CN108866215B - Real-time fluorescent quantitative PCR kit for detecting enterobacter aerogenes - Google Patents
Real-time fluorescent quantitative PCR kit for detecting enterobacter aerogenes Download PDFInfo
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Abstract
The utility model relates to a real-time fluorescent quantitative PCR kit for detecting enterobacter aerogenes, which comprises PCR reaction liquid, enzyme mixed liquid, a Cad gene standard product of the enterobacter aerogenes, a negative reference substance and a positive reference substance. The method can achieve the aim of accurately quantifying the content of the enterobacter aerogenes in the sample to be detected by extracting the genome DNA of the enterobacter aerogenes and combining the real-time fluorescence quantitative PCR detection technology, has the characteristics of rapidness, sensitivity and good specificity, and has important significance for judging the content of the active bacteria of the enterobacter aerogenes and subsequently researching the urinary tract infection.
Description
Technical Field
The utility model relates to the technical field of molecular biology and microbial detection, in particular to a real-time fluorescent quantitative PCR kit for detecting enterobacter aerogenes.
Background
Enterobacter aerogenes (A)Enterobacter aerogenes，EAE) belongs to Enterobacteriaceae, is a gram-negative facultative anaerobe, and is a conditional pathogen. When the host body state changes or bacteria enter parts except intestinal tracts, the host body can cause respiratory tract infection, urogenital tract infection, blood infection and the like, and is also a common pathogenic bacterium for elderly people infection or nosocomial pneumonia. Enterobacter aerogenes can produce B-lactamase, and the probability of causing nosocomial infection of the bacteria is continuously increased due to abuse of antibiotics for years, so that the enterobacter aerogenes becomes common conditional pathogenic bacteria for nosocomial infection.
The traditional method for diagnosing the enterobacter aerogenes is mainly to classify the enterobacter aerogenes according to the physiological and biochemical characteristics of bacteria, needs a large amount of time to judge the result of physiological and biochemical reaction, and is not beneficial to finding out pathogeny in time and diagnosing the pathogeny. As a molecular biology tool, the PCR technology can selectively amplify DNA or RNA fragments in vitro, and has the characteristics of simple operation, strong specificity, rapidness, high sensitivity and the like. The Taq Man FAM probe method has strong detection specificity and high sensitivity, is convenient for optimizing reaction conditions, and can be used for quickly detecting enterobacter aerogenes.
Disclosure of Invention
The utility model aims to solve the technical problem of providing a real-time fluorescence quantitative PCR kit for detecting enterobacter aerogenes, which is rapid, sensitive and good in specificity.
In order to solve the problems, the real-time fluorescence quantitative PCR kit for detecting the enterobacter aerogenes comprises
PCR reaction solution consisting of 12mM Tris-HCl (pH 8.9), 100mM KCl, 0.12% Triton X-100, 0.12% sodium cholate, 0.6 mg/mL BSA, 1.8mM MgCl250nM dNTP, 500nM upstream primer, 500nM downstream primer, 300nM Taqman probe;
wherein the sequence of the upstream primer is 5'-CGCTGTACGCTTCATCTATT-3'; the sequence of the downstream primer is 5'-GACGCTGTATGCCGAGGTT-3'; the probe sequence is 5'-TTCCCTTTGCCTAACATATCTACCGTTT-3';
an enzyme mixture composed of 5U/. mu.L Taq DNA polymerase, 50% glycerol, 20mM Tris-HCl (pH8.0, 25 ℃), 100mM KCl, 0.1mM EDTA, 1mM DTT, 0.5% Tween ®20 and 0.5% NP-40;
the standard product of the Cad gene of Enterobacter aerogenes is 1.00 multiplied by 108copies/ml、1.00×107copies/ml、1.00×106copies/ml、1.00×105copies/ml of recombinant plasmid;
wherein the sequence of the Cad gene standard product is 5'-CGCTGTACGCTTCATCTATTTTCCCTTTGCCTAACATATCTACCGTTTTTATCTGGTAGAAAATCGCCATATCTTTAATGCCGGGCATTTTACCTACTTTTTCAACCTCGGCATACAGCGTC-3';
-a negative control consisting of water;
positive control, consisting of 1.00X 107copies/ml of recombinant plasmid.
The upstream primer and the downstream primer are complementary strand sequences including a primer sequence.
The upstream primer and the downstream primer are sequences obtained by extending one to several bases or deleting one to several bases in the directions of 5 'end and 3' end.
The fluorescence reporter group marked at the 5 'end in the probe is one of FAM, TET, JOE, HEX and VIC, and the fluorescence quencher group marked at the 3' end is one of BHQ, DABCYL and TAMRA.
Compared with the prior art, the utility model has the following advantages:
1. the utility model adopts gene cloning technology to insert Cad gene segments of Enterobacter aerogenes into a vector pMD18-T to obtain recombinant plasmids containing the Cad gene segments, and the recombinant plasmids are used as standard products. Designing and synthesizing a group of specific primers and probes according to coding gene sequences of Cad gene segments of the enterobacter aerogenes, optimizing PCR reaction conditions, establishing a detection method taking real-time fluorescent quantitative polymerase chain reaction as a platform, and evaluating the established method.
2. The method can achieve the aim of accurately quantifying the content of the enterobacter aerogenes in the sample to be detected by extracting the genome DNA of the enterobacter aerogenes and combining the real-time fluorescent quantitative PCR detection technology, and has important significance for judging the content of the active bacteria of the enterobacter aerogenes and subsequently researching the urinary tract infection.
3. The utility model is rapid, sensitive and specific.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows the results of the sensitivity test according to the present invention. a represents the plasmid concentration of 1.00X 1012copies/ml, b represents plasmid concentration of 1.00X 1011copies/ml, c represents plasmid concentration of 1.00X 1010copies/ml, d represents a plasmid concentration of 1.00X 109copies/ml, e represents plasmid concentration of 1.00X 108copies/ml, f represents plasmid concentration of 1.00X 107copies/ml, g represents plasmid concentration of 1.00X 106copies/ml, h represents a plasmid concentration of 1.00X 105copies/ml and negative control.
FIG. 2 shows the results of the probe specificity test of the present invention. Wherein a is the genome of the Enterobacter aerogenes on the standard amplification curve, b represents that the standard amplification curve does not appear and is respectively lactobacillus rhamnosus, Klebsiella pneumoniae, Escherichia coli, Haemophilus influenzae, enterococcus faecalis, Neisseria meningitidis, Staphylococcus aureus, Enterobacter cloacae, beta hemolytic streptococcus, Clostridium tetani, Pseudomonas aeruginosa, Lactobacillus casei, Vibrio parahaemolyticus, Streptococcus thermophilus, Proteus mirabilis, Salmonella typhimurium, Neisseria gonorrhoeae, Streptococcus pneumoniae, Shigella flexneri, Salmonella, Streptococcus pyogenes, helicobacter pylori and negative control.
FIG. 3 is an electrophoretogram of gel imaging of Enterobacter aerogenes of the present invention. Lactobacillus rhamnosus, Klebsiella pneumoniae, Escherichia coli, Haemophilus influenzae, enterococcus faecalis, Neisseria meningitidis, Staphylococcus aureus, Enterobacter cloacae, beta hemolytic streptococcus, Clostridium tetani, Pseudomonas aeruginosa, Lactobacillus casei, Marker of DL2000, Vibrio parahaemolyticus, Streptococcus thermophilus, Proteus mirabilis, Salmonella typhimurium, Neisseria gonorrhoeae, Streptococcus pneumoniae, Shigella flexneri, Enterobacter aerogenes, Salmonella, Streptococcus pyogenes, helicobacter pylori and negative control are sequentially arranged from left to right.
FIG. 4 is a standard curve of Enterobacter aerogenes of the present invention.
Detailed Description
A real-time fluorescent quantitative PCR kit for detecting enterobacter aerogenes comprises
PCR reaction solution consisting of 12mM Tris-HCl (pH 8.9), 100mM KCl, 0.12% Triton X-100, 0.12% sodium cholate, 0.6 mg/mL BSA, 1.8mM MgCl250nM dNTP, 500nM upstream primer, 500nM downstream primer, 300nM Taqman probe;
wherein the sequence of the upstream primer is 5'-CGCTGTACGCTTCATCTATT-3'; the sequence of the downstream primer is 5'-GACGCTGTATGCCGAGGTT-3'; the probe sequence is 5'-TTCCCTTTGCCTAACATATCTACCGTTT-3';
an enzyme mixture composed of 5U/. mu.L Taq DNA polymerase, 50% glycerol, 20mM Tris-HCl (pH8.0, 25 ℃), 100mM KCl, 0.1mM EDTA, 1mM DTT, 0.5% Tween ®20 and 0.5% NP-40;
the standard product of the Cad gene of Enterobacter aerogenes is 1.00 multiplied by 108copies/ml、1.00×107copies/ml、1.00×106copies/ml、1.00×105copies/ml of recombinant plasmid;
wherein the sequence of the Cad gene standard product is 5'-CGCTGTACGCTTCATCTATTTTCCCTTTGCCTAACATATCTACCGTTTTTATCTGGTAGAAAATCGCCATATCTTTAATGCCGGGCATTTTACCTACTTTTTCAACCTCGGCATACAGCGTC-3' (SEQ ID NO. 2);
-a negative control consisting of water;
positive control, consisting of 1.00X 107copies/ml of recombinant plasmid.
Wherein: the forward primer and the reverse primer may be complementary strand sequences including primer sequences.
The forward primer and the reverse primer may be sequences obtained by extending one to several bases or deleting one to several bases in the directions of the 5 'end and the 3' end.
The fluorescence reporter group marked at the 5 'end in the probe is one of FAM, TET, JOE, HEX and VIC, and the fluorescence quencher group marked at the 3' end is one of BHQ, DABCYL and TAMRA.
The methods used in the following examples are conventional unless otherwise specified, and the primers, probes and sequence determination work used were synthesized and carried out by Biotechnology engineering (Shanghai) Ltd.
EXAMPLE 1 preparation of Cad Gene Standard
To establish a real-time fluorescent quantitative PCR method, an external standard substance required by the method must be prepared, and the standard substance should contain a highly conserved and specific sequence to ensure high specificity of the reaction. The gene is widely existed in enterobacter aerogenes and has high conservation. The study employed the Enterobacter aerogenes Cad gene as the target sequence. The part mainly adopts PCR technology to amplify the Cad gene of the enterobacter aerogenes, utilizes gene recombination technology to connect the Cad gene to a plasmid vector pMD18-T to construct a recombinant plasmid pMD18-T-Cad, carries out corresponding PCR identification and sequencing identification, and finally takes the quantitative result as a standard product of a method to be established, thereby laying a foundation for the next method and evaluation.
First, preparation of template DNA
Genomic DNA of Enterobacter aerogenes (standard strain) was extracted and used as a template for PCR amplification of the Cad gene.
Firstly, 1ml of bacterial suspension is taken and added into a 1.5ml centrifuge tube, and centrifuged for 2 minutes at 8000r/min, and supernatant is discarded.
② adding 400ul Buffer diagnostic suspension liquid for precipitation, mixing uniformly, putting into 65 ℃ water bath for 1h until the cells are completely lysed.
③ adding 200ul Buffer PB, mixing uniformly, standing for 5min at-20 ℃, and centrifuging to obtain the supernatant.
Fourthly, 600ul of isopropanol is added and mixed evenly, and the mixture is kept stand for 5min at the temperature of 20 ℃. The supernatant was discarded by centrifugation.
Fifthly, adding 1ml of 75 percent ethanol and standing for 5min at the temperature of 20 ℃. The supernatant was discarded by centrifugation.
Sixthly, washing the precipitate by using 75 percent ethanol and drying.
Dissolving in 50ul TE buffer, and storing at-20 deg.C.
Second, PCR amplification of Cad Gene fragment
1. Design and Synthesis of primers
According to the utility model, bioinformatics comparison analysis is carried out on the Cad gene complete sequence of the enterobacter aerogenes in the NCBI database, a conservative fragment sequence suitable for designing a Primer and a probe is selected as a target, and a group of real-time fluorescence quantitative PCR primers and probes and a group of peripheral primers of related sequences are designed by applying Primer express 3 software, Primer Premier 5 software and Beacon Designer 7 software.
The selected amplification sequence has a nucleotide sequence shown from 1 st site to 1526 th site of SEQ ID NO.1 in the sequence table.
SEQ ID NO.1
1 TTATTCTGAA GCGAGGAAAT TGTCGAGATA CGGTACTACG CGGGTGACGG AGGTCTGGAA
61 TACTCCGTTT TCAATCCAGT ACAAGGTGTT TTCGCCAGGG CGTAAATTAA ACGCCGTCAG
121 GTAGGCATCG GCAGCTTCGC GGTTCTGTCC TTTCATCTCA TAAACTTTTC CGAGCAGCAC
181 GTAATTCATC CACGACATTT CTAAATCAAT GCCCGTGTTG ATCGCGCTGT ACGCTTCATC
241 TATTTTCCCT TTGCCTAACA TATCTACCGT TTTTATCTGG TAGAAAATCG CCATATCTTT
301 AATGCCGGGC ATTTTACCTA CTTTTTCAAC CTCGGCATAC AGCGTCGTAA TTGTTTTTCA
361 TCCAACGGCT GCTGCGAATG ACGTAATACA TCCACCAGCG CTTTTTCGGC GTAAGCATAA
421 ATAAAGTCCG GCGATTGTTT AATGACGTCA TCAAGTATAT CGCTGGCTTT ACCTAATGAA
481 TCGACATCAC CTTTCATTAA CAGCTGATGG GCCTGATAAA GACGGGTTAA TGAAGGGCTT
541 TGCGCGGGTT GATACTGTTT CAGCATCGCT TTCATGCGAT CCGGCCACGG CTGCATTAGC
601 GCGGTTGACA GACTATCCAG CAGGTCATTC TGAATGGTGA GCTGGTTATC CATGGTAATA
661 AAGTAGCGTT TATCGAGCAT CGTCGAGCCG TCGGCGTTAT CGACCAGTTG CACCGACATA
721 AAGCACTGCT GGGCGCGATA GTGGCGCTGA TTAACGAATT CGATGGTCAA CGTTTTACCC
781 GAACTGCTGG GCTCGTTGAT GTTGTAGTTC GTTTTATCGT GGACCATAAA GGTCGAGAAA
841 GTGTTGAGCG AGGTGGTTAC CAGGCTGCCC AACCCTACCG CATAGGCATG CTGCGATGCC
901 CAGTTGTTGC ATGAACTGCC GTTTACCAGG TGTATGTCGA TATCGCGCGG GTTGAGCAGC
961 AGGCGAGTTT TGGTTTCCGG CGGCCGCGAT TCTATCGACG ACAAAGCAAC TAACGCGACG
1021 CAACTGCCCA ACGCCAGCAG GAATAGCGTC CATACCCAAA AGGTGGTGAT CCGTTTACGT
1081 TTTTCCGGCA CTGCCGCCGC CGCTGGCGCA GCAGATGAAG GAGAAATAGT CGGTGGCGAC
1141 GTTTCAGGAA CGGTGACAGG GGCCACTGGC GGGACAACCG GCGCCAACTC TTCGCCCTCT
1201 TCCGTACACC AAATTACCGG GACCGTCAGC TTGTAGCCGC GTTTGGGCAC CGTCGCAATA
1261 TATTCCATGC TGCCGTCATC ACCGTCTTTC AGTGATTTAC GCAACTCGGA AATACTTTGG
1321 GTGACGACAT GGCTGGTCAC CACGTTGCGC GTCCAGACGT GATCGATAAG CTCGTCCCGG
1381 CTAAGTACCT CGCCAGGATG TTGAGCAAAG TAGACCAGAA GATCGATTAA ACGCGGTTCG
1441 AGAGTAAGCT GACGTCCGTT CCGGCTAATT TGGTTTACAG AAGGAGTAAC GAGCCATTCC
1501 CCAACGCGAA CAACAGGTTG TTGCAT
The sequence of the peripheral primer is as follows:
an upstream primer: Cad-225F 5'-CGCTGTACGCTTCATCTATT-3'
A downstream primer: Cad-346R 5'-GACGCTGTATGCCGAGGTT-3'
The amplified fragment size was: 122 bp.
2. PCR reaction system and reaction conditions
PCR amplification was carried out using DNA as a template and the above-mentioned peripheral primer Cad-225F/Cad-346R as an amplification primer, using the following system and reaction conditions. The PCR system is shown in Table 1.
TABLE 1
Wherein the primer adopts Cad-225F/Cad-346R, the Taq enzyme adopts Beijing Baitag Power Taq Plus DNA polymerase, and the PCR amplification instrument is MG series 96 gradient PCR instrument of Hangzhou Langzhou scientific instrument Limited.
Amplification procedure/reaction conditions:
pre-denaturation at 94 ℃ for 5 min; 30 cycles of 94 ℃ for 30s, 50 ℃ for 30s and 72 ℃ for 1 min; and (3) carrying out 2% agarose electrophoresis on 5 mu L of the amplification product at 72 ℃ for 10min, detecting the size of the PCR product, and then purifying and recovering the residual PCR amplification product by adopting a DNA gel recovery kit produced by Shanghai's chemical company.
Thirdly, construction and transformation of recombinant plasmid pMD18-T-Cad
1. And (3) connection reaction: the PCR amplification product obtained by the above purification was ligated with pMD18-T (Dalianbao Biopsis Co.) using the following ligation system (Table 2):
TABLE 2
After the preparation was completed, the ligation reaction was carried out overnight at 16 ℃.
2. Transformation and PCR identification of pMD18-T-Cad plasmid
The cryopreserved DH5 α competent cells were removed from the-70 ℃ ultra-low temperature refrigerator and placed on an ice box to allow natural thawing.
Adding 10. mu.L of the ligation product into 100. mu.L of DH5 alpha competent cells.
② heat shock is carried out for 90 s in 42 ℃ water bath, and the mixture is immediately placed on ice to be cooled for 30min after the heat shock.
③ to a 1.5ml EP tube, 800. mu.l of precooled LB liquid medium (without ampicillin) was added and mixed, followed by culturing at 37 ℃ with gentle shaking at 140 rpm for 1 hour.
Fourthly, centrifuging the culture solution at 8000 rpm for 1min, discarding the supernatant, coating the cell heavy suspension absorption residue on an LB flat plate with 0.1ng Amp, placing the plate with the front side upward for 30min, and after the bacterial solution is completely absorbed by the culture medium, inverting the culture dish and culturing the plate in a constant temperature box at 37 ℃ overnight.
The next day, monoclonal colonies were picked from the plate and cultured in 100. mu.L of a PCR tube in LB liquid medium (ampicillin-containing) for 2-3 hours at 37 ℃ with shaking. 2. mu.L of the suspension was used as a template for PCR identification, and the remaining suspension was added to 20ml of LB liquid medium for amplification.
Sixthly, amplifying the diluted bacterium solution by using a gas-producing intestinal specific primer Cad-225F/Cad-346R, carrying out 2% agarose gel electrophoresis on a PCR product, and identifying a positive transformant by detecting the size of the PCR product.
The primer Cad-225F/Cad-346R has the following sequence:
an upstream primer; Cad-225F 5'-CGCTGTACGCTTCATCTATT-3'
A downstream primer; Cad-346R 5'-GACGCTGTATGCCGAGGTT-3'
The system is shown in Table 3:
TABLE 3
Amplification procedure/reaction conditions: pre-denaturation at 94 ℃ for 5 min; 30 cycles of 94 ℃ for 30s, 50 ℃ for 30s and 72 ℃ for 1 min; 10min at 72 ℃.
Extracting positive recombinant plasmid by adopting a plasmid preparation kit produced by Baitach company, determining concentration and purity, simultaneously sucking a part of purified plasmid, sending the purified plasmid to Shanghai bioengineering limited company for sequencing, and determining that the gene sequence of the insert is consistent with the target sequence.
EXAMPLE 2 preliminary establishment of a method for real-time fluorescent quantitative PCR detection of Enterobacter aerogenes
Design and synthesis of specific primer and probe
A group of real-time fluorescence quantitative PCR primers and probes are designed by using the selected conserved segment of the Cad gene of the enterobacter aerogenes as a target and applying Primer express 3 software, Primer Premier 5 software and Oligo 7 software.
As the core of the utility model, a group of primers and probe nucleotide sequences for real-time fluorescence PCR detection of enterobacter aerogenes are as follows:
an upstream primer: Cad-225F 5'-CGCTGTACGCTTCATCTATT-3'
A downstream primer: Cad-346R 5'-GACGCTGTATGCCGAGGTT-3'
And (3) probe: cad 225-3465 '-TTCCCTTTGCCTAACATATCTACCGTTT-3'
The primer and the probe have the following amplification target nucleotide sequences:
5’-CGCTGTACGCTTCATCTATTTTCCCTTTGCCTAACATATCTACCGTTTTTATCTGGTAGAAAATCGCCATATCTTTAATGCCGGGCATTTTACCTACTTTTTCAACCTCGGCATACAGCGTC-3’
second, obtaining and quantifying standard substance
1. The frozen E.coli DH 5. alpha.100. mu.L containing the recombinant plasmid was transferred to 5mL of LB liquid medium and shaken overnight at 37 ℃ and 200 rpm.
2. Transferring 1ml of overnight-cultured bacterial liquid into 10ml of LB liquid medium, increasing the bacterial speed at 200rpm, culturing for 2-3 hours, and extracting plasmids by adopting a plasmid preparation kit produced by Beijing Baitaike company.
3. The extracted plasmid was measured by an ultramicro ultraviolet-visible spectrophotometer (ND 5000) of Baitach Biotechnology Ltd, Beijing, to determine A260 and A280, and the purity of the plasmid was judged from A260/A280.
4. Calculation of plasmid pMD18-T-Cad concentration (copy number)
Molecular weight of the plasmid =2814bp × 660 (average molecular weight per base pair)
In the next step, the plasmid concentration is measured to be 194.22 ng/. mu.l, the plasmid purity is A260/A280=1.82, and the unit needs to be converted into copies/ml because the unit needs to be the copy number when the real-time fluorescence quantitative PCR is carried out.
Plasmid copies/ml = avogalois constant x number of moles of plasmid
Wherein the Avogastron constant =6.02 × 1023copies/mol。
Thus the extracted plasmid concentration copies/ml =194.22 × 10-6g/ml×6.02×1023copies/mol÷（2814bp×660g/bp•mol）=6.29×1013copies/ml
10 u l plasmid +52.9 sterile water to obtain a concentration of 1.00X 1012Plasmid of copies/ml, then the plasmid is diluted 10 times to obtain a series of concentrations of plasmid, and stored at-20 ℃ for use.
[ method evaluation ]
1. Sensitivity test
Diluting the plasmid obtained above by 10 times to obtain a series of plasmids with concentration of 1.00 × 1012copies/ml、1.00×1011copies/ml、1.00×1010copies/ml、1.00×109copies/ml、1.00×108copies/ml、1.00×107copies/ml、1.00×106copies/ml、1.00×105copies/ml, etc. as gradient templates. The reaction system is shown in Table 4:
TABLE 4
Reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min, 10 sec at 94 ℃ and 30s at 60 ℃ and collection of fluorescence signal, 40 cycles. The fluorescence curve obtained by software processing according to the fluorescence signal detected by the instrument, the signal of the fluorescence curve observed, the results refer to FIG. 1, the data show when the plasmid concentration reaches 1.00X 106Fluorescence signal was still observed at copies/ml, but the plasmid concentration reached 1.00X 105No fluorescence signal was detected at copies/ml, so the sensitivity of the method was 1.00X 106copies/ml。
2. Experiment of specificity
To confirm the specificity of the present invention for detecting Enterobacter aerogenes, we selected other tests specific to the common clinically infected microorganisms, including: lactobacillus rhamnosus, Klebsiella pneumoniae, Escherichia coli, Haemophilus influenzae, enterococcus faecalis, Neisseria meningitidis, Staphylococcus aureus, Enterobacter cloacae, beta hemolytic streptococcus, Clostridium tetani, Pseudomonas aeruginosa, Lactobacillus casei, Vibrio parahaemolyticus, Streptococcus thermophilus, Proteus mirabilis, Salmonella typhimurium, Neisseria gonorrhoeae, Streptococcus pneumoniae, Shigella flexneri, Salmonella, Streptococcus pyogenes, helicobacter pylori.
The specificity test includes a test using the genomic DNA of the above-mentioned sample as a template. The DNA extraction of the above microorganisms was carried out by using a kit for rapid extraction of genomic DNA from Shanghai's bacteria (C317 KA 2364) and a2 × real-time PCR premix (probe) real-time fluorescent quantitative PCR kit from Bori, in the reaction system shown in Table 5:
TABLE 5
Reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min, 10 sec at 94 ℃ and 30s at 60 ℃ and collection of fluorescence signal, 40 cycles. And processing the fluorescence signal detected by the instrument by software to obtain a fluorescence curve, observing the signal of the fluorescence curve, and analyzing the specificity. As for the results shown in FIG. 2, the genomic DNA was used as a template to detect only Enterobacter aerogenes as positive, and the other microorganisms were negative, indicating that the present invention has excellent specificity. The results are shown in Table 6.
TABLE 6
3. Gel imaging electrophoresis chart of enterobacter aerogenes probe
To confirm the specificity of the present invention for detecting Enterobacter aerogenes, we selected other tests specific to the common clinically infected microorganisms, including: lactobacillus rhamnosus, Klebsiella pneumoniae, Escherichia coli, Haemophilus influenzae, enterococcus faecalis, Neisseria meningitidis, Staphylococcus aureus, Enterobacter cloacae, beta hemolytic streptococcus, Clostridium tetani, Pseudomonas aeruginosa, Lactobacillus casei, Vibrio parahaemolyticus, Streptococcus thermophilus, Proteus mirabilis, Salmonella typhimurium, Neisseria gonorrhoeae, Streptococcus pneumoniae, Shigella flexneri, Salmonella, Streptococcus pyogenes, helicobacter pylori.
And preparing 2% agarose gel for detecting enterobacter aerogenes specific primer probe fluorescent quantitative results. And taking an amplification product of a specificity experiment as a template. Referring to FIG. 3, the results show that the present invention has very good specificity, as the genome DNA is used as the template, and the detection of only Enterobacter aerogenes is positive, and the other microorganisms are negative.
4. Preparation of Standard Curve
Taking the logarithm of the positive template with different copy numbers as an abscissa and the initial cycle number (Ct) reaching the fluorescence threshold in the PCR reaction process as an ordinate to obtain a standard curve for detecting enterobacter aerogenes as a reference standard for quantitative determination of a sample to be detected, and the result is shown in fig. 4. The original equation of the standard curve is y = a + bx, and the equation of the standard curve at this time is y =50.343-1.70 x.
<110> Suzhou Baiyuan Gene technology Co., Ltd
<120> real-time fluorescence quantitative PCR kit for detecting enterobacter aerogenes
<160>2
<210>1
<211>1526
<212>DNA
1 TTATTCTGAA GCGAGGAAAT TGTCGAGATA CGGTACTACG CGGGTGACGG AGGTCTGGAA
61 TACTCCGTTT TCAATCCAGT ACAAGGTGTT TTCGCCAGGG CGTAAATTAA ACGCCGTCAG
121 GTAGGCATCG GCAGCTTCGC GGTTCTGTCC TTTCATCTCA TAAACTTTTC CGAGCAGCAC
181 GTAATTCATC CACGACATTT CTAAATCAAT GCCCGTGTTG ATCGCGCTGT ACGCTTCATC
241 TATTTTCCCT TTGCCTAACA TATCTACCGT TTTTATCTGG TAGAAAATCG CCATATCTTT
301 AATGCCGGGC ATTTTACCTA CTTTTTCAAC CTCGGCATAC AGCGTCGTAA TTGTTTTTCA
361 TCCAACGGCT GCTGCGAATG ACGTAATACA TCCACCAGCG CTTTTTCGGC GTAAGCATAA
421 ATAAAGTCCG GCGATTGTTT AATGACGTCA TCAAGTATAT CGCTGGCTTT ACCTAATGAA
481 TCGACATCAC CTTTCATTAA CAGCTGATGG GCCTGATAAA GACGGGTTAA TGAAGGGCTT
541 TGCGCGGGTT GATACTGTTT CAGCATCGCT TTCATGCGAT CCGGCCACGG CTGCATTAGC
601 GCGGTTGACA GACTATCCAG CAGGTCATTC TGAATGGTGA GCTGGTTATC CATGGTAATA
661 AAGTAGCGTT TATCGAGCAT CGTCGAGCCG TCGGCGTTAT CGACCAGTTG CACCGACATA
721 AAGCACTGCT GGGCGCGATA GTGGCGCTGA TTAACGAATT CGATGGTCAA CGTTTTACCC
781 GAACTGCTGG GCTCGTTGAT GTTGTAGTTC GTTTTATCGT GGACCATAAA GGTCGAGAAA
841 GTGTTGAGCG AGGTGGTTAC CAGGCTGCCC AACCCTACCG CATAGGCATG CTGCGATGCC
901 CAGTTGTTGC ATGAACTGCC GTTTACCAGG TGTATGTCGA TATCGCGCGG GTTGAGCAGC
961 AGGCGAGTTT TGGTTTCCGG CGGCCGCGAT TCTATCGACG ACAAAGCAAC TAACGCGACG
1021 CAACTGCCCA ACGCCAGCAG GAATAGCGTC CATACCCAAA AGGTGGTGAT CCGTTTACGT
1081 TTTTCCGGCA CTGCCGCCGC CGCTGGCGCA GCAGATGAAG GAGAAATAGT CGGTGGCGAC
1141 GTTTCAGGAA CGGTGACAGG GGCCACTGGC GGGACAACCG GCGCCAACTC TTCGCCCTCT
1201 TCCGTACACC AAATTACCGG GACCGTCAGC TTGTAGCCGC GTTTGGGCAC CGTCGCAATA
1261 TATTCCATGC TGCCGTCATC ACCGTCTTTC AGTGATTTAC GCAACTCGGA AATACTTTGG
1321 GTGACGACAT GGCTGGTCAC CACGTTGCGC GTCCAGACGT GATCGATAAG CTCGTCCCGG
1381 CTAAGTACCT CGCCAGGATG TTGAGCAAAG TAGACCAGAA GATCGATTAA ACGCGGTTCG
1441 AGAGTAAGCT GACGTCCGTT CCGGCTAATT TGGTTTACAG AAGGAGTAAC GAGCCATTCC
1501 CCAACGCGAA CAACAGGTTG TTGCAT
<210>2
<211>122
<212>DNA
1 CGCTGTACGC TTCATCTATT TTCCCTTTGC CTAACATATC TACCGTTTTT ATCTGGTAGA
61 AAATCGCCAT ATCTTTAATG CCGGGCATTT TACCTACTTT TTCAACCTCG GCATACAGCG
121 TC
<210>3
<211>20
<212>DNA
1 CGCTGTACGC TTCATCTATT
<210>4
<211>19
<212>DNA
1 GACGCTGTAT GCCGAGGTT
<210>5
<211>28
<212>DNA
1 TTCCCTTTGC CTAACATATC TACCGTTT
Claims (2)
1. A real-time fluorescent quantitative PCR kit for detecting enterobacter aerogenes comprises
The PCR reaction solution was composed of 12mM Tris-HCl, 100mM KCl, 0.12% Triton X-100, 0.12% sodium cholate, 0.6 mg/mL BSA, 1.8mM MgCl250nM dNTP, 500nM upstream primer, 500nM downstream primer, 300nM Taqman probe;
wherein the sequence of the upstream primer is 5'-CGCTGTACGCTTCATCTATT-3'; the sequence of the downstream primer is 5'-GACGCTGTATGCCGAGGTT-3'; the probe sequence is 5'-TTCCCTTTGCCTAACATATCTACCGTTT-3';
enzyme mixture consisting of 5U/. mu.L Taq DNA polymerase, 50% glycerol, 20mM Tris-HCl, 100mM KCl, 0.1mM EDTA, 1mM DTT, 0.5% Tween®20 and 0.5% NP-40;
the standard product of the Cad gene of the enterobacter aerogenes consists of 1.00 multiplied by 108copies/ml、1.00×107copies/ml、1.00×106copies/ml、1.00×105copies/ml of recombinant plasmid;
wherein the sequence of the Cad gene standard product is 5'-CGCTGTACGCTTCATCTATTTTCCCTTTGCCTAACATATCTACCGTTTTTATCTGGTAGAAAATCGCCATATCTTTAATGCCGGGCATTTTACCTACTTTTTCAACCTCGGCATACAGCGTC-3';
a negative control consisting of water;
positive control substance, 1.00X 107copies/ml of recombinant plasmid.
2. The real-time fluorescent quantitative PCR kit for detecting enterobacter aerogenes according to claim 1, wherein the kit comprises: the fluorescence reporter group marked at the 5 'end in the probe is one of FAM, TET, JOE, HEX and VIC, and the fluorescence quencher group marked at the 3' end is one of BHQ, DABCYL and TAMRA.
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