CN1281763C - Process for detecting Enterobacter sakazakii by employing fluorescence PCR technology - Google Patents

Abstract

The present invention discloses a method for detecting Enterobacter sakazakii by a fluorescence PCR technology, which belongs to a bacterial detection technology. The technical scheme is to utilize the specific DNA sequences of the Enterobacter sakazakii to design specific oligonucleotide primers and probes; an excision enzyme probe fluorescence PCR technology and a fluorescence dye tabled PCR technology are respectively used for directly amplifying bacterial target genes from samples. The present invention comprises six specific oligonucleotide sequences of the designed Enterobacter sakazakii, two report plasmid amplification sequences for comparison, and matched fluorescence PCR reaction conditions. The present invention provides a standard method for detecting the Enterobacter sakazakii from foodstuff and clinical samples, and has the advantages that the method omits bacterial repeated cultivation, dispenses with observation and consequently saves time. In addition, the method has high sensitivity and high detection speed, and is capable of carrying out quantitative analysis.

Description

A method for detecting Enterobacter sakazakii using fluorescent PCR technology

technical field

The invention relates to a bacterium inspection technology, in particular to using fluorescent PCR reaction to detect pathogenic bacteria, especially Enterobacter sakazakii.

Background technique

Enterobacter sakazakii is a Gram-negative bacterium belonging to the genus Enterobacteriaceae. Before being named Enterobacter sakazakii in 1980, the bacterium was known as Enterobacter cloacae producing yellow pigment. It is a clinically rare pathogen. The meningitis, sepsis, and necrotizing enterocolitis caused by it have been reported all over the world, and there is an increasing trend. Most of the cases occur in infants and young children, and the course of the disease is short. , and sometimes the fatality rate can reach 80%. Although the source of E. sakazakii infection is unclear, infant formula acts as a vector. On November 26, 2002, the General Administration of Quality Supervision, Inspection and Quarantine issued Announcement No. 120 of 2002, the main content of which was to suspend the customs clearance and related inspection and quarantine procedures for certain batches of formula milk powder produced by Wyeth Company of the United States. The relevant products were destroyed under supervision because the US Food and Drug Administration (FDA) detected Enterobacter sakazakii in the above products; on March 6, 2003, Mead Johnson Company also voluntarily recalled a series of milk powder containing Enterobacter sakazakii Certain batches of infant milk powder.

At present, there is no standard method for the detection of Enterobacter sakazakii in food and clinically in my country. The method for the isolation and counting of Enterobacter sakazakii released by the US FDA in August 2002 still uses the "three-tube" enrichment method to detect Enterobacter sakazakii. Its disadvantage is that it needs to detect the target bacteria through classic inspection methods such as pre-enrichment, isolation and culture, and biochemical identification. Not only does the test take a long time, but also the sensitivity and specificity are relatively low.

Contents of the invention

In view of the above situation, the present invention overcomes the shortcomings in the prior art and provides a method for detecting Enterobacter sakazakii using fluorescent PCR technology, specifically, by detecting exonuclease probe PCR reaction or fluorescent dye chimeric PCR reaction The emitted fluorescent signal can be used to determine whether the sample contains Enterobacter sakazakii.

Technical scheme of the present invention is as follows:

1. Detection of Enterobacter sakazakii by exonuclease probe PCR reaction (Taqman probe method, hereinafter referred to as probe method), the sequences of primers, probes and reporter plasmid amplified fragments used in it are as follows:

Primers:

Probe primer sequence 1: CCGGAACAAGCTGAAAATTCT

Probe primer sequence 2: GTCTTCGTGCTGCGATTAC

Probe:

Probe Method Probe Sequence 1: ACTCTGACACCGCGCATTCCTG

Probe Method Probe Sequence 2: CCTTTGGCGTTTCCCGATGTCCGT

Amplified fragment of probe method reporter plasmid: GGAGGGATTGCAGCGTGTTTTTAATGAGGTCATCACGGGATCCCATGTGCGTGACGGACATCGGGAAACGCCAAAGGAGATT

It also includes oligonucleotide sequences derived from the above five oligonucleotide sequences with a difference of not more than 8 bases, and the antisense complementary sequences of each sequence, or their variants, or their partial sequences.

Components of the PCR reaction system:

Composition Concentration Concentration Sample Amount

PCR system premix 2 times 12.5μL

Probe primer sequence 1 10μmol/L 0.5μL

Probe primer sequence 2 10μmol/L 0.5μL

Probe method Probe sequence 1 3μmol/L 1μL

Probe Method Probe Sequence 2 3μmol/L 1μL

DNA sample 1μL

Reporter plasmid 10 ³ /μL 1 μL

Double distilled water 7.5μL

Total volume 25μL

PCR amplification program:

(1) 95°C for 10 seconds

(2) 95°C for 5 seconds

(3) 62°C for 20 seconds

(4) Go back to step (2) and repeat 45 times.

Detection process:

(1) Design specific oligonucleotide primers and probes, label the probe sequence 1 of the probe method with a FAM fluorophore, and label the probe sequence 2 of the probe method with a HEX fluorophore for external use. Dicer probe fluorescent PCR detection;

(2) Construction can use probe method primer sequence 1 and probe method primer sequence 2 amplification, and its amplified product can hybridize with probe method probe sequence 2 reporter plasmid, as positive control plasmid, prevent false negative from occurring result.

(3) using the probe method primer sequence 1 and the probe method primer sequence 2 as primers, and using the Enterobacter sakazakii genomic DNA as a template to perform specific amplification of the target gene;

(4) During the amplification process, a fluorescent PCR instrument is used for real-time fluorescence light intensity measurement, and the data is transmitted to the computer for analysis through supporting software. The FAM fluorescence signal of a specific wavelength generated by the specific amplification of Enterobacter sakazakii can be observed , proves that Enterobacter sakazakii exists in the sample to be tested; if only HEX fluorescence signal of another wavelength is observed but no FAM fluorescence signal, it proves that Enterobacter sakazakii does not exist in the sample to be tested; if no fluorescent signal is observed, then It indicates that the test failed, and the presence or absence of Enterobacter sakazakii cannot be determined, and a new test is required.

The present invention uses the specific primers of the Enterobacter sakazakii gene, through exonuclease probe PCR reaction, can observe the specific fluorescent signal, and no false positive is found in the 30 kinds of other pathogenic bacteria of different genus or the same genus that have been tested result.

2. The method for detecting Enterobacter sakazakii by fluorescent dye chimeric PCR reaction (referred to as fluorescent chimeric method), the sequence of the primers used and the sequence of the reporter plasmid amplified fragment are as follows:

Fluorescent chimeric primer sequence 1: GGGTTGTCTGCGAATCGCTT

Fluorescent chimeric primer sequence 2: GTCTTCGTGCTGCGTCAAAC

Amplified fragment of fluorescent chimeric reporter plasmid: TCCGTTTCTTCTTCGTCATAACTTAATGTTTTTATTTAAAATACCCTCTGAAAAGAAAGGA

It also includes oligonucleotide sequences derived from the above three oligonucleotide sequences with a difference of not more than 8 bases, and the antisense complementary sequences of each sequence, or their variants, or their partial sequences.

Components of the PCR reaction system:

Composition Concentration Concentration Sample Amount

PCR system premix 2 times 12.5μL

Primer sequence 1 for fluorescent chimeric method: 10μmol/L 0.5μL

Primer sequence 2 used for fluorescent chimeric method: 10μmol/L 0.5μL

DNA sample 1μL

Reporter plasmid 10 ³ /μL 1 μL

Double distilled water 9.5μL

Total volume 25μL

PCR amplification program:

(1) 95°C for 10 seconds

(2) 95°C for 5 seconds

(3) A certain temperature between 58°C and 65°C for 20 seconds

(4) Go back to step (2) and repeat 45 times.

(5) 95°C for 2 minutes

(6) Gradient temperature increase from 55°C to 95°C, increasing by 1°C every 25 seconds.

Detection process:

(1) Design specific oligonucleotide primers for fluorescent dye chimeric fluorescent PCR detection;

(2) Construct a reporter plasmid that can be amplified using the fluorescent chiseled primer sequence 1 and the fluorescent chiseled primer sequence 2 as a positive control plasmid to prevent false negative results.

(3) using the fluorescent chimeric primer sequence 1 and the fluorescent chimeric primer sequence 2 as primers, and using the Enterobacter sakazakii genomic DNA as a template to perform specific amplification of the target gene;

(4) During the amplification process, a fluorescent PCR instrument is used for real-time fluorescence light intensity measurement, and the melting temperature of the synthesized DNA fragment is measured after the PCR reaction is completed. Transfer the data to the computer and analyze it through supporting software. The fluorescence generated by the specific amplification of Enterobacter sakazakii can be observed, and the melting curve unique to the specific fragment generated by the amplification of Enterobacter sakazakii can be obtained (main peak 87.1°C ±0.5°C; secondary peak 85.0±0.5°C), it proves that there is Enterobacter sakazakii in the sample to be tested; if only the fluorescent signal is observed, but the melting curve does not conform to the specific melting curve of Enterobacter sakazakii, and the reporter plasmid can be observed The fluorescent signal and the unique melting curve (peak value 79.0±0.5°C) produced by the specific fragment of the amplified reporter plasmid prove that Enterobacter sakazakii does not exist in the sample to be tested; other results indicate that the test failed, and it cannot be determined whether The presence of Enterobacter sakazakii requires retesting.

The invention uses the specific primers of the Enterobacter sakazakii gene to detect the Enterobacter sakazakii through the fluorescent chimeric PCR reporter fluorescent signal, and no false positive results are found in the 30 tested different genus or other pathogenic bacteria of the same genus.

3. Culture method of Enterobacter sakazakii in the sample to be tested:

(1) Configure special medium: tryptone 20g, lactose 5g, K ₂ HPO ₄ 2.75g, KH ₂ PO ₄ 2.75g, NaCl 34.22g, sodium lauryl sulfate 0.1g, vancomycin 10mg, add sterile water to 1 liter, adjust the pH value to 6.8±0.2;

(2) Take 100 g of sample powder to be tested, dissolve or suspend it in 1 liter of special medium, and incubate overnight at 37°C;

(3) Take 2 mL of the sample culture to be tested obtained above, add it to 50 mL of brain heart infusion, shake and cultivate for 3 hours on a shaker at 200 rpm at 37°C.

Compared with the prior art, the beneficial effect of the present invention is that the identification method based on fluorescent PCR is suitable for directly amplifying target genes from clinical samples such as patient's bacteria-containing body fluid, food and body fluid culture, so as to detect Enterobacter sakazakii. The fluorescent PCR method has the characteristics of accurate detection, strong specificity and high sensitivity, and can quickly and accurately identify specific target bacteria. It uses PCR to amplify bacterial target genes, avoiding repeated cultivation and saving time; the PCR identification method is not affected by the culture conditions and the physiological state of bacteria, and is more accurate than the physiological and biochemical identification method. Compared with ordinary PCR technology, fluorescent PCR technology does not require electrophoresis observation, so it has the characteristics of higher sensitivity, faster detection speed and quantitative analysis. The invention fills up the blank of detecting Enterobacter sakazakii by the fluorescent PCR method.

Description of drawings

Figure 1: Exonuclease probe fluorescence PCR detection results of a milk powder sample (FAM fluorescence signal intensity)

Figure 2: Detection results of a milk powder sample exonuclease probe fluorescent PCR reporter plasmid (FAM fluorescence signal intensity)

Figure 3: Detection results of a milk powder sample exonuclease probe fluorescent PCR reporter plasmid (HEX fluorescence signal intensity)

Figure 4: Fluorescent dye chimeric fluorescent PCR detection results (fluorescent signal intensity) of a milk powder sample

Figure 5: The peak melting temperature of the DNA amplification product of a fluorescent dye chimeric fluorescent PCR test sample for a milk powder sample

Figure 6: Fluorescent signal intensity of fluorescent dye chimeric fluorescent PCR reporter plasmid in a milk powder sample

Figure 7: The peak melting temperature of the amplified product of fluorescent dye chimeric fluorescent PCR reporter plasmid in a milk powder sample

Figure 8: FAM fluorescence signal intensity of non-Enterobacter sakazakii DNA in exonuclease probe fluorescent PCR

Figure 9: HEX fluorescence signal intensity of non-Enterobacter sakazakii DNA in exonuclease probe fluorescent PCR

Figure 10: Fluorescent signal intensity of non-Enterobacter sakazakii DNA in fluorescent dye chimeric PCR

Figure 11: Melting temperature of non-Enterobacter sakazakii DNA amplified by reporter plasmid in fluorescent dye chimeric PCR

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

Example 1

Sample: An imported brand of infant formula milk powder. The suspected colony of Enterobacter sakazakii was detected by conventional physiological and biochemical methods, and then Enterobacter sakazakii was detected by fluorescent signal exonuclease probe PCR:

1. Sample Culture

(1) Configure special medium: tryptone 20g, lactose 5g, K ₂ HPO ₄ 2.75g, KH ₂ PO ₄ 2.75g, NaCl 34.22g, sodium lauryl sulfate 0.1g, vancomycin 10mg, add sterile water to 1 liter, adjust the pH value to 6.8±0.2;

(2) Take 100g of milk powder and dissolve it in 1 liter of special culture medium, and cultivate overnight at 37°C;

(3) Take 2 mL of the sample culture to be tested obtained above, add it to 50 mL of brain heart infusion, and incubate on a shaker at 37° C. at 200 rpm for 3 hours.

2. DNA extraction

Take 1 mL of brain heart infusion enrichment solution, let it stand in an ice bath for 5 minutes, then centrifuge at 12,000 rpm for 5 minutes at room temperature, discard the supernatant, add 100 μL lysozyme solution, keep warm at 37°C for 10 minutes, add TE buffer 500μL, shake and mix. Add the same volume of Tris-saturated phenol (pH 8.0), shake vigorously, centrifuge at 12,000 rpm for 3 minutes, take the supernatant, and repeat the phenol extraction. Take the supernatant, add 0.1 times the volume of sodium acetate (2mol/L), mix well, add an equal volume of ice ethanol, mix well, let stand at low temperature for 30 minutes, centrifuge at 12000 rpm for 5 minutes, discard the supernatant , washed once with 70% cold ethanol, centrifuged at 12,000 rpm for 5 minutes at room temperature, discarded the supernatant, added 50 μL TE solution, and stored at -20°C.

3.PCR amplification

PCR reaction system:

Composition Concentration Sample Amount

PCR system premix 2 times 12.5μL

Primer sequence used in probe method 1 10μmol/L 0.5μL

Primer sequence used in probe method 2 10μmol/L 0.5μL

Probe sequence used in probe method 1 3 μmol/L 1 μL

Probe sequence used in probe method 2 3 μmol/L 1 μL

DNA sample 1μL

Reporter plasmid 10 ³ /μL 1 μL

Double distilled water 7.5μL

Total volume 25μL

PCR amplification program:

(1) 95°C for 10 seconds

(2) 95°C for 5 seconds

(3) 62°C for 20 seconds

(4) Go back to step (2) and repeat 45 times.

A total of 7 tubes of PCR experiments were carried out, in which the DNA samples were: Enterobacter sakazakii standard strain ATCC12868 positive control; original concentration DNA sample; 10-fold diluted DNA sample; 10 ² -fold diluted DNA sample; 10 ³ -fold diluted DNA sample; 10 ⁴ -fold Diluted DNA sample; negative control with no sample for reporter plasmid only.

4. Observation of the fluorescence PCR pattern of the tested sample

During the fluorescent PCR process, it can be observed that the positive control of the standard strain and the samples of various concentrations produced obvious FAM fluorescence at cycles of 18.36; 22.25; 25.83; 29.75; 33.5;

Figure 1: Exonuclease probe fluorescence PCR detection results of a milk powder sample (FAM fluorescence signal intensity)

The FAM fluorescence intensity signals represented by each curve in the figure are:

1. Enterobacter sakazakii standard strain ATCC12868;

2. Original concentration DNA sample;

3. 10 times diluted DNA sample;

4.10 ² -fold diluted DNA sample;

5.10 ³ times diluted DNA sample;

6.10 ⁴ times diluted DNA samples;

In the negative control, no FAM fluorescence was generated during the PCR process, and the results are shown in Figure 2.

Figure 2: Detection results of a milk powder sample exonuclease probe fluorescent PCR reporter plasmid (FAM fluorescence signal intensity)

The FAM fluorescence intensity signal represented by the curve in the figure is:

1. Negative control with only reporter plasmid and no sample.

However, the HEX fluorescence of the reporter plasmid was generated at cycle 34.19, and the results are shown in Figure 3.

Figure 3: Detection results of a milk powder sample exonuclease probe fluorescent PCR reporter plasmid (HEX fluorescence signal intensity)

The HEX fluorescence intensity signal represented by the curve in the figure is:

1. Negative control with only reporter plasmid and no sample.

The above experiments showed that the samples contained Enterobacter sakazakii.

After 2 days, routine microbial culture and biochemical testing proved that 98% of the tested target colonies were Enterobacter sakazakii, and one of the strains was named gq103. The fluorescent signal exonuclease probe PCR test results were consistent with the biochemical test results.

Example 2

Sample: A domestic brand of infant formula milk powder. The suspected colony of Enterobacter sakazakii was detected by conventional physiological and biochemical methods, and then Enterobacter sakazakii was detected by the chimera pathway of fluorescent signal fluorescent dye:

1. Sample culture:

With embodiment 1.

2. DNA extraction

With embodiment 1.

3.PCR amplification

The composition ratio of each component in the PCR reaction system is as follows:

Composition Concentration Concentration Sample Amount

PCR system premix 2 times 12.5μL

Primer sequence 1 used for fluorescent chimeric method: 10μmol/L 0.5μL

Primer sequence 2 used for fluorescent chimeric method: 10μmol/L 0.5μL

DNA sample 1μL

Reporter plasmid 10 ³ /μL 1 μL

Double distilled water 9.5μL

Total volume 25μL

In the PCR method, the amplification program and the melting temperature program for determining the amplified fragment are:

(1) 95°C for 10 seconds

(2) 95°C for 5 seconds

(3) A certain temperature between 58°C and 65°C for 20 seconds

(4) Go back to step (2) and repeat 45 times.

(5) 95℃ for 2 minutes

(6) Gradient temperature increase from 55°C to 95°C, increasing by 1°C every 25 seconds.

A total of 7 tubes of PCR experiments were carried out, in which the DNA samples were: Enterobacter sakazakii standard strain ATCC29004 positive control; original concentration DNA sample; 10-fold diluted DNA sample; 10 ² -fold diluted DNA sample; 10 ³ -fold diluted DNA sample; 10 ⁴ -fold Diluted DNA sample; negative control with no sample for reporter plasmid only.

4. Observation of fluorescence PCR spectrum and melting curve of the tested sample

During the fluorescent PCR process, it can be observed that the positive control of the standard strain and samples of various concentrations produced obvious fluorescence, as shown in Figure 4.

Figure 4: Fluorescent dye chimeric fluorescent PCR detection results (fluorescent signal intensity) of a milk powder sample

The fluorescence intensity signals represented by each curve in the figure are:

1. Enterobacter sakazakii standard strain ATCC29004;

2. Original concentration DNA sample;

3. 10 times diluted DNA sample;

4.10 ² -fold diluted DNA sample;

5.10 ³ times diluted DNA sample;

6.10 ⁴ times diluted DNA samples;

Its melting temperature conforms to the characteristics of Enterobacter sakazakii, as shown in Figure 5.

Figure 5: The peak melting temperature of the DNA amplification product of a fluorescent dye chimeric fluorescent PCR test sample for a milk powder sample

The peak coincidence of multiple curves in the figure means:

1. Report the melting temperature of the amplified fragment of the plasmid

2. Sub-peak of melting temperature of Enterobacter sakazakii

3. Main peak of melting temperature of Enterobacter sakazakii

The reporter plasmid in the negative control also produced fluorescent results during the PCR process, as shown in Figure 6.

Figure 6: Fluorescent signal intensity of fluorescent dye chimeric fluorescent PCR reporter plasmid in a milk powder sample

The fluorescence intensity signal represented by the curve in the figure is:

1. Negative control with only reporter plasmid and no sample.

Its melting temperature conforms to the characteristics of the plasmid, as shown in Figure 7.

Figure 7: The peak melting temperature of the amplified product of fluorescent dye chimeric fluorescent PCR reporter plasmid in a milk powder sample

The melting temperature represented by the curve in the figure is:

1. Report the melting temperature of the amplified fragment of the plasmid

The above experiments showed that the samples contained Enterobacter sakazakii.

After 2 days, routine microbial culture and biochemical testing proved that 98% of the tested target colonies were Enterobacter sakazakii, and one of the strains was named gq105. Fluorescent dye chimeric PCR test results were consistent with biochemical test results.

Example 3

Sample: 5 strains of Enterobacteriaceae that are closely related to Enterobacter sakazakii were cultured, the strains used were Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii and Haffney hive Subbacteria.

1. DNA extraction

Take 1 mL of the enrichment solution and let it stand in an ice bath for 5 minutes, then centrifuge at 12,000 rpm for 5 minutes at room temperature, discard the supernatant, add 100 μL of lysozyme solution, incubate at 37°C for 10 minutes, and add 500 μL of TE buffer , shake to mix. Add the same volume of Tris-saturated phenol (pH 8.0), shake vigorously, centrifuge at 12,000 rpm for 3 minutes, take the supernatant, and repeat the phenol extraction. Take the supernatant, add 0.1 times the volume of sodium acetate (2mol/L), mix well, add an equal volume of ice ethanol, mix well, let stand at low temperature for 30 minutes, centrifuge at 12000 rpm for 5 minutes, discard the supernatant , washed once with 70% cold ethanol, centrifuged at 12,000 rpm for 5 minutes at room temperature, discarded the supernatant, added 50 μL TE solution, and stored at -20°C.

2. Fluorescent PCR amplification reports fluorescent signals through two ways

(1) The fluorescent signal is reported through the exonuclease probe approach, and the composition ratio of each component in the PCR reaction system is as follows:

Composition Concentration Concentration Sample Amount

PCR system premix 2 times 12.5μL

Primer sequence used in probe method 1 10μmol/L 0.5μL

Primer sequence used in probe method 2 10μmol/L 0.5μL

Probe sequence used in probe method 1 3 μmol/L 1 μL

Probe sequence used in probe method 2 3 μmol/L 1 μL

DNA sample 1μL

Reporter plasmid 10 ³ /μL 1 μL

Double distilled water 7.5μL

Total volume 25μL

PCR amplification program:

(1) 95°C for 10 seconds

(2) 95°C for 5 seconds

(3) 62°C for 20 seconds

(4) Go back to step (2) and repeat 45 times.

A total of 5 tubes of PCR experiments were carried out, in which the DNA samples were: Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii and Hafnia alveoli. The fluorescence PCR spectrum of the tested sample showed that no FAM fluorescence was produced, and the results are shown in FIG. 8 .

Figure 8: FAM fluorescence signal intensity of non-Enterobacter sakazakii DNA in exonuclease probe fluorescent PCR

The FAM fluorescence intensity signals represented by the curves in the figure are: five superimposed curves whose fluorescence signal is 0. However, the HEX fluorescence of the reporter plasmid was generated around 35 cycles, and the results are shown in Figure 9.

Figure 9: HEX fluorescence signal intensity of non-Enterobacter sakazakii DNA in exonuclease probe fluorescent PCR

The HEX fluorescence intensity signal represented by the curve in the figure is: the HEX fluorescence intensity signal produced by five nearly overlapping reporter plasmids, which proves that there is no Enterobacter sakazakii in the tested sample. Experiments have shown that the detection of exonuclease probe fluorescent PCR will not produce positive signals for Enterobacter sakazakii for these five bacteria.

(2) Fluorescent signal is reported by fluorescent dye chimeric PCR reaction, and the PCR reaction system is as follows:

Composition Concentration Sample Amount

PCR system premix 2 times 12.5μL

Primer sequence for fluorescent chimeric method 1 10μmol/L 0.5μL

Primer sequence for fluorescent chimeric method 2 10μmol/L 0.5μL

DNA sample 1μL

Reporter plasmid 10 ³ /μL 1 μL

Double distilled water 9.5μL

Total volume 25μL

Amplification procedure and the melting temperature procedure of measuring amplified fragment in the PCR method:

(1) 95°C for 10 seconds

(2) 95°C for 5 seconds

(3) 62°C for 20 seconds

(4) Go back to step (2) and repeat 45 times.

(5) 95℃ for 2 minutes

(6) Gradient temperature increase from 55°C to 95°C, increasing by 1°C every 25 seconds.

A total of 6 PCR experiments were carried out, in which the DNA samples were Escherichia coli; Escherichia coli O157H7; Enterobacter cloacae; Enterobacter aerogenes; Citrobacter freundii; Hafnia hives. Observation of fluorescent PCR patterns and melting curves of the tested samples showed that during the fluorescent PCR process, the reporter plasmids in each negative bacteria could be observed to produce HEX fluorescence during the PCR process. The results are shown in Figure 10.

Figure 10: Fluorescent signal intensity of non-Enterobacter sakazakii DNA in fluorescent dye chimeric PCR

The fluorescence intensity signal represented by the curve in the figure is: the superposition curve of the fluorescence signal produced by the reporter plasmid in each negative bacteria experiment. But the melting temperature conforms to the characteristics of the plasmid, as shown in Figure 11.

Figure 11: Melting temperature of non-Enterobacter sakazakii DNA amplified by reporter plasmid in fluorescent dye chimeric PCR

The curve in the figure represents the melting temperature of the amplified fragment of the reporter plasmid. Experiments showed that the positive signals of Enterobacter sakazakii would not be generated for these five bacteria by fluorescent dye chimeric PCR detection.

sequence list

SEQUENCE LISTING

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Claims (3)

1. The method for detecting Enterobacter sakazakii using fluorescent PCR technology is characterized in that the fluorescent signal sent in the detection fluorescent dye chimeric PCR reaction is detected, and its primer sequence and reporter plasmid amplification sequence are as follows: Fluorescent chimeric primer sequence 1: GGGTTGTCTGCGAATCGCTT Fluorescent chimeric primer sequence 2: GTCTTCGTGCTGCGTCAAAC Fluorescence chimeric reporter plasmid amplified sequence: TCCGTTTCTTCTTCGTCATAACTTAATGTTTTTTAT TTAAAATACCCTCTGAAAAGAAAGGA. 2. the method for utilizing fluorescent PCR technology to detect Enterobacter sakazakii according to claim 1, is characterized in that, in the PCR reaction system, each component constituent ratio is as follows: Composition Concentration Concentration Sample Amount PCR system premix 2 times 12.5μL Fluorescent Chimeric Primer Sequence 1: 10μmol/L 0.5μL Fluorescent Chimeric Primer Sequence 2: 10μmol/L 0.5μL DNA sample 1μL Reporter plasmid 10 ³ /μL 1 μL Double distilled water 9.5μL The total volume is 25 μL. 3. the method for utilizing fluorescent PCR technology to detect Enterobacter sakazakii according to claim 1, is characterized in that, in the PCR method, amplification program and the melting temperature program of measuring amplified fragment are: (1) 95°C for 10 seconds (2) 95°C for 5 seconds (3) A certain temperature between 58°C and 65°C for 20 seconds (4) Go back to step (2) and repeat 45 times. (5) 95°C 2 minutes (6) Gradient temperature increase from 55°C to 95°C, increasing by 1°C every 25 seconds.

Images