CN1900708A - PCR quick detecting method for heat resistance bacteria - Google Patents

Abstract

Technical principle of the disclosed quick test method is that using PCR technique to amplify nucleic acid rapidly and specifically so as to amplify nucleic acid, which is infinitesimal in condensed juice, to millions times within hours; selecting suitable primer, PCR can amplify fragment of target DNA specifically to level of easy to be detected so as to reach purpose of detecting heat resisting bacterium quickly. Operation procedure is as following: concentrating objective bacterium (heat resisting bacterium), pre-amplifying bacterium, releasing nucleic acid from bacterium with wall broken, picking-up nucleic acid, and carrying out PCR amplification, gel electrophoresis test for amplified production, determining result. Comparing with traditional method, the disclosed method shortens detecting time form 4-5 days to 10 hours. The method meets requirement of production including export trade specified as 'heat resisting bacterium not be allowed'.

Description

The PCR method for quick of thermoduric bacteria

Technical field

The invention belongs to the detection method of food microorganisms, be specifically related to a kind of PCR method for quick thermoduric bacteria in the Apple juice concentrate.

Background technology

Sour soil ring grease bacillus (Alicyclobacillus acidoterrestris) in the Apple juice concentrate is a member in the cyclic fatty acid bacillus (Alicyclobacillus), and it is commonly called as and is thermoduric bacteria, be a kind of thermophilic, have a liking for acid bacterium.The gemma of thermoduric bacteria can stand the pasteurization processes under the acid condition and survive, under optimum conditions, when thermoduric bacteria is bred in fruit juice in a large number, do not produce gas, do not change the pH value of fruit juice, but can produce the special odor compound, also make fruit juice produce slight precipitation or vaporific muddiness sometimes, the fruit juice organoleptic quality is had a significant impact.Although thermoduric bacteria and not pathogenic does not produce toxin yet, the bad change of fruit juice sensory properties that it causes, even cause that fruit juice ruins, destroyed the consumption and the commodity value of fruit juice fully.So, very tight in the international trade to the thermoduric bacteria content requirement in the Apple juice concentrate, thermoduric bacteria is the essential items for inspection of Apple juice concentrate product, also usually becomes the technology barriers in the Apple juice concentrate trade, thereby has proposed new requirement for the production of Apple juice concentrate, trade and commodity inspection.For thermoduric bacteria is effectively controlled, at first require to detect thermoduric bacteria is rapid and precise, so that in time to the production line feedback information taking corresponding strick precaution, control and cleaning measure, improve in the Apple juice concentrate production run real-time monitoring capacity to thermoduric bacteria.The thermoduric bacteria detection method of continuing to use at present is conventional cultivation detection method, sense cycle is very long, generally needed 4～5 days just can go out examining report, the hysteresis quality of testing result makes it in time to instruct production, so Apple juice concentrate presses for a kind of detection method of thermoduric bacteria fast and accurately and kit in producing.

Summary of the invention

The objective of the invention is to overcome the long drawback of thermoduric bacteria traditional detection method sense cycle, and a kind of sensitivity to thermoduric bacteria in the Apple juice concentrate, easy, detection method accurately and rapidly are provided.

Know-why of the present invention is PCR (polymerase chain reaction) technology that is based upon on the molecular biology basis, it is the method for external quick specific amplified nucleic acid, the nucleic acid that it can make denier in the sample be expanded within a few hours original more than millions of times, as long as select suitable primer, PCR just can increase the target dna segment specifically to easy detection level, thereby realizes the fast detecting purpose to objective microbe.

Operation steps of the present invention is as follows:

Purpose bacterium (thermoduric bacteria) bacterium (the pre-amplification of the thermoduric bacteria) → thalline broken wall that concentrates → increase in advance discharges nucleic acid → extraction nucleic acid and carries out pcr amplification → amplified production detected through gel electrophoresis → result and judge.

Embodiment

Concrete operations step of the present invention is as follows:

Concentrating of a, purpose bacterium (thermoduric bacteria)

Get 10g Apple juice concentrate to be checked, after the dilution of sterilized water appropriateness, filter with syringe filter.The filter membrane diameter of syringe filter is 25mm, membrane aperture 0.22 μ m, because the diameter of bacterium is much larger than 0.22 μ m, so thermoduric bacteria and other all diameters all are retained in the filter membrane surface greater than the microorganism of 0.22 μ m;

B, increase bacterium (the pre-amplification of thermoduric bacteria) in advance

The little filter membrane that has filtered cider is gone in 402 nutrient solutions of 50mL, 45 ℃ of constant-temperature shaking culture 6h behind 70 ℃ of thermal treatment 10min,

70 ℃ of heat treated purposes are to make heat labile other assorted bacterium inactivation, and thermal treatment simultaneously can also impel the thermoduric bacteria gemma of dormancy to sprout, and the purpose of 45 ℃ of constant-temperature shaking culture is to make the thermoduric bacteria number obtain propagation, thereby reaches the limit that PCR detects,

402 nutrient solutions are good culture matrixes of thermoduric bacteria, and its constituent is:

CaCl ₂·2H ₂O： 0.25g MgSO ₄： 0.50g

(NH4) ₂SO ₄： 0.20g Yeast?extract： 2.00g

Glucose： 5.00g KH ₂PO ₄： 5.00g

Distilled water:1000mL 10%H ₂SO ₄Adjust pH to 4.0;

C, thalline broken wall discharge nucleic acid

With the centrifugal 10min of nutrient solution 12000rpm of shaken cultivation 6h in 402 nutrient solutions to collect thalline, the thalline of collecting is presented in the 1.5mL small test tube, add the 0.5mL aseptic double-distilled water, DNA is extracted in immersion method (100 ℃ boiling water bath 5 minutes) cracking, the centrifugal 5min of 8000rpm, the thermoduric bacteria genomic DNA promptly is present in the supernatant

Get above-mentioned clear liquid 10 μ L as template, carry out pcr amplification;

D, extraction nucleic acid carry out pcr amplification

50 μ L pcr amplification systems are formed: Taq enzyme 2U, Mg ²⁺Concentration 2.0mmol/L, dNTP concentration 0.2mmol/L, primer concentration 0.2 μ mol/L, 10 * Buffer damping fluid, 5 μ L, template 10 μ L, all the other volumes are supplied with aseptic tri-distilled water.

The primer structure is:

The left end primer: 5 '-ACGGGTAGGCATCTACTTGT-3 ';

The right-hand member primer: 5 '-AGGAGCTTTCCACTCTCCTTGT-3 ';

The pcr amplification program is: 58 ℃ of optimum annealing temperatures; Loop program is: enter the PCR circulation behind 94 ℃ of sex change 4min, and 94 ℃ of 30S, 58 ℃ of 30S, 72 ℃ of 30S, 5min is supplied in 35 the back 72 ℃ of extensions of circulation of increasing;

E, amplified production detected through gel electrophoresis

Get 10 μ L amplified productions, add 2 μ L sample-loading buffers (glycerine-bromophenol blue solution), fully behind the mixing, 1.7% agarose carries out gel electrophoresis, about 80V voltage electrophoresis 30min, the gel behind the electrophoresis is taken out from electrophoresis tank, Ultraviolet Detector is observed electrophoresis result;

F, result judge

The amplified production of 300bp occurs if having an appointment behind the pcr amplification, and then illustrating has thermoduric bacteria in the raw sample, and the result should be judged to be " the thermoduric bacteria positive ", otherwise should be judged to be " thermoduric bacteria feminine gender ".

When being used to detect, this method to do the pcr amplification of positive control and negative control simultaneously.The amplified production of 300bp occurs if positive control is had an appointment, negative control does not have the amplified production appearance simultaneously, just can carry out the result and judge; Otherwise the result who " false positive " or " false negative " occurred is described, should detects again.

Utilize the present invention to carry out PCR and detect thermoduric bacteria in the Apple juice concentrate, the lowest detection limit is 1 thermoduric bacteria in the 10ml sample, and its result is expressed as " having " (〉=1CFU/10mL, thermoduric bacteria positive) or " nothing " (＜1CFU/10mL, thermoduric bacteria feminine gender).Because the requirement to thermoduric bacteria in the Apple juice concentrate export trade is " must not detect ", so the testing result of this method satisfies requirements of actual production fully.The method detection time is 10 hours, cultivates 4～5 days sense cycle of detection method than tradition and shortens greatly, and detect when can realize various product.

Claims (2)

1. the PCR method for quick of thermoduric bacteria is characterized in that operation steps is as follows:

Purpose bacterium (thermoduric bacteria) bacterium (the pre-amplification of the thermoduric bacteria) → thalline broken wall that concentrates → increase in advance discharges nucleic acid → extraction nucleic acid and carries out pcr amplification → amplified production detected through gel electrophoresis → result and judge.

2. the PCR method for quick of thermoduric bacteria according to claim 1 is characterized in that being undertaken by following concrete steps:

Concentrating of purpose bacterium 2.a (thermoduric bacteria)

Get 10g Apple juice concentrate to be checked, after the dilution of sterilized water appropriateness, filter with syringe filter.The filter membrane diameter of syringe filter is 25mm, membrane aperture 0.22 μ m, because the diameter of bacterium is much larger than 0.22 μ m, so thermoduric bacteria and other all diameters all are retained in the filter membrane surface greater than the microorganism of 0.22 μ m;

2.b increase bacterium (the pre-amplification of thermoduric bacteria) in advance

The little filter membrane that has filtered cider is gone in 402 nutrient solutions of 50mL, 45 ℃ of constant-temperature shaking culture 6h behind 70 ℃ of thermal treatment 10min,

70 ℃ of heat treated purposes are to make heat labile other assorted bacterium inactivation, and thermal treatment simultaneously can also impel the thermoduric bacteria gemma of dormancy to sprout, and the purpose of 45 ℃ of constant-temperature shaking culture is to make the thermoduric bacteria number obtain propagation, thereby reaches the limit that PCR detects,

402 nutrient solutions are good culture matrixes of thermoduric bacteria, and its constituent is:

CaCl ₂·2H ₂O：?0.25g MgSO ₄： 0.50g

(NH4) ₂SO ₄： 0.20g Yeast?extract： 2.00g

Glucose： 5.00g KH ₂PO ₄： 5.00g

Distilled water:1000mL 10%H ₂SO ₄Adjust pH to 4.0;

2.c the thalline broken wall discharges nucleic acid

With the centrifugal 10min of nutrient solution 12000rpm of shaken cultivation 6h in 402 nutrient solutions to collect thalline, the thalline of collecting is presented in the 1.5mL small test tube, add the 0.5mL aseptic double-distilled water, DNA is extracted in immersion method (100 ℃ boiling water bath 5 minutes) cracking, the centrifugal 5min of 8000rpm, the thermoduric bacteria genomic DNA promptly is present in the supernatant

Get above-mentioned clear liquid 10 μ L as template, carry out pcr amplification;

2.d extraction nucleic acid carries out pcr amplification

50 μ L pcr amplification systems are formed: Taq enzyme 2U, Mg ²⁺Concentration 2.0mmol/L, dNTP concentration 0.2mmol/L, primer concentration 0.2 μ mol/L, 10 * Buffer damping fluid, 5 μ L, template 10 μ L, all the other volumes are supplied with aseptic tri-distilled water.

The primer structure is:

The left end primer: 5 '-ACGGGTAGGCATCTACTTGT-3 ';

The right-hand member primer: 5 '-AGGAGCTTTCCACTCTCCTTGT-3 ';

The pcr amplification program is: 58 ℃ of optimum annealing temperatures; Loop program is: enter the PCR circulation behind 94 ℃ of sex change 4min, and 94 ℃ of 30S, 58 ℃ of 30S, 72 ℃ of 30S, 5min is supplied in 35 the back 72 ℃ of extensions of circulation of increasing;

2.e amplified production detected through gel electrophoresis

Get 10 μ L amplified productions, add 2 μ L sample-loading buffers (glycerine-bromophenol blue solution), fully behind the mixing, 1.7% agarose carries out gel electrophoresis, about 80V voltage electrophoresis 30min, the gel behind the electrophoresis is taken out from electrophoresis tank, Ultraviolet Detector is observed electrophoresis result;

2.f the result judges

The amplified production of 300bp occurs if having an appointment behind the pcr amplification, and then illustrating has thermoduric bacteria in the raw sample, and the result should be judged to be " the thermoduric bacteria positive ", otherwise should be judged to be " thermoduric bacteria feminine gender ".