CN102382888B - Immunomagnetic bead -FQ-PCR detection method for Shewanella putrefaciens - Google Patents

Abstract

The invention discloses an immunomagnetic bead-FQ-PCR (Fluorescence Quantitive Polymerase Chain Reaction) detection method for Shewanella putrefaciens. The immunomagnetic bead-FQ-PCR detection method comprises the following steps: preparing an antibody solution through Shewanella putrefaciens; preparing immunomagnetic bead by using magnetic bead; enriching bacteria from a sample containing Shewanella putrefaciens; concentrating, purifying and then boiling and decomposing Shewanella putrefaciens to obtain a template DNA solution; mixing the template DNA solution in a reaction system containing TaqTM II mixing solution, FQR primer solution and FQF primer solution to carry out PCR; and judging that whether the sample solution to be detected contains Shewanella putrefaciens or not according to the response of a fluorescent signal in the PCR. The reaction is an FQ-PCR fluorescent reaction carried out on specific primers of Shewanella putrefaciens, the detection is convenient, simple and sensitive, the quantitive detection time is shorter, the detection efficiency is higher, and therefore, the immunomagnetic bead-FQ-PCR detection method for Shewanella putrefaciens of the invention is shorter in detection time and higher in detection efficiency and sensitivity.

Description

Immunomagnetic beads-FQ-PCR detection method of Shewanella putrefaciens

Technical field

The present invention relates to the detection technique of Shewanella putrefaciens, be specifically related to the immunity of Shewanella putrefaciens

Magnetic bead-FQ-PCR detection method.

Background technology

Shewanella putrefaciens ( shewanella putrefaciens, MacDonell and Colwell), once name pseudomonas putrefaciens, was genus Shewanella, belonged to Gram-negative, amphimicrobian, non-fermented type bacterium, it,, for the main spoilage organism of refrigeration fish, is also the dominant bacteria of ocean Lu Yuan sewage draining exit.Shewanella putrefaciens is distributed widely in food, mud, fresh water and seawater, it is the typical spoilage organism that hydrocoles is relevant with being rich in egg-white food, it is corrupt active strong, it can be reduced to Trimethylamine 99 (TMA) trimethylamine oxide (TMAO), produce volatile sulfur, when corrupt, occur Hydrogen Sulphide odour or acid smell.The dominant bacteria of the cultured large yellow croaker of 0 ℃, 5 ℃ refrigeration is Shewanella putrefaciens; Penaeus vannamei is preserved 4 d and is just produced Shewanella under ice temperature condition, and it also causes imitative stichopus japonicus " Beancurd sheet syndromes ".Quantitative fluorescent PCR (FQ-PCR) technology is that a fluorescence technique of utilizing of releasing in 1996 Nian You U.S. Applied Biosystems companies is carried out the emerging technology of absolute quantitation to nucleic acid.This technology is by add fluorescence dye in PCR reaction system, by dipole-dipole interaction between acceptor chromophoric group, energy is transferred to acceptor chromophoric group from donor chromophoric group, in whole PCR process, utilize the accumulation of fluorescent signal to carry out Real-Time Monitoring, finally, by typical curve, final realization carried out quantitative analysis to template.Compare with conventional PCR detection technique, it has, and specificity is stronger, accurate quantitative analysis, effectively solve PCR pollution problem, level of automation high.In numerous modern detecting, show one's talent, become a kind of important rapid detection means in Bacteria Detection field.If the patent No. is ZL200710031324, the patent of invention that name is called the test kit of fluorescence quantitative PCR detection of bacillus pyocyaneus discloses the test kit being comprised of reagent such as DNA extraction liquid, pcr amplification reaction liquid, positive and negative quality control products, wherein in pcr amplification reaction liquid, has hot resistant DNA polymerase, deoxyribonucleoside triphosphate, specific forward and reverse primer and probe; Can detect the special thing of blood, urine, sputum, pus, water, gas containing Pseudomonas aeruginosa.

Magnetic bead (Fe ₃o ₄) as absorption with in conjunction with the carrier of various molecules, form immunomagnetic beads with the antibodies that contains corresponding antigens, antibody antigen in sample on immunomagnetic beads is combined, pass through magnetic resolution, concentrated, the pure testing sample containing corresponding antigens, shorten detection time, improve detection efficiency and sensitivity.

Summary of the invention

Technical problem to be solved by this invention be to provide a kind of detection time lower short, detection efficiency and sensitivity

Immunomagnetic beads-FQ-PCR detection method of high Shewanella putrefaciens.

The present invention solves the problems of the technologies described above adopted technical scheme: immunomagnetic beads-FQ-PCR detection method of Shewanella putrefaciens, comprises the steps:

1, antibody preparation: be 10 by concentration ⁹the Shewanella putrefaciens liquid 0.20mL of cfu/mL is injected in first immunisation in mouse peritoneal, and once, booster immunization is three times altogether for the Shewanella putrefaciens liquid booster immunization of the one week same dose in interval, and the Shewanella putrefaciens liquid concentration of each booster immunization is 10 ¹⁰cfu/mL, after last booster immunization the 4th day, to extract eyeball of mouse and get blood, blood is placed in incubation 0.5h at 37 ℃, and the centrifugal 15min of 3000 r/min at 4 ℃, gets supernatant, obtains Shewanella putrefaciens antibody-solutions; This Shewanella putrefaciens antibody-solutions can utilize the bright indiscriminate method of coomassie to detect protein content;

2, immunomagnetic beads preparation: the Fe that gets 48mg ₃o ₄magnetic strain successively use ethanol and distilled water ultrasonic cleaning clean, with intermediate water, be settled to 20mL and obtain magnetic bead solution, in 10mL magnetic strain solution, adding mass concentration is 10% glutaraldehyde solution 2mL, under room temperature, stir 3h, after solid-liquid separation, with phosphoric acid tween damping fluid (PBST), clean magnetic strain, formulated by phosphoric acid buffer and polysorbas20 in described phosphoric acid tween damping fluid, described in described phosphoric acid tween damping fluid, the concentration expressed in percentage by volume of polysorbas20 is 0.03～0.07%, again the magnetic strain after cleaning is suspended in 2mL phosphoric acid buffer (PBS), the pH of described phosphoric acid buffer is 7～7.5, then add the above-mentioned Shewanella putrefaciens antibody-solutions of 50 μ L, at 4 ℃, stir gently 12h, obtain Shewanella putrefaciens immunomagnetic beads solution, 4 ℃ save backup,

3, testing sample enrichment: add 1mL analyte sample fluid in filling the above-mentioned Shewanella putrefaciens immunomagnetic beads solution of 0.1mL, the light and slow rotational oscillation 30min of room temperature, test tube is placed in to 3min on Magnetic cell sorting frame, shifts out liquid in pipe, with the above-mentioned phosphoric acid buffer of 1.0mL, clean Shewanella putrefaciens immunomagnetic beads at every turn, repeated washing 3～5 times, clean completely, add 0.5～1mL sterilized water, magnetic resolution, take out after Shewanella putrefaciens immunomagnetic beads, obtain the sample solution to be checked of enrichment;

4, template DNA: by above-mentioned sample solution to be checked, boil 10 min at 100 ℃ of temperature, the centrifugal 5min of 12000 r/min, gets supernatant, obtains template DNA solution;

5, PCR reaction: get above-mentioned template DNA solution 5 μ L, join by containing fluorescence dye taqtM II mixed solution (Dalian precious raw biological company limited) 10 μ L, primer concentration are that the FQF primer solution 0.5 μ L of 10 μ M is, in the reaction system of FQR primer solution 0.5 μ L that primer concentration is 10 μ M, with sterilized water sterilized water, be settled to 20 μ L, response procedures: 95 ℃ of denaturation 5min; 94 ℃ of sex change 5s, 50 ℃ of annealing 15s, 72 ℃ of extensions 15s, totally 45 circulations; If there is fluorescent signal response in PCR reaction, sample solution to be checked contains Shewanella putrefaciens; Described FQF primer solution contains the peculiar property primer that sequence is GTGAAACTCA TTTGGGTTGT AT, and described FQR primer solution contains the peculiar property primer that sequence is CCGTTCGGAA ATCGTTAG.

Above-mentioned FQF, FQR primer are synthesized by Shanghai Sheng Gong biotechnology company limited.

After PCR reaction finishes, also can carry out solubility curve analysis, then carry out the analysis of CT value with the software of Rotor-Gene, the concentration gradient of goal gene and standard example approaches situation per sample, carries out the content analysis of Shewanella putrefaciens in sample.

Compared with prior art, the invention has the advantages that immunomagnetic beads-FQ-PCR detection method of Shewanella putrefaciens, by Shewanella putrefaciens antibody-solutions, prepare, be prepared into immunomagnetic beads with magnetic bead again, can carry out Enrichment of bacteria to the sample that contains Shewanella putrefaciens, make Shewanella putrefaciens concentrated, pure, then boil to decompose and obtain template DNA solution, and by containing fluorescence dye taqin TM II mixed solution, FQR primer solution and FQF primer solution reaction system, carry out PCR reaction, according to the fluorescent signal in PCR reaction, have or not response, judge whether sample solution to be checked contains Shewanella putrefaciens, this reaction is the FQ-PCR fluorescent reaction that Shewanella putrefaciens Auele Specific Primer is carried out, detect easy, sensitive, the qualitative detection time is lower short, detection efficiency is high, so the present invention is a kind of detection time of lower short, detection efficiency and immunomagnetic beads-FQ-PCR detection method of the higher Shewanella putrefaciens of sensitivity.

Embodiment

Below in conjunction with embodiment, the present invention is described in further detail.

Embodiment

1, by concentration, be 10 ⁹the Shewanella putrefaciens liquid 0.20mL of cfu/mL is injected in first immunisation in mouse peritoneal, and once, booster immunization is three times altogether for the Shewanella putrefaciens liquid booster immunization of the one week same dose in interval, and the Shewanella putrefaciens liquid concentration of each booster immunization is 10 ¹⁰cfu/mL, after last booster immunization the 4th day, to extract eyeball of mouse and get blood, blood is placed in 37 ℃ of incubation 0.5h, and the centrifugal 15min of 3000 r/min at 4 ℃, obtains supernatant, is Shewanella putrefaciens antibody-solutions;

2, immunomagnetic beads preparation: get 48 mg Fe ₃o ₄magnetic strain successively use ethanol and distilled water ultrasonic cleaning clean, with intermediate water, be settled to 20mL and obtain magnetic bead solution, get 10mL magnetic strain solution, adding mass concentration is 10% glutaraldehyde solution 2mL, under room temperature, stir 3h, after solid-liquid separation, with PBST solution, (the PBS damping fluid and the polysorbas20 that by pH, are 7～7.5 are prepared, wherein the concentration expressed in percentage by volume of polysorbas20 is 0.03～0.07%) strain of cleaning magnetic, (pH is 7～7.5 again the magnetic strain after cleaning to be suspended in to 2mL PBS damping fluid, can autogamy, also can be commercially available) in, then add the above-mentioned Shewanella putrefaciens antibody-solutions of 50 μ L, at 4 ℃, stir gently 12h, obtain Shewanella putrefaciens immunomagnetic beads solution, 4 ℃ save backup,

3, testing sample enrichment: add 1mL analyte sample fluid in filling the above-mentioned Shewanella putrefaciens immunomagnetic beads solution of 0.1mL, the light and slow rotational oscillation 30min of room temperature, test tube is placed in to 3min on Magnetic cell sorting frame, shifts out liquid in pipe, use 1.0mL PBS buffer solution for cleaning immunomagnetic beads at every turn, repeated washing 3～5 times, clean completely, add 0.5～1mL sterilized water, magnetic resolution, take out after immunomagnetic beads, obtain the sample solution to be checked of enrichment;

4, template DNA: by above-mentioned sample solution to be checked, boil 10min at 100 ℃ of temperature, the centrifugal 5min of 12000 r/min, gets supernatant, obtains template DNA solution;

5, PCR reaction: get above-mentioned template DNA solution 5 μ L, join by containing fluorescence dye taqtM II mixed solution (SYBR Premix Ex taqtM II) 10 μ L, primer concentration are the FQF primer solution 0.5 μ L of 10 μ M, in the reaction system of FQR primer solution 0.5 μ L that primer concentration is 10 μ M, be settled to 20 μ L, response procedures: 95 ℃ of denaturation 5min with sterilized water sterilized water; 94 ℃ of sex change 5s, 50 ℃ of annealing 15s, 72 ℃ of extensions 15s, totally 45 circulations; If there is fluorescent signal response in PCR reaction, sample solution to be checked contains Shewanella putrefaciens.After PCR reaction finishes, also can carry out solubility curve analysis, then carry out the analysis of CT value with the software of Rotor-Gene, the concentration gradient that analyzes sample goal gene and standard example approaches situation.

Standard example

Standard solubility curve and CT value are set up: after the activation of Shewanella putrefaciens reference culture (ATCC) reference culture, at 100 ℃ of temperature, boil 10min, the centrifugal 5min of 12000 r/min, gets supernatant, obtain bacterium liquid template DNA, the bacterial concentration of DNA profiling is respectively: 5.2 * 10 ¹-5.2 * 10 ⁸cfu/μ L, does 10 doubling dilutions with sterilized water, obtains 5.4 * 10 ⁰-5.4 * 10 ⁷the template of 8 gradients of copies/μ L, and do negative control; According to following reaction system, carry out pcr amplification: bacterium liquid DNA profiling or negative control 5 μ L, with SYBR Premix Ex taqtM II 10 μ L, primer concentration are that the above-mentioned FQF primer solution 0.5 μ L of 10 μ M is, the reaction system of above-mentioned FQR primer solution 0.5 μ L that primer concentration is 10 μ M is carried out PCR reaction, with sterilized water sterilized water, be settled to 20 μ L, response procedures: 95 ℃ of denaturation 5min; 94 ℃ of sex change 5s, 50 ℃ of annealing 15s, 72 ℃ of extensions 15s, totally 45 circulations; After PCR reaction finishes, first carry out solubility curve analysis, with the software of Rotor-Gene, carry out the analysis of CT value again, software can analyze the house-keeping gene Ct(Comparative Delta-delta of DNA profiling) method is carried out relative quantitative assay, and the relative concentration of gene is in the ratio of control group; Its relation conefficient is 0.989, and slope is-3.32, and intercept is 40.29, thereby can show that the linear relationship expression formula between bacterial strain number and CT is: CT=-3.32 * log(bacterium number)+40.291.

<110> University Of Ningbo

Immunomagnetic beads-FQ-PCR detection method of <120> Shewanella putrefaciens

<160> 2

<170> PatentIn version 3.1

<210> 1

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> is according to the Auele Specific Primer of Shewanella putrefaciens gene design

<400> 1

GTGAAACTCA TTTGGGTTGT AT 22

<210>2

<211>18

<212> DNA

<213> artificial sequence

<220>

<223> is according to the Auele Specific Primer of Shewanella putrefaciens gene design

<400>2

CCGTTCGGAA ATCGTTAG 18

Claims (1)

1. immunomagnetic beads-FQ-PCR detection method of Shewanella putrefaciens, is characterized in that comprising the steps:

1), antibody preparation: be 10 by concentration ⁹the Shewanella putrefaciens liquid 0.20mL of cfu/mL is injected in first immunisation in mouse peritoneal, and once, booster immunization is three times altogether for the Shewanella putrefaciens liquid booster immunization of the one week same dose in interval, and the Shewanella putrefaciens liquid concentration of each booster immunization is 10 ¹⁰cfu/mL, after last booster immunization the 4th day, to extract eyeball of mouse and get blood, blood is placed in incubation 0.5h at 37 ℃, and the centrifugal 15min of 3000r/min at 4 ℃, gets supernatant, obtains Shewanella putrefaciens antibody-solutions;

2), immunomagnetic beads preparation: the Fe that gets 48mg ₃o ₄magnetic bead successively use ethanol and distilled water ultrasonic cleaning clean, with distilled water, be settled to 20mL and obtain magnetic bead solution, in 10mL magnetic bead solution, adding mass concentration is 10% glutaraldehyde solution 2mL, under room temperature, stir 3h, after solid-liquid separation, use phosphoric acid tween buffer solution for cleaning magnetic bead, formulated by phosphoric acid buffer and polysorbas20 in described phosphoric acid tween damping fluid, described in described phosphoric acid tween damping fluid, the concentration expressed in percentage by volume of polysorbas20 is 0.03～0.07%, again the magnetic bead after cleaning is suspended in 2mL phosphoric acid buffer, the pH of described phosphoric acid buffer is 7～7.5, then add the above-mentioned Shewanella putrefaciens antibody-solutions of 50 μ L, at 4 ℃, stir gently 12h, obtain Shewanella putrefaciens immunomagnetic beads solution, 4 ℃ save backup,

3), testing sample enrichment: add 1mL analyte sample fluid in filling the above-mentioned Shewanella putrefaciens immunomagnetic beads solution of 0.1mL, light and slow rotational oscillation 30min under room temperature, test tube is placed in to 3min on Magnetic cell sorting frame, shift out liquid in pipe, each with the above-mentioned phosphoric acid buffer cleaning of 1.0mL Shewanella putrefaciens immunomagnetic beads, repeated washing 3-5 time, clean complete, add 0.5～1mL sterilized water, magnetic resolution, take out after Shewanella putrefaciens immunomagnetic beads, obtain the sample solution to be checked of enrichment;

4), template DNA: by above-mentioned sample solution to be checked, boil 10min at 100 ℃ of temperature, the centrifugal 5min of 12000r/min, gets supernatant, obtains template DNA solution;

5), PCR reaction: get above-mentioned template DNA solution 5 μ L, join in the reaction system of the FQF primer solution 0.5 μ L that is 10 μ M by the TaqTM II mixed solution 10 μ L that contain fluorescence dye, primer concentration, FQR primer solution 0.5 μ L that primer concentration is 10 μ M, with sterilized water, be settled to 20 μ L, response procedures: 95 ℃ of denaturation 5min; 94 ℃ of sex change 5s, 50 ℃ of annealing 15s, 72 ℃ of extensions 15s, totally 45 circulations; If there is fluorescent signal response in PCR reaction, sample solution to be checked contains Shewanella putrefaciens; Described FQF primer solution contains the peculiar property primer that sequence is GTGAAACTCATTTGGGTTGT AT, and described FQR primer solution contains the peculiar property primer that sequence is CCGTTCGGAAATCGTTAG;

6) method of above-mentioned steps 1-5 is the non-method diagnosing the illness.