CN101613744A - A kind of soil Salmonellas fast quantitative measurement method for detecting that is used for - Google Patents
A kind of soil Salmonellas fast quantitative measurement method for detecting that is used for Download PDFInfo
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Abstract
A kind of soil Salmonellas fast quantitative measurement method for detecting that is used for, it may further comprise the steps successively: the gradient dilution of (1) pedotheque; (2) selective enrichment is cultivated; (3) DNA extraction; (4) pcr amplification and amplified production detect; (5) look into MPN table and calculate the method that the present invention has as a result adopted MPN, realized the needs of Salmonellas detection by quantitative in the environmental sample according to formula; Adopt the method that selective enrichment is cultivated and specific PCR detects, avoided other microorganisms interference, improved the sensitivity and the specificity of detection, compare with existing method, sensitivity can improve two more than the order of magnitude.In addition, the present invention has also shortened the time of detecting, has alleviated workload, and can carry out the detection of batch samples.
Description
One, technical field
The invention belongs to soil microorganisms detection range in the biotechnology, the invention discloses a kind of method that detects at Salmonellas (Salmonella) fast quantification in soil and the environment.
Two, background technology
Salmonellas is one of The main pathogenic fungi that causes the human gastrointestinal inflammation, making it to become the public health problem of The World Health Organization's extensive concern owing to the ecological characteristic of salmonella infecting both domestic animals and human and the food source property diarrhea disease percentage that caused thereof rise in recent years, is universally acknowledged important pathogen.U.S.'s moscow' saint paul in 2008 infects epidemic situation and breaks out, and Salmonella infection spreads to 23 states, and 228 people infect, and 25 people are in hospital, 1 people's death.It is that cayenne is subjected to due to the Salmonella infection that Food and Drug Administration of the United States Federal (FDA) confirms.The Salmonellas survival time in the soil is long, and relevant document points out that the Salmonellas survival time reaches three months in the soil.Therefore detect the Salmonellas in soil and the environment accurately and rapidly, for preventing that salmonella-polluted diffusion has crucial meaning.
Salmonellas detects and adopts health care and the used method of food inspection always in the soil, also promptly utilizes differential medium to identify in conjunction with Physiology and biochemistry.When but this method is applied to the pedotheque detection, on the one hand owing to the soil constitution complexity, contain a large amount of microorganisms and the sensitivity that detects produced disturb, there are shortcomings such as sense cycle is long, loss is high, program is complicated, required reagent is various on the other hand, waste time and energy, can not satisfy the requirement of a large amount of soil or environmental sample rapid detection.
Shortcomings such as detection method provided by the present invention can overcome sense cycle length, complex steps that traditional microorganism culturing detection method exists, waste time and energy, make only to shorten to detection time and need 2 days, operation steps and labour intensity also greatly reduce, reach save time, laborsaving, quick, highly sensitive requirement.The inventive method mainly is MPN method (the maximum most probable number (MPN) that soil microorganisms is detected, most probable number) and modern molecular biology technique polymerase chain reaction (Polymerase Chain Reaction) combine, make detected result quantitatively can have the higher detection susceptibility again, provide stable, a practical detection method for Salmonellas in soil and the environment detects.
Three, summary of the invention
Goal of the invention: the invention provides the detection method that a kind of energy fast quantification detects Salmonellas in soil and the environmental sample.
Technical scheme: a kind of soil Salmonellas fast quantitative measurement method for detecting that is used for is characterized in that it may further comprise the steps successively: the gradient dilution of (1) pedotheque; (2) selective enrichment is cultivated; (3) DNA extraction; (4) pcr amplification and amplified production detect; (5) look into MPN table and calculate the result according to formula; The PCR reaction system is reaction conditions (25ul) in the described step (4): 10 * PCR damping fluid 2.5ul, 25mmol/L Mg
2+1.5ul, 2.5mmol/L dNTP 2.0ul, each 1.0ul of 10.0umol/L primer, 5U/ul Taq archaeal dna polymerase 0.5ul, Salmonellas template 1.0ul to be measured, wherein Auele Specific Primer is:
Forward primer I ttrC-13; 5 '-[ACTGCCGATAAATGCACGTT]-3 '
Reverse primer II ttrB-1; 5 '-[CTTTTTTCCGCCAGTGAAGA]-3 '
。
The gradient dilution process of pedotheque comprises: take by weighing the adding of 5.00g soil sample and fill in the 250ml triangular flask of 45ml sterilized water, vibrated 10 minutes, soil sample is evenly dispersed in becomes soil supension in the diluent, thereby prepares serial gradient dilution liquid.The selective enrichment culturing process comprises: each extent of dilution is got the 900 μ L magnesium chloride Victoria Green WPB liquid nutrient mediums that 100 μ L suspensions join sterilization, and 18-24h is cultivated in 42 ± 1 ℃ of water-baths, and each extent of dilution is established three repetitions.
The leaching process of DNA may further comprise the steps: cultured bacteria suspension is transferred in the 1.5ml centrifuge tube of sterilization, the centrifugal 2min of 13000r/min, abandoning supernatant; Add the 1ml sterilized water again, cover the pipe lid completely, put upside down centrifuge tube repeatedly for several times, with the mixed content thing; The centrifugal 2min of 13000r/min, abandoning supernatant adds the 10ul ultrapure water, and boiling water boiled 7 minutes, and the centrifugal 2min of 13000r/min gets supernatant liquor then, finishes DNA extraction, uses 4 ℃ of preservations in the short period of time, and long-time the use needs-20 ℃ of preservations.
The pcr amplification process may further comprise the steps: 25ul PCR reaction system: 10 * PCR damping fluid 2.5ul, 25mmol/L Mg
2+1.5ul, 2.5mmol/L dNTP 2.0ul, each 1.0ul of 10.0umol/L primer, 5U/ul Taq archaeal dna polymerase 0.5ul.Amplification program is: 95 ℃ of pre-sex change 1min; 33 circulations (72 ℃ are extended 30sec for 95 ℃ of sex change 30sec, 60 ℃ of annealing 30sec); Mend flat 4min for 72 ℃.
Adopt three pipes or five pipe parallel methods behind the pedotheque gradient dilution, each extent of dilution is done three or five parallel samples.
Soil or environmental sample are diluted with sterilized water, carry out a times gradient dilution after vibrating into suspension, be inoculated in then in the selective enrichment nutrient solution magnesium chloride malachite green culture-medium, after 18-24h is cultivated in 42 ± 1 ℃ of water-baths, adopt the DNA in the pyrolysis method extraction enrichment liquid.With Salmonellas specific gene sequence is primer, is that template is carried out pcr amplification with the DNA that extracts, and amplification detects with 1.0% agarose electrophoresis, produces the positive that is designated as of specific amplification band, does not produce the feminine gender that is designated as of specific band.Then according to the MPN method of calculation, positive and the pairing extent of dilution of negative reaction, the content of Salmonellas in the calculation sample to occur.
Beneficial effect: the present invention has adopted the method for MPN, has realized the needs of Salmonellas detection by quantitative in the environmental sample; Adopt the method that selective enrichment is cultivated and specific PCR detects, avoided other microorganisms interference, improved the sensitivity and the specificity of detection, compare with existing method, sensitivity can improve two more than the order of magnitude.In addition, the present invention has also shortened the time of detecting, has alleviated workload, and can carry out the detection of batch samples.
Four, description of drawings
Salmonellas MPN-PCR testing process figure in Fig. 1 soil and the environmental sample
This flow process mainly comprises five links such as gradient dilution, selective enrichment cultivation, DNA extraction, pcr amplification and MPN calculating of sample.
Salmonellas MPN-PCR detected result example (pcr amplification result) in Fig. 2 pedotheque
Can judge the band approximation of serial dilution concentration according to the distribution of band from figure, and then calculate the quantity of Salmonellas in the soil.
Five, concrete embodiment
1, the gradient dilution of sample:
Take by weighing the adding of 5.00g soil sample and fill in the 250ml triangular flask of 45ml sterilized water, vibrated 10 minutes, soil sample is evenly dispersed in becomes soil supension in the diluent.After soil disperses, draw the 1ml soil supension and in the 9ml diluent, mix, be diluted to suitable extent of dilution by 10 times successively.Extent of dilution is decided according to concrete sample.
2, selective enrichment is cultivated: selective enrichment medium is a magnesium chloride Victoria Green WPB liquid nutrient medium (prescription is seen attached list).Each extent of dilution is got the 900 μ L magnesium chloride Victoria Green WPB liquid nutrient mediums that 100 μ L suspensions join sterilization, and 18-24h is cultivated in 42 ± 1 ℃ of water-baths, and each extent of dilution is established three repetitions.
3, DNA extraction:
Cultured bacteria suspension is transferred in the 1.5ml centrifuge tube of sterilization, the centrifugal 2min of 13000r/min, abandoning supernatant; Add the 1ml sterilized water again, cover the pipe lid completely, put upside down centrifuge tube repeatedly for several times, with the mixed content thing; The centrifugal 2min of 13000r/min, abandoning supernatant adds the 10ul ultrapure water, and boiling water boiled 7 minutes, and the centrifugal 2min of 13000r/min gets supernatant liquor then, finishes DNA extraction, uses 4 ℃ of preservations in the short period of time, and long-time the use needs-20 ℃ of preservations.
4, pcr amplification:
Salmonellas Auele Specific Primer: ttrC-13/ttrB-1
Primer I ttrC-13; 5 '-[ACTGCCGATAAATGCACGTT]-3 '
Primer I I ttrB-1; 5 '-[CTTTTTTCCGCCAGTGAAGA]-3 '
Reaction system (25ul):
Composition consumption (ul)
Ultrapure water dH
2O 15.5
10×PCR?buffer 2.5
25mmol/L Mg
2+ 1.5
2.5mmol/L?dNTP 2.0
10.0umol/L primer each 1.0
5U/ul Taq archaeal dna polymerase 0.5
Salmonellas template 1.0 to be measured
Cumulative volume 25.0
The pcr amplification condition:
95 ℃ of pre-sex change 1min; 33 circulations (72 ℃ are extended 30sec for 95 ℃ of sex change 30sec, 60 ℃ of annealing 30sec); Mend flat 4min for 72 ℃
Pcr amplification product is identified: get amplified production 4ul, point sample is in 1.0% agarose gel electrophoresis, with 1500bp Maker as the standard molecule reference, strength of electric field electrophoresis in 0.5 * TAE electrophoretic buffer with 5V/cm, use the 0.5ug/ml ethidium bromide staining, utilize the ultraviolet gel imaging system to take pictures.The results are shown in accompanying drawing 2.
5, MPN calculates:
The pcr amplification result detects with 1.0% agarose electrophoresis, will produce positive (being designated as+) of specific amplification band, negative (being designated as-) that does not produce specific band.Then according to the MPN method of calculation, positive and the pairing extent of dilution of negative reaction, the content of Salmonellas in the calculation sample to occur.
The MPN counting is according to having or not microorganism band to be measured quantitative index to occur drawing in each weaker concn in the dilution series gradient, finding the bacterium approximation according to presenting the male number in each number of iterations (present method adopts three pipes to repeat) in corresponding dilution method survey number cartogram again.When using the dilution method counting, all present feminine gender between all repetitions in must last weaker concn in concentration dilution series.All are got in the concentration dilution series by set amount index system, and to repeat all to present the high dilution of male be first figure place of quantitative index, and then determine the order of magnitude of bacterial concentration to be measured.
Concrete computation of table lookup method is: will present male pipe number by the ascending order of dilution gradient, and select back 3 serial dilution gradients (10
x, 10
X+1, 10
X+2), it is arranged in one three figure place, and (as X, X+1 X+2) is referred to as band approximation (abc).Wherein, first extent of dilution (promptly 10
x) be last extent of dilution that repeats all to be positive for three times, if the 3rd extent of dilution (10
X+2) more down gradient still be positive, the number that is positive in this extent of dilution can being repeated be added to the 3rd extent of dilution (10
X+2) on.Consult " dilution method is surveyed the number cartogram " then, draw corresponding numerical value, calculate the sum (referring to " soil microorganisms research method ", Science Press goes out 1985 editions publication) that 1ml detects Salmonellas in the pedotheque, calculation formula is as follows:
In the implementation case, select 10
7, 10
8, 10
9Three extent of dilution, band approximation (320) are consulted " dilution method is surveyed the number cartogram ", and drawing corresponding numerical value is 110.Thereby draw in the implementation case in the soil
In present case, we also utilize traditional HE cultural method to carry out the MPN counting.The result shows that the MPN-PCR detection method has improved about 100 times than MPN-HE culture method limit of detection, more can react the quantity of the Salmonellas of reality in the pedotheque.
The used PCR primer sequence of table 1 present method feature
Magnesium chloride malachite green culture-medium prescription:
Tryptones 5.0g/L, sodium-chlor 8.0g/L, potassium primary phosphate 1.6g/L, malachite green 0.036g/L faces the time spent, adds sterilization 40% magnesium chloride solution
The approximation of every milliliter of diluent bacterium of table 2
Sequence table
＜110〉Nanjing Soil Inst., Chinese Academy of Sciences
＜120〉a kind of soil Salmonellas fast quantitative measurement method for detecting that is used for
<130>
<160>2
<170>PatentIn?version?3.2
<210>1
<211>20
<212>DNA
＜213〉primer
<400>1
actgccgata?aatgcacgtt 20
<210>2
<211>20
<212>DNA
＜213〉primer
<400>2
cttttttccg?ccagtgaaga 20
Claims (6)
1, a kind of soil Salmonellas fast quantitative measurement method for detecting that is used for is characterized in that it may further comprise the steps: the gradient dilution of (1) pedotheque; (2) selective enrichment is cultivated; (3) DNA extraction; (4) pcr amplification and amplified production detect; (5) look into MPN table and calculate the result according to formula; The PCR reaction system is reaction conditions (25ul) in the described step (4): 10 * PCR damping fluid 2.5ul, 25mmol/LMg
2+1.5ul, 2.5mmol/L dNTP 2.0ul, each 1.0ul of 10.0umol/L primer, 5U/ul TaqDNA polysaccharase 0.5ul, Salmonellas template 1.0ul to be measured, wherein Auele Specific Primer is:
Forward primer I ttrC-13; 5 '-[ACTGCCGATAAATGCACGTT]-3 '
Reverse primer II ttrB-1; 5 '-[CTTTTTTCCGCCAGTGAAGA]-3 '.
2, a kind of soil Salmonellas fast quantitative measurement method for detecting that is used for according to claim 1, the gradient dilution process that it is characterized in that (1) pedotheque comprises: take by weighing the adding of 5.00g soil sample and fill in the 250ml triangular flask of 45ml sterilized water, vibrated 10 minutes, soil sample is evenly dispersed in becomes soil supension in the diluent, thereby prepares serial gradient dilution liquid.
3, a kind of soil Salmonellas fast quantitative measurement method for detecting that is used for according to claim 1, it is characterized in that (2) selective enrichment culturing process comprises: each extent of dilution is got the 900 μ L magnesium chloride Victoria Green WPB liquid nutrient mediums that 100 μ L suspensions join sterilization, 18-24h is cultivated in 42 ± 1 ℃ of water-baths, and each extent of dilution is established three repetitions.
4, a kind of soil Salmonellas fast quantitative measurement method for detecting that is used for according to claim 1, the leaching process that it is characterized in that (3) .DNA may further comprise the steps: cultured bacteria suspension is transferred in the 1.5ml centrifuge tube of sterilization, the centrifugal 2min of 13000r/min, abandoning supernatant; Add the 1ml sterilized water again, cover the pipe lid completely, put upside down centrifuge tube repeatedly for several times, with the mixed content thing; The centrifugal 2min of 13000r/min, abandoning supernatant adds the 10ul ultrapure water, and boiling water boiled 7 minutes, and the centrifugal 2min of 13000r/min gets supernatant liquor then, finishes DNA extraction, uses 4 ℃ of preservations in the short period of time, and long-time the use needs-20 ℃ of preservations.
5, a kind of soil Salmonellas fast quantitative measurement method for detecting that is used for according to claim 1 is characterized in that (4) pcr amplification process may further comprise the steps: 25ul PCR reaction system: 10 * PCR damping fluid 2.5ul, 25mmol/L Mg
2+1.5ul, 2.5mmol/L dNTP 2.0ul, each 1.0ul of 10.0umol/L primer, 5U/ul Taq archaeal dna polymerase 0.5ul.Amplification program is: 95 ℃ of pre-sex change 1min; 33 circulations (72 ℃ are extended 30sec for 95 ℃ of sex change 30sec, 60 ℃ of annealing 30sec); Mend flat 4min for 72 ℃.
6, a kind of soil Salmonellas fast quantitative measurement method for detecting that is used for according to claim 1 is characterized in that adopting three pipes or five pipe parallel methods behind the pedotheque gradient dilution, and each extent of dilution is done three or five parallel samples.
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| CN101993909A (en) * | 2010-12-01 | 2011-03-30 | 江南大学 | Quantitative detection method for salmonellas in municipal surplus sludge |
| CN102154472A (en) * | 2011-01-18 | 2011-08-17 | 西安建筑科技大学 | Quantitative detection method of salmonella living body in water |
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| CN106636437A (en) * | 2017-02-14 | 2017-05-10 | 江西省疾病预防控制中心 | Rapid quantitative method for salmonellas in food based on MPN (most probable number) and PCR (polymerase chain reaction) |
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| CN101993909A (en) * | 2010-12-01 | 2011-03-30 | 江南大学 | Quantitative detection method for salmonellas in municipal surplus sludge |
| CN101993954A (en) * | 2010-12-01 | 2011-03-30 | 江南大学 | Fast quantitative detection method of shigella contained in sludge |
| CN102154472B (en) * | 2011-01-18 | 2013-02-06 | 西安建筑科技大学 | Quantitative detection method of salmonella living body in water |
| CN102154472A (en) * | 2011-01-18 | 2011-08-17 | 西安建筑科技大学 | Quantitative detection method of salmonella living body in water |
| CN102732595A (en) * | 2011-04-12 | 2012-10-17 | 廖兴广 | Salmonella microscale MPN detection method |
| CN102337344A (en) * | 2011-11-04 | 2012-02-01 | 南京农业大学 | Quantitative detection method of escherichia coli in soil and assay kit thereof |
| CN102337344B (en) * | 2011-11-04 | 2013-08-21 | 南京农业大学 | Quantitative detection method of escherichia coli in soil and assay kit thereof |
| CN102864204A (en) * | 2012-09-07 | 2013-01-09 | 四川大学 | Enrichment culture solution for detection of Salmonella in poultry eggs |
| CN102864204B (en) * | 2012-09-07 | 2014-02-05 | 四川大学 | Enrichment culture solution for detection of Salmonella in poultry eggs |
| CN104789636A (en) * | 2015-05-12 | 2015-07-22 | 广西壮族自治区梧州食品药品检验所 | Quantitative dilution method for microorganism detection |
| CN104946754A (en) * | 2015-06-09 | 2015-09-30 | 南京农业大学 | Method and detection kit for quantitatively detecting ralstonia solanacearum in soil |
| CN106520969A (en) * | 2016-11-24 | 2017-03-22 | 江苏省农业科学院 | Quantitative detection method for pseudomonadaceae in soil on basis of standard soil sample |
| CN106636437A (en) * | 2017-02-14 | 2017-05-10 | 江西省疾病预防控制中心 | Rapid quantitative method for salmonellas in food based on MPN (most probable number) and PCR (polymerase chain reaction) |
| CN114457176A (en) * | 2022-03-10 | 2022-05-10 | 澜途集思(深圳)数字科技有限公司 | Ecological organism identification method based on MPN algorithm |
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