniae recombinant plasmid, and the negative control substance comprises human genome DNA; the reaction solution comprises the following detection primers:
primer MCF: GTAGGTTATACCGAAGGTGC, SEQ ID NO: 1;
and (3) primer MCR: ATTCACCGCAAAGTTTACATTC, SEQ ID NO: 2;
primer TUF: GAAGAATTGCTTGAATTGGT, SEQ ID NO: 3;
primer TUR: CCACGGTCGATACGTCCTGA, SEQ ID NO: 4;
primer FKYF: ACCTGCGGTTGGATCACC, SEQ ID NO: 5;
primers YGR: CCATCGTAGTTATCTCTCTA, SEQ ID NO: 6;
primer FKR: GAAGATGAGTTTTGAGATACA, SEQ ID NO: 7;
primer ECF: GTCTGCAATTGCCACCACTG, SEQ ID NO: 8;
primer ECR: AAGTCGCATCCGTTTGTTGC, SEQ ID NO: 9;
primer 23 SF: CACGGTGGATGCCYWGGC, SEQ ID NO: 10;
primer 23 SR: GTTTGAGATTTTGAGAGACTC, SEQ ID NO: 11;
primer ICF: TATGGTTGGGATAAGGCTGG, SEQ ID NO: 12;
primer ICR: CGAGCTTAGTGATACTTGTG, SEQ ID NO: 13;
preferably, in the above-mentioned kit for detecting 11 types of lower respiratory tract pathogenic bacteria quantum dots nucleic acids, the capture probe comprises:
GCTTGGTGTGAGACACACC for probe MCP, SEQ ID NO: 14;
a probe HNP: ATTACAGGTCGTGGTACAGTC, SEQ ID NO: 15;
probe FLP: TCAGCACTTAAAGCTCTTGAAG, SEQ ID NO: 16;
probe FKP: AGTGCTCACACAGATTGTCT, SEQ ID NO: 17;
the probe YGP: GCTCAGGTGGTTAGAGCGC, SEQ ID NO: 18;
probe SAP: TAGCATATCAGAAGGCACACC, SEQ ID NO: 19;
and (3) probe BMP: ATGTATACTTTGTATACGTG, SEQ ID NO: 20;
a probe TLP: GTGTCACGTAAGTGACGCG, SEQ ID NO: 21;
probe SMYP: TCGCGCAGTAAAGGGTGAT, SEQ ID NO: 22;
probe LGP: GGAAGCACAATCAAAGAGG, SEQ ID NO: 23;
and (3) probe ECP: GGTCATTAGCGCCACTCACTGCA, SEQ ID NO: 24;
preferably, in the above-mentioned kit for detecting 11 types of lower respiratory tract pathogenic bacteria quantum dots nucleic acid, the detection membrane strip further comprises an internal control probe for monitoring sample nucleic acid extraction and amplification:
ICP: TTTGCTAATCATGTTCATACC, SEQ ID NO: 25;
preferably, in the above quantum dot nucleic acid detection kit for 11 types of lower respiratory tract pathogenic bacteria, 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 carbon chain and an oligo dT (n), the length of the main chain of the fatty acid carbon chain is 1 to 12 carbon atoms, and n in the oligo dT (n) is an integer of 1 to 30.
Preferably, in the above quantum dot nucleic acid detection kit for 11 types of lower respiratory tract pathogenic bacteria, a biotin label is modified at the 5' end of a reverse primer in the detection primers, an intermediate arm is connected between the detection primers and the biotin, the intermediate arm is one or a combination of two of a fatty acid carbon chain and oligo dT (n), the main chain length of the fatty acid carbon chain is 1-12 carbon atoms, and n in the oligo dT (n) is an integer of 1-30.
Preferably, in the above-mentioned kit for detecting 11 types of lower respiratory tract pathogenic bacteria quantum dots nucleic acid, the excitation wavelength of the quantum dots is 200-500nm, the emission wavelength of the quantum dots is 400-700nm, and the size of the quantum dots is 1-200 nm.
Preferably, the quantum dot is a CdSe/ZnS core-shell quantum dot used in the quantum dot nucleic acid detection kit for the 11 lower respiratory tract pathogenic bacteria.
Preferably, in the above-mentioned quantum dot nucleic acid assay kit for 11 lower respiratory tract pathogenic bacteria, the positive control comprises a Klebsiella pneumoniae recombinant plasmid (obtained by T-cloning SEQ ID NO:26 into PGMET vector).
The Klebsiella pneumoniae DNA fragment sequence is (SEQ ID NO: 26):
ACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATG CTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATG GGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTA CCACTTTGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAACCGTAGG GGAACCTGCGGTTGGATCACCTCCTTACCTTAAAGAACCTGCCTTTGTAGT GCTCACACAGATTGTCTGATGAAAGTAAAGAAGCAAGGCGTCTTGCGATT GAGACTTCAGTGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTTTCACG GCGGTAACAGGGGTTCGAATCCCCTAGGGGACGCCACTTGCTGGTTCGTG AGTGAAAGACGCGTGCCGATGTATCTCAAAACTCATCTTCGGGTGATGTT TGAGATATTTGCTCTTTAAAAATCTGGATCAAGCTGAAAATTGAAACGAC ACACTGTTTAAGTGTGTTCGAGTCTCTCAAATTTTCGCAATCAGAAGTGAA ACATCTTCGGGTTGTGAGGTTAAGCGACTAAGCGTACACGGTGGATGCCC TGGCAGTCAGAGGCGATGAAGGACGTGCTAATCTGCGAA
preferably, in the above detection kit for simultaneously detecting 24 respiratory pathogenic bacteria quantum dots nucleic acid, the contents of the reaction liquid components are (per part):
10 buffer 2.5ul, 20mM dN (U) TP 0.25ul, 2U/ul UDG 0.5ul, 10 uM nucleotide sequence as shown in SEQ ID No: 1-13, 0.75. mu.L each, 5U/ul Hotstart taq 0.25ul, H2O was made up to 21 ul.
Preferably, the above-mentioned detection method for simultaneously detecting 11 species of respiratory tract respiratory pathogenic bacteria quantum dots nucleic acid is characterized by being applicable to clinical sample types: pharyngeal swab, nasal swab, nasopharyngeal aspirate, deep expectoration, respiratory aspirate, bronchial lavage, alveolar lavage, lung biopsy, and the like. The specific detection steps are as follows:
1) each sample reaction system to be detected is 21ul of reaction liquid and 4ul of nucleic acid sample;
2) placing the sample in a real-time fluorescent PCR instrument for PCR amplification detection;
3) the real-time fluorescent PCR amplification program was set up as follows:
first, stage 1: the treatment is carried out for 2min at the temperature of 50 ℃,
and (2) stage: treating at the temperature of 95 ℃ for 3min,
and (3) stage: 45 cycles of 95 ℃ for 15s, 56 ℃ for 30-60s and 72 ℃ for 30 s.
Preferably, in the above detection method for simultaneously detecting 11 lower respiratory tract pathogenic bacteria quantum dots, the detected respiratory tract pathogenic bacteria are:
staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, stenotrophomonas maltophilia, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus pneumoniae, Klebsiella pneumoniae, Enterobacter cloacae, and Moraxella catarrhalis.
Compared with the prior art, the invention has the following characteristics:
1) compared with the existing chromogenic gene chip, the kit has the advantages of fewer detection steps, obviously shortened detection time, lower equipment cost (low light source requirement) than that of an organic fluorescent gene chip, complex preparation process and complex and fussy detection process because the carrier of the existing fluorescent gene chip is glass, and the detection instrument needs a laser scanner with high cost, thus being not beneficial to clinical popularization.
2) The invention provides a quantum dot nucleic acid detection kit and a method for simultaneously detecting 11 lower respiratory tract pathogenic bacteria, which are used for simultaneously detecting staphylococcus aureus, acinetobacter baumannii, pseudomonas aeruginosa, escherichia coli, stenotrophomonas maltophilia, haemophilus influenzae, streptococcus pyogenes, streptococcus pneumoniae, klebsiella pneumoniae, enterobacter cloacae and moraxella catarrhalis, avoiding abuse of antibiotics and simultaneously providing a basis for early diagnosis of patients with mixed infection or early clinical manifestation which is not obvious.
3) The invention simultaneously adopts an independent competitive internal standard to monitor the whole process of extraction and amplification, thereby preventing false negative of each reaction tube.
4) The detection of the quantum dot nucleic acid detection kit for simultaneously detecting 11 lower respiratory tract pathogenic bacteria can use a simple ultraviolet imaging instrument to carry out fluorescence-signal detection, and errors caused by artificial interpretation are avoided to the greatest extent.
6) The detection sensitivity was 500 copies/ml.
Drawings
FIG. 1 shows the results of sensitivity detection of each pathogen amplified in the reaction solution.
FIG. 2 shows the results of the clinical sputum specimen test.
Detailed Description
The most key concept of the invention is as follows: a detection kit for detecting respiratory tract pathogenic bacteria by quantum dot nucleic acid with high flux, high sensitivity and high specificity is established by utilizing the optical characteristics and gene chip characteristics of quantum dot materials, and can simultaneously detect 11 common lower respiratory tract infection pathogenic bacteria.
The detection spectrum of the 11 respiratory tract pathogenic bacteria quantum dot nucleic acid detection kit provided by the invention is as follows: staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, stenotrophomonas maltophilia, Haemophilus influenzae, Streptococcus pyogenes, Streptococcus pneumoniae, Klebsiella pneumoniae, Enterobacter cloacae, and Moraxella catarrhalis. Example 1: preparation and application of quantum dot nucleic acid detection kit for respiratory tract pathogenic bacteria
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 of the detection membrane strip has a light signal through a fluorescence detector to judge whether the probe is hybridized with the nucleic acid product, so as to determine 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 carbon chain and oligo dT (n), the length of the main chain of the fatty acid carbon chain is 1-12 carbon atoms, and n in the oligo dT (n) is an integer of 1-30.
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 a 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 an intermediate arm, the intermediate arm is one or a combination of two of a fatty acid carbon chain and oligo dT (n), the length of the main chain of the fatty acid carbon chain is 1-12 carbon atoms, and n in the oligo dT (n) 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, a plurality of pairs of primers are used for nucleic acid amplification, biotin is modified at the 5' end of one primer in a pair of primers for certain gene amplification, the primer and the biotin are connected with an intermediate arm, the intermediate arm is one or the combination of two of a fatty acid carbon chain and oligo dT (n), the length of the main chain of the fatty acid carbon chain is 1-12 carbon atoms, and n in the oligo dT (n) is an integer of 1-30.
2) After the nucleic acid amplification, the product is subjected to a nucleic acid denaturation treatment by alkali denaturation.
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.
Design of primers and probes of quantum dot nucleic acid detection kit for 11 respiratory tract pathogenic bacteria
According to genome sequences disclosed by staphylococcus aureus, acinetobacter baumannii, pseudomonas aeruginosa, escherichia coli, stenotrophomonas maltophilia, haemophilus influenzae, streptococcus pyogenes, streptococcus pneumoniae, klebsiella pneumoniae, enterobacter cloacae and moraxella catarrhalis in a GENBANK database, nucleic acid sequence comparison is carried out, conserved regions are screened, a detection probe and a primer are designed by using a primer 5.0, and Tm values of all amplification primers are as close as possible; primers with sensitivity meeting the requirements are screened by a large number of experimental tests (single amplification and multiple combined amplification). The detection primer related sequences are as follows:
primer MCF: GTAGGTTATACCGAAGGTGC (SEQ ID NO:1)
And (3) primer MCR: ATTCACCGCAAAGTTTACATTC (SEQ ID NO:2)
Primer TUF: GAAGAATTGCTTGAATTGGT (SEQ ID NO:3)
Primer TUR: CCACGGTCGATACGTCCTGA (SEQ ID NO:4)
Primer FKYF: ACCTGCGGTTGGATCACC (SEQ ID NO:5)
Primers YGR: CCATCGTAGTTATCTCTCTA (SEQ ID NO:6)
Primer FKR: GAAGATGAGTTTTGAGATACA (SEQ ID NO:7)
Primer ECF: GTCTGCAATTGCCACCACTG (SEQ ID NO:8)
Primer ECR: AAGTCGCATCCGTTTGTTGC (SEQ ID NO:9)
Primer 23 SF: CACGGTGGATGCCYWGGC (SEQ ID NO:10)
Primer 23 SR: GTTTGAGATTTTGAGAGACTC (SEQ ID NO:11)
Primer ICF: TATGGTTGGGATAAGGCTGG (SEQ ID NO:12)
Primer ICR: CGAGCTTAGTGATACTTGTG (SEQ ID NO:13)
Each reverse primer was modified with a biotin tag and an oligodT5 was placed between the biotin and oligonucleotide strands.
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 is shown in Table 1:
TABLE 1
| 10p/ul primer SEQ ID No: 1-13 | 0.75ul |
| 10*buffer | 2.5ul |
| 20mM dN(U)TP | 0.25ul |
| 2U/ul UDG | 0.5ul |
| 5U/ul hotstart taq | 0.25ul |
| H2O | Up to 21ul |
4ul of extracted DNA template was added to each amplification system, for a total volume of 25 ul.
Fourth, reaction procedure determination
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 500 copies/ml. The specific procedure is as follows in table 2:
TABLE 2
Design of capture probe
The nucleic acid sequences of all pathogenic bacteria are inquired and downloaded in a NCBI database of a bioinformatics website, the regions with the highest target specificity are found out through BLAST comparison, meanwhile, the capture probes can be subjected to hybridization test design probes at the same hybridization temperature, and the probe sequences of the specific genes of all pathogenic bacteria are determined through a large number of sensitivity test tests and specificity tests, wherein the specific sequences and the serial numbers are as follows:
probe MCP: GCTTGGTGTGAGACACACC (SEQ ID NO:14)
A probe HNP: ATTACAGGTCGTGGTACAGTC (SEQ ID NO:15)
Probe FLP: TCAGCACTTAAAGCTCTTGAAG (SEQ ID NO:16)
Probe FKP: AGTGCTCACACAGATTGTCT (SEQ ID NO:17)
The probe YGP: GCTCAGGTGGTTAGAGCGC (SEQ ID NO:18)
Probe SAP: TAGCATATCAGAAGGCACACC (SEQ ID NO:19)
And (3) probe BMP: ATGTATACTTTGTATACGTG (SEQ ID NO:20)
A probe TLP: GTGTCACGTAAGTGACGCG (SEQ ID NO:21)
Probe SMYP: TCGCGCAGTAAAGGGTGAT (SEQ ID NO:22)
Probe LGP: GGAAGCACAATCAAAGAGG (SEQ ID NO:23)
And (3) probe ECP: GGTCATTAGCGCCACTCACTGCA (SEQ ID NO:24)
Probe ICP: TTTGCTAATCATGTTCATACC (SEQ ID NO:25)
Wherein the ICP probe is an internal control probe of an amplification system and is used for monitoring false negative of sample nucleic acid extraction and amplification reaction.
The 5' end of each probe was labeled with an amino group, and an oligo dT5 was located between the amino group and the oligonucleotide chain.
Sixth, preparation of membrane strip
Each capture probe is synthesized by a primer synthesis unit, then diluted to the required concentration by adopting a diluent, and then fixed on a nylon membrane through the condensation reaction of amino and carboxyl to prepare the detection membrane strip.
The layout of the test strip is shown in table 3 below:
TABLE 3
The pathogens corresponding to the upper points of the membrane strip are shown in table 4:
TABLE 4
Seventh, determination of hybridization conditions
After PCR amplification is finished, alkali denaturation treatment is carried out on the amplification product, 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 incubation time and the concentration of SA-QD in the incubation solution will also have the same effect on the results.
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 incubation liquid for incubation at room temperature and shaking 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.
Example 2
The invention is used for detecting the performance of the quantum dot nucleic acid detection kit for 11 types of lower respiratory tract pathogenic bacteria:
1. sensitivity detection
The reaction systems of example 1 were each filled in 21ul portions, and 4ul portions of genomic DNA were added to each reaction system at 5000copies/ml and 500 copies/ml.
PCR amplification procedure PCR amplification was performed according to the procedure described in example 1, as follows:
quantum dot gene chip detection procedures the kit use procedure in example 1 was followed for the relevant detection. The detection results are shown in FIG. 1. The fluorescence detection result shows that the detection target sensitivity of the kit can reach 500 copies/ml.
2. Specificity detection
The kit specificity detection is carried out by using a circulating free nucleic acid extraction kit produced by Hangzhou Qianji biotechnology limited to extract nucleic acid of streptococcus pyogenes, streptococcus agalactiae, viridian streptococcus group, streptococcus pneumoniae, staphylococcus saprophyticus, staphylococcus epidermidis, staphylococcus hominis, hemolytic staphylococcus, enterobacter cloacae, klebsiella pneumoniae, proteus mirabilis, proteus vulgaris, candida albicans, candida glabrata, candida tropicalis, candida krusei, candida parapsilosis, staphylococcus aureus, enterococcus faecium, enterococcus faecalis, enterobacter aerogenes, pseudomonas aeruginosa, acinetobacter baumannii, escherichia coli, klebsiella oxytoca, haemophilus influenzae, serratia marcescens, burkholderia cepacia and stenotrophomonas maltophilia.
The amplification system was prepared as in example 1, with PCR amplification according to the following procedure:
quantum dot gene chip detection procedures the kit use procedure in example 1 was followed for the relevant detection. The result shows that only the corresponding detection site has a fluorescent signal, which indicates that the specificity of the capture probe meets the requirement.
Example 3: sputum clinical sample detection
24 sputum samples suspected of lower respiratory tract infection are collected according to the clinical microorganism sample collection standard, a circulating free nucleic acid extraction kit produced by Hangzhou Chiji Biotechnology limited company is adopted to extract nucleic acid, then the reaction system of the embodiment 1 is used to detect the nucleic acid sample, and the specific amplification process and the detection process refer to the embodiment 1. The specific detection results are as follows:
note: ND identifies detection of the associated pathogen.
In the embodiment, the sample is detected and analyzed by one-generation sequencing, and the detection result of all the embodiments is consistent with the sequencing result.
Sequence listing
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HANGZHOU BOXIN BIOTECHNOLOGY Co.,Ltd.
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<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
ccatcgtagt tatctctcta 20
<210>7
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
gaagatgagt tttgagatac a 21
<210>8
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
gtctgcaatt gccaccactg 20
<210>9
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
aagtcgcatc cgtttgttgc 20
<210>10
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
cacggtggat gccywggc 18
<210>11
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>11
gtttgagatt ttgagagact c 21
<210>12
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
tatggttggg ataaggctgg 20
<210>13
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>13
cgagcttagt gatacttgtg 20
<210>14
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>14
gcttggtgtg agacacacc 19
<210>15
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>15
attacaggtc gtggtacagt c 21
<210>16
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>16
tcagcactta aagctcttga ag 22
<210>17
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>17
agtgctcaca cagattgtct 20
<210>18
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>18
gctcaggtgg ttagagcgc 19
<210>19
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>19
tagcatatca gaaggcacac c 21
<210>20
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>20
atgtatactt tgtatacgtg 20
<210>21
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>21
gtgtcacgta agtgacgcg 19
<210>22
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>22
tcgcgcagta aagggtgat 19
<210>23
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>23
ggaagcacaa tcaaagagg 19
<210>24
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>24
ggtcattagc gccactcact gca 23
<210>25
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>25
tttgctaatc atgttcatac c 21
<210>26
<211>639
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>26
actcgactcc atgaagtcgg aatcgctagt aatcgtagat cagaatgcta cggtgaatac 60
gttcccgggc cttgtacaca ccgcccgtca caccatggga gtgggttgca aaagaagtag 120
gtagcttaac cttcgggagg gcgcttacca ctttgtgatt catgactggg gtgaagtcgt 180
aacaaggtaa ccgtagggga acctgcggtt ggatcacctc cttaccttaa agaacctgcc 240
tttgtagtgc tcacacagat tgtctgatga aagtaaagaa gcaaggcgtc ttgcgattga 300
gacttcagtg tccccttcgt ctagaggccc aggacaccgc cctttcacgg cggtaacagg 360
ggttcgaatc ccctagggga cgccacttgc tggttcgtga gtgaaagacg cgtgccgatg 420
tatctcaaaa ctcatcttcg ggtgatgttt gagatatttg ctctttaaaa atctggatca 480
agctgaaaat tgaaacgaca cactgtttaa gtgtgttcga gtctctcaaa ttttcgcaat 540
cagaagtgaa acatcttcgg gttgtgaggt taagcgacta agcgtacacg gtggatgccc 600
tggcagtcag aggcgatgaa ggacgtgcta atctgcgaa 639
Claims (10)
1. A quantum dot nucleic acid detection kit for simultaneously detecting 11 lower respiratory tract pathogenic bacteria is characterized by comprising a reaction solution, a positive reference substance, a negative reference substance, a fluorescence detection solution, a denaturant, a neutral agent and a membrane strip; wherein, the membrane strip comprises a nylon membrane and a specific capture probe coupled 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 denaturant comprises 0.01N-1.5N sodium hydroxide solution; the neutralizers comprise 0.01N-1.5N hydrochloric acid solution; the positive control substance comprises a Klebsiella pneumoniae recombinant plasmid, and the negative control substance comprises human genome DNA; the reaction solution comprises the following detection primers:
primer MCF: GTAGGTTATACCGAAGGTGC, SEQ ID NO: 1;
and (3) primer MCR: ATTCACCGCAAAGTTTACATTC, SEQ ID NO: 2;
primer TUF: GAAGAATTGCTTGAATTGGT, SEQ ID NO: 3;
primer TUR: CCACGGTCGATACGTCCTGA, SEQ ID NO: 4;
primer FKYF: ACCTGCGGTTGGATCACC, SEQ ID NO: 5;
primers YGR: CCATCGTAGTTATCTCTCTA, SEQ ID NO: 6;
primer FKR: GAAGATGAGTTTTGAGATACA, SEQ ID NO: 7;
primer ECF: GTCTGCAATTGCCACCACTG, SEQ ID NO: 8;
primer ECR: AAGTCGCATCCGTTTGTTGC, SEQ ID NO: 9;
primer 23 SF: CACGGTGGATGCCYWGGC, SEQ ID NO: 10;
primer 23 SR: GTTTGAGATTTTGAGAGACTC, SEQ ID NO: 11;
primer ICF: TATGGTTGGGATAAGGCTGG, SEQ ID NO: 12;
primer ICR: CGAGCTTAGTGATACTTGTG, SEQ ID NO: 13.
2. The quantum dot nucleic acid detection kit for simultaneously detecting 11 lower respiratory tract pathogenic bacteria according to claim 1, wherein the specific capture probe comprises:
GCTTGGTGTGAGACACACC for probe MCP, SEQ ID NO: 14;
a probe HNP: ATTACAGGTCGTGGTACAGTC, SEQ ID NO: 15;
probe FLP: TCAGCACTTAAAGCTCTTGAAG, SEQ ID NO: 16;
probe FKP: AGTGCTCACACAGATTGTCT, SEQ ID NO: 17;
the probe YGP: GCTCAGGTGGTTAGAGCGC, SEQ ID NO: 18;
probe SAP: TAGCATATCAGAAGGCACACC, SEQ ID NO: 19;
and (3) probe BMP: ATGTATACTTTGTATACGTG, SEQ ID NO: 20;
a probe TLP: GTGTCACGTAAGTGACGCG, SEQ ID NO: 21;
probe SMYP: TCGCGCAGTAAAGGGTGAT, SEQ ID NO: 22;
probe LGP: GGAAGCACAATCAAAGAGG, SEQ ID NO: 23;
and (3) probe ECP: GGTCATTAGCGCCACTCACTGCA, SEQ ID NO: 24.
3. The quantum dot nucleic acid detection kit for simultaneously detecting 11 lower respiratory tract pathogenic bacteria according to claim 2, wherein the membrane strip further comprises an internal control probe for simultaneously monitoring the extraction and amplification of sample nucleic acid:
ICP: TTTGCTAATCATGTTCATACC, SEQ ID NO: 25.
4. The quantum dot nucleic acid detection kit for simultaneously detecting 11 lower respiratory tract pathogenic bacteria according to claim 2, wherein the capture probe is 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 carbon chain and oligo dT (n), the main chain length of the fatty acid carbon chain is 1-12 carbon atoms, and n in the oligo dT (n) is an integer from 1-30.
5. The quantum dot nucleic acid detection kit for simultaneously detecting 11 lower respiratory tract pathogenic bacteria according to claim 1, wherein a biotin label is modified at the 5' end of a reverse primer in the detection primers, 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 carbon chain and oligo dT (n), the length of the main chain of the fatty acid carbon chain is 1-12 carbon atoms, and n in the oligo dT (n) is an integer from 1-30.
6. The kit as claimed in 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 simultaneously detecting 11 lower respiratory tract pathogenic bacteria quantum dots nucleic acid according to claim 1, wherein the quantum dots are CdSe/ZnS core-shell quantum dots.
8. The quantum dot nucleic acid detection kit for simultaneously detecting 11 lower respiratory tract pathogenic bacteria according to claim 1, wherein the positive control comprises a klebsiella pneumoniae recombinant plasmid;
the DNA fragment sequence of Klebsiella pneumoniae is SEQ ID NO: 26:
ACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAACCGTAGGGGA ACCTGCGGTTGGATCACCTCCTTACCTTAAAGAACCTGCCTTTGTAGTGCTCACACAGATTGTCTGATGAAAGTAAAGAAGCAAGGCGTCTTGCGATTGAGACTTCAGTGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTTTCACGGCGGTAACAGGGGTTCGAATCCCCTAGGGGACGCCACTTGCTGGTTCGTGAGTGAAAGACGCGTGCCGATGTATCTCAAAACTCATCTTCGGGTGATGTTTGAGATATTTGCTCTTTAAAAATCTGGATCAAGCTGAAAATTGAAACGACACACTGTTTAAGTGTGTTCGAGTCTCTCAAATTTTCGCAATCAGAAGTGAAACATCTTCGGGTTGTGAGGTTAAGCGACTAAGCGTACACGGTGGATGCCCTGGCAGTCAGAGGCGATGAAGGACGTGCTAATCTGCGAA。
9. the quantum dot nucleic acid detection kit for simultaneously detecting 11 lower respiratory tract pathogenic bacteria according to claim 1, wherein the reaction solution comprises the following components (in parts by weight):
10 buffer 2.5ul, 20mM dN (U) TP 0.25ul, 2U/ul UDG 0.5ul, 10 uM nucleotide sequence as shown in SEQ ID NO: 1-13, 0.75. mu.L each, 5U/ul Hotstart taq 0.25ul, H2O was made up to 21 ul.
10. The detection method for simultaneously detecting 11 types of lower respiratory tract pathogenic bacteria quantum dot nucleic acid is characterized in that the detection kit according to claim 1 is adopted, and the types of the applicable clinical samples are as follows: a pharyngeal swab, a nasal swab, a nasopharyngeal aspirate, a deep cough sputum, a respiratory tract aspirate, a bronchial lavage, an alveolar lavage, or a lung tissue biopsy specimen;
the specific detection steps are as follows:
1) each sample reaction system to be detected is 21ul of reaction liquid and 4ul of nucleic acid sample;
2) placing the sample in a real-time fluorescent PCR instrument for PCR amplification detection;
3) the real-time fluorescent PCR amplification program was set up as follows:
first, stage 1: treating at 50 deg.C for 2 min;
and (2) stage: treating at 95 deg.C for 5 min;
and (3) stage: performing two cycles every 1 ℃ reduction at 95 ℃ for 15-30s, 60-56 ℃ for 30-60s, 72 ℃ for 30s and 60-56 ℃ respectively;
and (4) stage: 15-30s at 95 ℃, 30-60s at 55 ℃, 30s at 72 ℃ and 30 cycles.
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| US20120058487A1 (en) * | 1999-09-28 | 2012-03-08 | Bergeron Michel G | Compositions and methods for detecting klebsiella pneumoniae |
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| CN108624653A (en) * | 2018-05-30 | 2018-10-09 | 杭州千基生物科技有限公司 | A kind of kit of quantum dot detection of nucleic acids for bloodstream infection pathogen |
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