CN1900307B

CN1900307B - Process for preparing competitive template for quick quantitatively detecting heatstable bacterium QC-PCR

Info

Publication number: CN1900307B
Application number: CN2005100429785A
Authority: CN
Inventors: 李建科; 冯再平; 仇农学; 常玉华
Original assignee: Shaanxi Normal University
Current assignee: Shaanxi Normal University
Priority date: 2005-07-22
Filing date: 2005-07-22
Publication date: 2010-11-03
Anticipated expiration: 2025-07-22
Also published as: CN1900307A

Abstract

The process of preparing competitive template for quick quantitative QC-PCR detection of thermotolerant bacteria includes the following steps: capturing target bacteria and extracting DNA, designing competitive template primer, PCR amplification of competitive template, gel electrophoresis detection, recovering competitive template and detecting the concentration in the recovered competitive template. The present invention obtains a competitive template of 247 bp less than the target temperature of 298 bp by 51 bp in the middle through PCR of primer 1 and primer 3 in the 50muLPCR system and subsequent separation and purification with the DNA kit, and amplifies the competitive template and the target temperature simultaneously. The competitive template of the present invention may be used in the quick quantitative QC-PCR detection of thermotolerant bacteria.

Description

The QC-PCR fast quantification of thermoduric bacteria detects the preparation method of used competitive template

Technical field

The invention belongs to the food microorganisms detection range, the QC-PCR fast quantification that particularly relates to thermoduric bacteria detects the preparation method of used competitive template.

Background technology

The sour soil ring grease genus bacillus (Alicyclobacillusacidoterrestris) that contains in the apple condensed juice, it is a member of cyclic fatty acid bacillus (Alicyclobacillus), be commonly called as thermoduric bacteria, be a kind of thermophilic, have a liking for the bacterium of acid, its gemma can stand the pasteurization processes under the acidic conditions 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 the fruit juice corruption, completely destroy the consumption and the commodity value of fruit juice.So, very tight in the international trade to the thermoduric bacteria content requirement in the apple condensed juice, thermoduric bacteria is the essential items for inspection of apple condensed juice product, also usually becomes the technology barriers in the apple condensed juice trade, thereby has proposed new challenge for the production of apple condensed juice, trade and commodity inspection.

China's apple annual production occupies the first in the world, the apple condensed juice production development is rapid, turnout and export volume all rank first in the world, China's apple condensed juice working ability was for per hour handling 1500～2000 tons of fresh fruit amounts in 2002, product enters the world market more than 85%, about 300,000 tons of annual export volumes, export goods and earn foreign currency 1.5 hundred million dollars.Yet thermoduric bacteria content severe overweight usually in China's apple condensed juice because of the thermoduric bacteria economy and trade dispute that causes that exceeds standard happens occasionally, has had a strong impact on the foreign exchange earning of China's apple condensed juice, thereby has brought enormous economic loss for the apple industry in foreign trade.Having exceeded standard of thermoduric bacteria into a bottleneck of China's apple condensed juice quality safety.One of key that addresses this problem is that quick, special, sensitive detection technique means will be arranged, so that in time feed back to production line, implements effectively control, to guarantee quality product and safety.

The universal method that both at home and abroad thermoduric bacteria in the apple condensed juice is detected is that the bacterium plate is cultivated number scale at present, and sense cycle is 4-5 days, can not timely and effective control product safety quality.About the PCR detection method, the thermoduric bacteria among the RT-PCR detection AJC is arranged at present, and PCR in real time detects the thermoduric bacteria among the AJC.But the former can only be qualitative, can not be quantitative, furthermore because the leaching process of RNA requires harshness, reverse transcription makes the uncertain factor of detected result increase again, the latter requires also very high to plant and instrument except that complicated operating process.

Summary of the invention

QC-PCR (competition quantitative PCR) fast quantification that the objective of the invention is to disclose a kind of thermoduric bacteria detects the preparation method of used competitive template.

Operation steps of the present invention is as follows:

Capture object bacteria and carry out the competitive template concentration that the pcr amplification → detected through gel electrophoresis → recovery competitive template of extraction → competitive template design of primers → competitive template of DNA → mensuration reclaims.

Embodiment

The present invention is undertaken by following concrete steps:

A. capture object bacteria and carry out the extraction of DNA

B. competitive template design of primers

Adopt the principle that makes up competitive template, design the primer 1 and the primer 3 of amplification thermoduric bacteria competitive template:

Primer 1:5 '-ACGGGTAGGCATCTACTTGT-3 '; Primer 3:5 '-AGGAGCTTTCCACTCTCCTTGTGCGGCGTTGCTCCGTCAGGCTT-3 ';

C. the pcr amplification of competitive template

C.1 carry out pcr amplification with primer 1 and primer 3, obtain to lack outside the 51bp competitive template that other parts are identical (247bp) than in the middle of the To Template (298bp);

C.2 50 μ L pcr amplification systems are formed:

Taq archaeal dna polymerase 2U, Mg ²⁺Concentration 2.0mmol/L,

DNTP concentration 0.2mmol/L, primer concentration 0.2 μ mol/L,

The glycerine of interpolation 10% is done promotor, 10 * Buffer damping fluid, 5 μ L,

The thermoduric bacteria dna profiling 10 μ L that extract,

All the other volumes are supplied with aseptic tri-distilled water;

C.3 warm start and touchdown PCR

C.3.1 on ice chest with each composition mixing of PCR reaction system

C.3.2 when PCR circulation instrument temperature rises to more than 70 ℃, place the circulation instrument to increase in each amplification sample again

C.3.3 annealing temperature drops to 50 ℃ by 60 ℃;

C.4 cycling program

Enter PCR circulation behind 94 ℃ of sex change 4min, 94 ℃ of 30S, 60 ℃ drop to 50 ℃ of 30S, 72 ℃ of 30S, and 5min is supplied in the back 72 ℃ of extensions that circulate of increase 35;

D. detected through gel electrophoresis

D.1 get 10 μ L amplified productions and DNA Marker, add 2 μ L sample-loading buffers (glycerine-tetrabromophenol sulfonphthalein solution), fully go up sample behind the mixing

D.1.1 2.0% sepharose

D.1.2 80V voltage

D.1.3 time 30-40min;

D.2 the gel behind the electrophoresis is taken out from electrophoresis chamber, Ultraviolet Detector is observed electrophoresis result;

E. reclaim competitive template

Reclaim competitive template with the separation and purification of dna gel separation and purification test kit,

F. measure the competitive template concentration that reclaims

F.1 nucleic acid/protein analyzer is measured the competitive template concentration that reclaims;

F.2 calculate the competitive template dna content according to the competitive template molecular weight;

F.3 the competitive template with preparation is diluted to 5 * 10 ⁰～5 * 10 ⁸The competitive template series of individual copy;

F.4-4 ℃ refrigerator is preserved standby.

Result of use of the present invention: with 50 μ L PCR systems, use primer 1, primer 3, PCR obtains except lacking the 51bp than in the middle of the To Template (298bp), and the competitive template that other parts are identical (247bp) obtains required competitive template with the separable purifying of DNA separation and purification kit again. Through competitive template and To Template coamplification, when competitive template was close with To Template concentration, the two had close amplification efficiency, and both amplified productions can clearly separate in gel electrophoresis. The prepared competitive template of this law can be successfully used in QC-PCR to the Quantitative detection of thermoduric bacteria.

Claims

1. the QC-PCR fast quantification of thermoduric bacteria detects the preparation method of used competitive template, it is characterized in that carrying out according to the following steps:

Carry out the extraction of DNA 1.a capture object bacteria

1.b competitive template design of primers

1.c the pcr amplification of competitive template

1.c.1 carry out pcr amplification with primer 1 and primer 3, obtain to lack outside the 51bp than the To Template centre of long 298bp, the long 247bp that other parts are identical strives template unexpectedly;

1.c.250 μ L pcr amplification system is formed:

Taq archaeal dna polymerase 2U, Mg ²⁺Concentration 2.0mmol/L,

DNTP concentration 0.2mmol/L, primer concentration 0.2 μ mol/L,

The thermoduric bacteria dna profiling 10 μ L that extract,

All the other volumes are supplied with aseptic tri-distilled water;

1.c.3 warm start and touchdown PCR

1.c.3.1 on ice chest with each composition mixing of PCR reaction system

1.c.3.2 when PCR circulation instrument temperature rises to more than 70 ℃, place the circulation instrument to increase in each amplification sample again

1.c.3.3 annealing temperature drops to 50 ℃ by 60 ℃;

1.c.4 cycling program

1.d detected through gel electrophoresis

1.d.1 get 10 μ L amplified productions and DNA Marker, add 2 μ L sample-loading buffer glycerine-tetrabromophenol sulfonphthalein solution, fully go up sample behind the mixing

The sepharose of 1.d.1.12.0%

1.d.1.280V voltage

1.d.1.3 time 30-40min;

1.d.2 the gel behind the electrophoresis is taken out from electrophoresis chamber, and Ultraviolet Detector is observed electrophoresis result;

1.e recovery competitive template

1.f measure the competitive template concentration that reclaims

1.f.1 nucleic acid/protein analyzer is measured the competitive template concentration that reclaims;

1.f.2 calculate the competitive template dna content according to the competitive template molecular weight;

1.f.3 the competitive template of preparation is diluted to 5 * 10 ⁰～5 * 10 ⁸The competitive template series of individual copy;

1.f.4-4 it is standby that ℃ refrigerator is preserved.