CN110747284A - Method for rapidly detecting content of escherichia coli based on fluorescent quantitative PCR technology - Google Patents
Method for rapidly detecting content of escherichia coli based on fluorescent quantitative PCR technology Download PDFInfo
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Abstract
A method for rapidly detecting the content of escherichia coli based on a fluorescent quantitative PCR technology. The invention takes Escherichia coli LacZ gene as a detection target gene, synthesizes a certain mass of standard target sequence with a plurality of nucleotides by a chemical synthesis method, quantifies the number of molecules contained in the target sequence with a certain mass, then dilutes the molecules to obtain LacZ gene series concentration standard products, takes the LacZ gene series concentration standard products and DNA of a sample to be detected as templates, utilizes a specific primer to carry out real-time fluorescence quantitative PCR to draw a standard curve, and judges the result according to the standard curve and the Ct value of the sample to be detected. Meanwhile, a group of specific primers is designed and synthesized according to the specific fragment sequence of the Escherichia coli LacZ gene, PCR reaction conditions are optimized, a detection method taking real-time fluorescence quantitative polymerase chain reaction as a platform is established, and the established method is evaluated.
Description
Technical Field
The invention belongs to the field of molecular biology and in-vitro diagnostic reagents, and particularly relates to a method for rapidly detecting escherichia coli content based on a fluorescent quantitative PCR technology.
Background
Coli is the predominant and most abundant bacterium in the intestine of humans and many animals, almost 1/3 on the dry weight of stool, and is one of the important food-borne pathogenic microorganisms responsible for human disease. Escherichia coli is discharged out of the body along with feces, and causes great pollution to the surrounding environment, water sources, food and the like. When the environment is monitored, the more escherichia coli in the sampled and checked sample indicates that the sample is more seriously polluted by the excrement, and the possibility that the enteropathogenic bacteria exist in the sample is higher. The sanitary standard specified in the sanitary Standard for Drinking Water GB5749-85 in China puts forward a definite requirement on the domestic Drinking water, and the total number of Escherichia coli in each liter of drinking water cannot exceed 3. The European and American drug administration and the national food and drug administration and administration shall not more than 10 ng/dose of residual amount of Escherichia coli DNA in biological products, and not more than 100 pg/dose of residual amount of DNA in Chinese hamster ovary cells (CHO cells). Therefore, how to rapidly and accurately detect the content of escherichia coli in an environment or a biological product still needs to be further researched and developed.
Disclosure of Invention
In order to rapidly and accurately detect the content of escherichia coli in an environment or a biological product, the invention provides a method for rapidly, accurately and highly sensitively detecting the content of escherichia coli in an environment sample, a biological product and the like, namely a method for rapidly detecting the content of escherichia coli based on a fluorescent quantitative PCR technology, which comprises the following specific steps:
(I) preparing LacZ gene series concentration standard substance
Using Escherichia coli β -galactosidase gene LacZ gene as detection target gene, synthesizing to obtain a certain mass of standard target sequence SEQ ID.1 with multiple nucleotides by chemical synthesis method, according to N ═ (M/M) NACalculating the number of molecules contained in a standard target sequence with certain mass, quantifying and diluting to obtain a LacZ gene series concentration standard substance; wherein N is the number of molecules contained in the standard target sequence, M is the mass of the standard target sequence, M is the molar mass of the standard target sequence, and NAIs 6.02 multiplied by 1023；
For example: the molar mass of the synthesized LacZ gene fragment was 30562.8g, and the mass was 32.6ug, according to the conversion formula: copy number (copies) of LacZ gene fragment (M/M) × 6.02 × 1023copies, from samples of the standard target LacZ gene fragment, were found to be: 6.42X 1014copies, diluted to 1.0X 10 with sterile deionized water8copies/ul, subpackaged, marked and stored at-20 ℃.
(II) drawing a standard curve
The real-time fluorescent quantitative PCR comprises the following components: 2 × SYBR Premix Ex TaqII Tli RNaseH Plus, upstream primer with final concentration of 5-20 μ M, downstream primer with final concentration of 5-20 μ M, LacZ gene series concentration standard prepared in the step (one) and ddH2O，ddH2O was used as a negative control;
and (5) taking the LacZ gene series concentration standard substance prepared in the step (I) as a template, and diluting the template by 10 times in a gradient manner. Wherein the series concentration standard substance comprises the following series concentrations: 1.0X 108copies/μl、1.0×107copies/μl、1.0×106copies/μl、 1.0×105copies/μl、1.0×104copies/μl、1.0×103Taking each dilution gradient template (0.2-1.5) to perform fluorescent quantitative PCR amplification by copies/mul, wherein the reaction system is 8-35 mul and is three multiple wells, and the amplification program is as follows: (1) (35-65) deg.C for 1.2-2.5 min; (2) (75-105) deg.C for 8-18 min; (3) (7-26) s at (85-106) DEG C; (4) (45-82) deg.C for 0.6-2.1 min; and (3) to (4) continuously performing 40 cycles, and establishing a standard curve for the LacZ fluorescent quantitative PCR detection by taking the logarithmic value of the concentration gradient dilution of the standard substance as the ordinate and the Ct value as the abscissa. The standard curve equation obtained by curve regression is: Y-4E +12E-0.715CtWherein Y is the template concentration, the unit copies/mu l, and the curve correlation coefficient R2 is 0.9968, which indicates that the standard curve has better linearity;
and (III) comparing the standard curve of the real-time fluorescent quantitative PCR detection obtained in the step (II) with the Ct value of the sample to be detected, evaluating and measuring the content of the escherichia coli.
As an improvement, the standard target sequence has 93 nucleotides, and the standard target sequence is shown as SEQ ID NO. 1; the sequence of the upstream primer is SEQ ID NO. 2: 5'-CGTACGGGGTATACATGTCTGA-3', downstream primer sequence SEQ ID NO. 3: 5'-GATTAGGGCCGCAAGAAAACT-3' are provided.
As an improvement, the standard target sequence has 114 nucleotides, and the standard target sequence is shown as SEQ ID NO. 4; the sequence of the upstream primer is SEQ ID NO. 5: 5'-GATACACTTGCTGATGCGGTG-3', downstream primer sequence SEQ ID NO. 6: 5'-CGCCATTTGACCACTACCATC-3' are provided.
As an improvement, the DNA template subjected to fluorescence quantitative PCR amplification in the step (II) is obtained by the following specific method: taking 0.25-1.75ml of bacteria culture solution, placing into a sterilized Eppendorf tube, and centrifuging at 1-6 deg.C and rotation speed (5000-; pouring out the supernatant, adding (100-300) mu l of buffer GA into the thallus precipitate, and oscillating until the thallus is completely suspended; adding (8-35) microliter protease K solution, and mixing; adding (100-350) microliter buffer GB, oscillating for 5-30 seconds, and standing at 40-100 ℃ for 10 min; adding 80-325 μ l of anhydrous ethanol, and shaking thoroughly for 5-30 s; adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3, placing the adsorption column CB3 in a collecting pipe, carrying out centrifugal treatment for 15-45 seconds at the rotating speed (8000-; adding (350-; adding (400-; then the adsorption column CB3 is put back into the collecting pipe, and the centrifugal treatment (0.5-3.5) min is carried out at the rotating speed (8000-; placing the adsorption column CB3 at room temperature for 1-30 minutes to thoroughly dry the residual rinsing liquid in the adsorption material; transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50-200 mu l of elution buffer TE into the middle part of the adsorption film, standing at room temperature for 2-5min, then carrying out centrifugation at the rotating speed (6000-.
Meanwhile, the primer for rapidly detecting the content of the escherichia coli by using the fluorescent quantitative PCR technology is also provided, and comprises an upstream primer and a downstream primer, wherein the sequence of the upstream primer and the sequence of the downstream primer are 5'-CGTACGGGGTATACATGTCTGA-3', 5'-GATTAGGGCCGCAAGAAAACT-3' independently, or are respectively and independently extended to the direction of 5 'end and/or 3' end on the basis of the sequences, or are obtained by deleting one to a plurality of bases.
Simultaneously, also provides a primer for rapidly detecting the content of the escherichia coli by using a fluorescent quantitative PCR technology, and is characterized in that: comprises an upstream primer and a downstream primer, wherein the upstream primer sequence and the downstream primer sequence are independently 5'-GATACACTTGCTGATGCGGTG-3', 5'-CGCCATTTGACCACTACCATC-3' or sequences obtained by respectively and independently extending one to a plurality of bases or deleting one to a plurality of bases in the direction of 5 'end and/or 3' end on the basis of the sequences.
β -D-galactosidase synthetic gene (LacZ gene) is widely existed in Escherichia coli, has high conservative property, becomes a common molecular target for rapidly detecting Escherichia coli, is used for biological products and in vitro diagnosis, on the basis, the invention takes Escherichia coli β -galactosidase gene (LacZ gene) as a target detection gene, carries out real-time fluorescence quantitative PCR detection on the environmental sample and the biological products so as to carry out Escherichia coli quantitative analysis, has simple and convenient operation, short time consumption and high sensitivity, simultaneously designs and synthesizes a group of specific primers according to the specific fragment sequence of the Escherichia coli LacZ gene, optimizes PCR reaction conditions, establishes a detection method taking real-time fluorescence quantitative polymerase chain reaction as a platform, and evaluates the established method.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.
Drawings
FIG. 1 is a standard curve of the fluorescent quantitative PCR assay of example 4 of the present invention.
Detailed Description
The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
Preparing a DNA template amplified by fluorescent quantitative PCR, wherein the specific method comprises the following steps: taking 0.25ml of the bacterial culture solution, putting the bacterial culture solution into a sterilized Eppendorf tube, and centrifuging for 1min at the temperature of 1 ℃ and the rotating speed of 5000 rpm; pouring out the supernatant, adding 100 mu l of buffer solution GA into the thallus precipitate, and oscillating until the thallus is completely suspended; adding 8 mul proteinase K solution, and mixing; adding 100 μ l buffer solution GB, shaking for 5-30 s, standing at 40 deg.C for 5-25 min; adding 80 mul of absolute ethyl alcohol, fully oscillating and uniformly mixing for 5-30 seconds; adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3, placing the adsorption column CB3 in a collecting pipe, carrying out centrifugal treatment for 15 seconds at the rotating speed of 8000rpm, pouring out waste liquid, and placing the adsorption column CB3 in the collecting pipe; adding 350 μ l buffer solution GD into adsorption column CB3, centrifuging at 8000rpm for 10 s, removing waste liquid, and placing adsorption column CB3 into a collection tube; adding 400 mul of rinsing liquid PW into the adsorption column CB3, before using the rinsing liquid PW, firstly checking whether absolute ethyl alcohol is added, then carrying out centrifugal treatment at the rotation speed of 8000rpm for 10 seconds, and pouring out waste liquid; then placing the adsorption column CB3 back into the collecting pipe, carrying out centrifugal treatment for 0.5min at the rotation speed of 8000rpm, and pouring out the waste liquid; placing the adsorption column CB3 at room temperature for 1 minute to thoroughly dry the residual rinsing liquid in the adsorption material; transferring the adsorption column CB3 into a clean centrifugal tube, suspending and dripping 50 mu l of elution buffer TE into the middle part of the adsorption film, standing at room temperature for 2min, then centrifuging at the rotating speed of 6000rpm for 1min, and collecting the solution into the centrifugal tube to obtain the genome DNA template.
Example 2
Preparing a DNA template amplified by fluorescent quantitative PCR, wherein the specific method comprises the following steps: taking 1ml of the bacterial culture solution, putting the bacterial culture solution into a sterilized Eppendorf tube, and centrifuging for 2min at the temperature of 4 ℃ and the rotation speed of 10000 rpm; pouring out the supernatant, adding 200 mu l of buffer solution GA into the thallus precipitate, and oscillating until the thallus is completely suspended; adding 20 mul proteinase K solution, and mixing; adding 220 μ l buffer GB, shaking for 15s, standing at 70 deg.C for 10 min; adding 220 mul of absolute ethyl alcohol, fully oscillating and uniformly mixing for 15 seconds; adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3, placing the adsorption column CB3 in a collecting pipe, carrying out centrifugal treatment for 30 seconds at the rotating speed of 12000rpm, pouring waste liquid, and placing the adsorption column CB3 in the collecting pipe; adding 500 μ l buffer solution GD into adsorption column CB3, centrifuging at 12000rpm for 10-45 s, pouring off waste liquid, and placing adsorption column CB3 into a collection tube; adding 600 μ l of rinsing liquid PW into adsorption column CB3, before using the rinsing liquid PW, firstly checking whether absolute ethyl alcohol is added, then carrying out centrifugal treatment for 30 seconds at the rotating speed (12000) rpm, and pouring out waste liquid; then placing the adsorption column CB3 back into the collecting pipe, carrying out centrifugal treatment for 2min at the rotating speed of 12000rpm, and pouring out the waste liquid; placing the adsorption column CB3 at room temperature for 1-30 minutes to thoroughly dry the residual rinsing liquid in the adsorption material; transferring the adsorption column CB3 into a clean centrifugal tube, suspending and dripping 50-200 mu l of elution buffer TE into the middle part of the adsorption membrane, standing at room temperature for 2-5min, centrifuging at the rotating speed of 12000rpm for 2min, and collecting the solution into the centrifugal tube to obtain the genome DNA template.
Example 3
Preparing a DNA template amplified by fluorescent quantitative PCR, wherein the specific method comprises the following steps: taking 1.75ml of the bacterial culture solution, putting into a sterilized Eppendorf tube, and centrifuging for 4min at the temperature of 6 ℃ and the rotation speed of 20000 rpm; pouring out the supernatant, adding 300 mu l of buffer solution GA into the thallus precipitate, and oscillating until the thallus is completely suspended; adding 35 mul protease K solution, and mixing; adding 350 μ l buffer solution GB, shaking for 30 s, standing at 100 deg.C for 25 min; adding 80 mul of absolute ethyl alcohol, fully oscillating and uniformly mixing for 30 seconds; adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3, placing the adsorption column CB3 in a collecting pipe, carrying out centrifugal treatment for 45 seconds at the rotating speed of 20,000) rpm, pouring the waste liquid, and placing the adsorption column CB3 in the collecting pipe; adding 750 ul of buffer GD into an adsorption column CB3, carrying out centrifugal treatment for 45 seconds at the rotating speed of 21,000rpm, pouring waste liquid, and putting the adsorption column CB3 into a collecting pipe; adding 800 mul of rinsing liquid PW into the adsorption column CB3, before using the rinsing liquid PW, firstly checking whether absolute ethyl alcohol is added, then carrying out centrifugal treatment for 50 seconds at the rotating speed of 23,000) rpm, and pouring out waste liquid; then placing the adsorption column CB3 back into the collecting pipe, carrying out centrifugal treatment for 3.4min at the rotating speed of 21,000) rpm, and pouring out waste liquid; placing the adsorption column CB3 at room temperature for 1-30 minutes to thoroughly dry the residual rinsing liquid in the adsorption material; transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 180 mu l of elution buffer TE into the middle part of the adsorption membrane, standing for 4min at room temperature, centrifuging for 3.5min at the rotating speed of 18,000rpm, and collecting the solution into the centrifuge tube to obtain the genome DNA template.
Example 4
Plotting real-time fluorescent quantitative PCR curve
The real-time fluorescent quantitative PCR comprises the following components: 2 × SYBR Premix Ex TaqII Tli RNaseH Plus, upstream primer with final concentration of 5 μ M, downstream primer with final concentration of 6 μ M, LacZ gene series concentration standard prepared in step (I) and ddH2O，ddH2O was used as a negative control;
taking the LacZ gene series concentration standard substance prepared in the step (I) as a template, and diluting the template by 10 times in a gradient manner, wherein the series concentration of the series concentration standard substance is 1.0 multiplied by 108copies/μl、1.0×107copies/μl、1.0×106copies/μl、 1.0×105copies/μl、1.0×104copies/μl、1.0×103Taking 0.4. mu.l of each dilution gradient template to carry out fluorescence quantitative PCR amplification, wherein the reaction system is 8. mu.l, and the reaction system is three multiple wells, and the amplification program is as follows: (1) 1.5min at 36 ℃; (2) 8min at 75 ℃; (3) 8s at 85 ℃; (4) 0.7min at 45 ℃; of these, 40 cycles of (3) to (4) were continued. And taking the logarithmic value of the concentration gradient dilution of the standard substance as the ordinate and the Ct value as the abscissa to establish a standard curve for the LacZ fluorescent quantitative PCR detection (see figure 1). The standard curve equation obtained by curve regression is: Y-4E +12E-0.715CtWherein Y is the template concentration, the unit is copies/mu l, and the curve correlation coefficient R2 is 0.9968, which shows that the standard curve has better linearity.
Example 5
Plotting real-time fluorescent quantitative PCR curve
The real-time fluorescent quantitative PCR comprises the following components: 2 × SYBR Premix Ex TaqII Tli RNaseH Plus, upstream primer with final concentration of 10 μ M, downstream primer with final concentration of 10 μ M, LacZ gene series concentration standard prepared in step (I) and ddH2O，ddH2O was used as a negative control;
taking the LacZ gene series concentration standard substance prepared in the step (I) as a template, and diluting the template by 10 times in a gradient manner, wherein the series concentration of the series concentration standard substance is 1.0 multiplied by 108copies/μl、1.0×107copies/μl、1.0×106copies/μl、 1.0×105copies/μl、1.0×104copies/μl、1.0×103Taking 1. mu.l of each dilution gradient template to carry out fluorescence quantitative PCR amplification, wherein the reaction system is 20. mu.l, and the reaction system is three multiple wells, and the amplification program is as follows: (1) 2min at 50 ℃; (2) 10min at 95 ℃; (3) 15s at 95 ℃; (4) 1min at 60 ℃; of these, 40 cycles of (3) to (4) were continued. And taking the logarithmic value of the concentration gradient dilution of the standard substance as the ordinate and the Ct value as the abscissa to establish a standard curve for the LacZ fluorescent quantitative PCR detection (see figure 1). The standard curve equation obtained by curve regression is: Y-4E +12E-0.715CtWherein Y is the template concentration, the unit is copies/mu l, and the curve correlation coefficient R2 is 0.9968, which shows that the standard curve has better linearity.
Example 6
Plotting real-time fluorescent quantitative PCR curve
The real-time fluorescent quantitative PCR comprises the following components: 2 × SYBR Premix Ex TaqII Tli RNaseH Plus, upstream primer with final concentration of 20 μ M, downstream primer with final concentration of 20 μ M, LacZ gene series concentration standard prepared in step (I) and ddH2O，ddH2O was used as a negative control;
using the LacZ gene series concentration standard substance prepared in the step (I) as a template, and diluting the LacZ gene series concentration standard substance by 10 times in a gradient manner (the series concentration of the series concentration standard substance is 1.0 multiplied by 10)8copies/μl、1.0×107copies/μl、1.0×106copies/μl、1.0×105copies/μl、1.0×104copies/μl、1.0×103copies/mul), taking 1.5 mul of each dilution gradient template to carry out fluorescent quantitative PCR amplification, wherein the reaction system is 30 mul, and each dilution gradient template is three multiple wells, and the amplification program is as follows: (1) 2min at 65 ℃; (2) 15min at 100 ℃; (3) 20s at 95 ℃; (4) 2min at 80 ℃; wherein, the (3) to (4) are continued for 40 cycles, the logarithmic value of the concentration gradient dilution of the standard substance is used as the ordinate, and the Ct value is used as the abscissa to establish the standard curve of the LacZ fluorescent quantitative PCR detection (see figure 1). The standard curve equation obtained by curve regression is: Y-4E +12E-0.715CtWherein Y is the template concentration, the unit is copies/mu l, and the curve correlation coefficient R2 is 0.9968, which shows that the standard curve has better linearity.
Example 7
Plotting real-time fluorescent quantitative PCR curve
The real-time fluorescent quantitative PCR comprises the following components: 2 × SYBR Premix Ex TaqII Tli RNaseH Plus, upstream primer with final concentration of 5-20 μ M, downstream primer with final concentration of 5-20 μ M, LacZ gene series concentration standard prepared in the step (one) and ddH2O，ddH2O was used as a negative control; taking the LacZ gene series concentration standard substance prepared in the step (I) as a template, and diluting the template by 10 times in a gradient manner, wherein the series concentration of the series concentration standard substance is 1.0 multiplied by 108copies/μl、1.0×107copies/μl、1.0×106copies/μl、1.0×105copies/μl、1.0×104copies/μl、 1.0×103Taking each dilution gradient template (0.2-1.5) to perform fluorescent quantitative PCR amplification by copies/mul, wherein the reaction system is 8-35 mul and is three multiple wells, and the amplification program is as follows: (1) (35-65) deg.C for 1.2-2.5 min; (2) (75-105) deg.C for 8-18 min; (3) (7-26) s at (85-106) DEG C; (4) (45-82) deg.C for 0.6-2.1 min; wherein, the (3) to (4) are continued for 40 cycles, the logarithmic value of the concentration gradient dilution of the standard substance is used as the ordinate, and the Ct value is used as the abscissa to establish the standard curve of the LacZ fluorescent quantitative PCR detection (see figure 1). The standard curve equation obtained by curve regression is: Y-4E +12E-0.715CtWherein Y is the template concentration, the unit is copies/mu l, and the curve correlation coefficient R2 is 0.9968, which shows that the standard curve has better linearity.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> university of Jiangsu
<120> method for rapidly detecting content of escherichia coli based on fluorescent quantitative PCR technology
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Claims (6)
1. The method for rapidly detecting the content of the escherichia coli based on the fluorescent quantitative PCR technology is characterized by comprising the following steps: the method comprises the following steps:
preparing a LacZ gene series concentration standard product:
using Escherichia coli β -galactosidase gene LacZ gene as detection target gene, synthesizing to obtain standard target sequence SEQ ID NO.1 with multiple nucleotides according to N ═ M/M) NACalculating the number of molecules contained in a standard target sequence with certain mass, quantifying and diluting to obtain a LacZ gene series concentration standard substance; wherein N is the number of copies contained in the standard target sequence, M is the mass of the standard target sequence, M is the molar mass of the standard target sequence, NAIs 6.02 multiplied by 1023；
(II) drawing a standard curve:
the real-time fluorescent quantitative PCR comprises the following components: 2 × SYBR Premix Ex TaqII Tli RNaseH Plus, upstream primer with final concentration of 5-20 μ M, downstream primer with final concentration of 5-20 μ M, LacZ gene series concentration standard prepared in the step (one) and ddH2O，ddH2O was used as a negative control; taking the LacZ gene series concentration standard product prepared in the step (I) as a template, diluting the template by 10 times in a gradient manner, taking each dilution gradient template (0.2-1.5) mu l for carrying out fluorescent quantitative PCR amplification, wherein the reaction system is 8-35 mu l, and each reaction system is three multiple wells, and the amplification program comprises the following steps: (1) (35-65) deg.C for 1.2-2.5 min; (2) (75-105) deg.C for 8-18 min; (3) (7-26) s at (85-106) DEG C; (4) (45-82) deg.C for 0.6-2.1 min; wherein, the (3) to (4) continuously carry out 40 cycles, and the logarithm value of the standard target sequence template concentration is used as an abscissa, and the Ct value is used as an ordinate to establish a standard curve of the LacZ fluorescent quantitative PCR detection;
(III) measuring the content of escherichia coli in the sample to be measured:
and (5) detecting the Ct value of the sample to be detected, comparing the standard curve of the real-time fluorescence quantitative PCR detection obtained in the step (II) with the Ct value of the sample to be detected, and calculating to obtain the content of the escherichia coli.
2. The method for rapidly detecting the content of the escherichia coli based on the fluorescent quantitative PCR technology as claimed in claim 1, wherein: the standard target sequence has 93 nucleotides, and is shown as SEQ ID NO. 1; the sequence of the upstream primer is SEQ ID NO. 2: 5'-CGTACGGGGTATACATGTCTGA-3', downstream primer sequence SEQ ID NO. 3: 5'-GATTAGGGCCGCAAGAAAACT-3' are provided.
3. The method for rapidly detecting the content of the escherichia coli based on the fluorescent quantitative PCR technology as claimed in claim 1, wherein: the standard target sequence has 114 nucleotides, and is shown in SEQ ID NO. 4: the sequence of the upstream primer is SEQ ID NO. 5: 5'-GATACACTTGCTGATGCGGTG-3', downstream primer sequence SEQ ID NO. 6: 5'-CGCCATTTGACCACTACCATC-3' are provided.
4. The method for rapidly detecting the content of the escherichia coli based on the fluorescent quantitative PCR technology as claimed in claim 1, wherein: the DNA template of the fluorescent quantitative PCR amplification in the step (II) is obtained by the following specific method: taking 0.25-1.75ml of bacteria culture solution, placing into a sterilized Eppendorf tube, and centrifuging at 1-6 deg.C and rotation speed (5000-; pouring out the supernatant, adding (100-300) mu l of buffer GA into the thallus precipitate, and oscillating until the thallus is completely suspended; adding (8-35) microliter protease K solution, and mixing; adding (100-350) μ l buffer solution GB, shaking for 5-30 seconds, standing at 40-100 deg.C for 5-25 min; adding 80-325 μ l of anhydrous ethanol, and shaking thoroughly for 5-30 s; adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3, placing the adsorption column CB3 in a collecting pipe, carrying out centrifugal treatment for 15-45 seconds at the rotating speed (8000-; adding (350-; adding (400-; then the adsorption column CB3 is put back into the collecting pipe, and the centrifugal treatment (0.5-3.5) min is carried out at the rotating speed (8000-; placing the adsorption column CB3 at room temperature for 1-30 minutes to thoroughly dry the residual rinsing liquid in the adsorption material; transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50-200 mu l of elution buffer TE into the middle part of the adsorption film, standing at room temperature for 2-5min, then carrying out centrifugation at the rotating speed (6000-.
5. The primer for rapidly detecting the content of the escherichia coli by using the fluorescent quantitative PCR technology is characterized in that: comprises an upstream primer and a downstream primer, wherein the upstream primer sequence and the downstream primer sequence are independently 5'-CGTACGGGGTATACATGTCTGA-3', 5'-GATTAGGGCCGCAAGAAAACT-3' or sequences obtained by respectively and independently extending one to a plurality of bases or deleting one to a plurality of bases in the direction of 5 'end and/or 3' end on the basis of the sequences.
6. The primer for rapidly detecting the content of the escherichia coli by using the fluorescent quantitative PCR technology is characterized in that: comprises an upstream primer and a downstream primer, wherein the upstream primer sequence and the downstream primer sequence are independently 5'-GATACACTTGCTGATGCGGTG-3', 5'-CGCCATTTGACCACTACCATC-3' or sequences obtained by respectively and independently extending one to a plurality of bases or deleting one to a plurality of bases in the direction of 5 'end and/or 3' end on the basis of the sequences.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112048454A (en) * | 2020-09-10 | 2020-12-08 | 兰州大学 | Microbial composition, kit and method for evaluating quality of slurry |
| CN113462786A (en) * | 2021-06-22 | 2021-10-01 | 江苏省菩德食品安全检测技术有限公司 | Method for identifying meat adulteration component based on DNA targeting gene fragment |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109055587A (en) * | 2018-09-21 | 2018-12-21 | 上海公安学院 | Detect kit, specific primer and probe and the application of E. CoIi content |
-
2019
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109055587A (en) * | 2018-09-21 | 2018-12-21 | 上海公安学院 | Detect kit, specific primer and probe and the application of E. CoIi content |
Non-Patent Citations (2)
| Title |
|---|
| MARIUS WEGENER: "The glpD gene is a novel reporter gene for E. coli that is superior to established reporter genes like lacZ and gusA", 《J MICROBIOL METHODS》 * |
| 安志远: "SD大鼠死后心血中大肠杆菌DNA 含量与早期死亡时间相关性的研究", 《中国司法鉴定》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112048454A (en) * | 2020-09-10 | 2020-12-08 | 兰州大学 | Microbial composition, kit and method for evaluating quality of slurry |
| CN112048454B (en) * | 2020-09-10 | 2023-01-03 | 兰州大学 | Microbial composition, kit and method for evaluating quality of serous fluid |
| CN113462786A (en) * | 2021-06-22 | 2021-10-01 | 江苏省菩德食品安全检测技术有限公司 | Method for identifying meat adulteration component based on DNA targeting gene fragment |
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