CN101029335A - PCR inspection of tubercle bacillus in dairy product - Google Patents

Abstract

A method for testing tuberculine composite bacterial population in dairy by real-time fluorescent PCR is carried out by adding BCG into emulsion as analog clinical testing samples, extracting DNA, real-time fluorescent PCR inspecting based on TaqMan technology, selecting ITS sequence as cloning sequence target sequence between tuberculine with code 16sRNA gene and 23sRNA gene. It's safe and has better sensitivity. It can be used to prevent human and domestic animal and diagnose cow tuberculosis.

Description

The PCR detection method of mycobacterium tuberculosis in the dairy products

Technical field:

The present invention relates to biological technical field.Be specifically related to the real-time fluorescence PCR detection method of mycobacterium tuberculosis complex in a kind of dairy products.

Background technology:

Tuberculosis is caused that by bacillus tuberculosis typus humanus, Mycobacterium bovis, mycobacterium africanum, mycobacterium microti these cause of diseases are referred to as mycobacterium tuberculosis mixture (or group).Up to the present, tuberculosis can also infect more than 50 kind of Mammals and more than 20 kind of bird except infected person.Ox is the most responsive animal, and prapes is mainly caused by Mycobacterium bovis.Ox is with human in close relations, and it is to be caused by Mycobacterium bovis that people's tuberculosis has more than 10%.Therefore developed country pays much attention to control lungy, and the U.S. is first basically eliminate country lungy in the world, and countries such as the Denmark in Europe, Belgium, Germany have also eliminated prapes substantially, and Britain and France are in state of a control.

The tuberculosis popular gesture that is rapid rise in the world has 1,000 ten thousand New Development patients every year approximately since the eighties in 20th century, and 300 die ten thousand deaths dies, and is one of case fatality rate soprano in the infectious diseases due to single factor.China is the country that second largest in the world tuberculosis is in a bad way.According to the data that the Ministry of Health announces, China has pulmonary tuberculosis patient about 4,500,000 at present, and wherein the infectivity pulmonary tuberculosis patient is about 1,500,000, and tuberculosis patient quantity occupies the second place of the world.There are 1,450,000 new cases in China every year approximately, is one of the high burden of 22 tuberculosis in whole world country, and is annual because of the tuberculosis death toll reaches 130,000, substantially exceeds the summation of other transmissible disease death tolls.Infected tubercule bacillus and do not send out the patient, more nearly 5.5 hundred million people account for 45% of national population.The relevant expert estimates at 10 years from now on, if do not take appropriate measures, estimates that the whole nation will increase tuberculosis patient 2,000 ten thousand to 3,000 ten thousand people newly.This is healthy with the serious threat broad masses of the people's, brings heavy burden for country and society, seriously restricts the development of China's economic and society.Tuberculosis is an ancient infecting both domestic animals and human disease, but its more popular new characteristics are given in the development of society.Along with the proportion of milk in resident's normal diet strengthens gradually, people's incidence of tuberculosis is also rising, and social epidemiology studies show that the two is tangible epidemiology dependency.

Therefore, strengthening food safety is to strengthen primary work in the public health prevention system, but Chinese for a long time quality of dairy products standards system is unsound.The standard of existing former milk is very low, and Chinese at present one-level milk standard all is judged to be at majority state can not be as the raw material of liquid state milk, and tubercule bacillus detects in the dairy products does not still have effective means.

The gene diagnosis technology is compared with other diagnostic methods has following outstanding advantage: 1, can thoroughly disclose disease pathogen 2 from gene level, can significantly shorten the time 3 of diagnosis, need not tissue, organ or cell are selected 4, quick and precisely, safe and reliable, therefore be a widely used mature technology in the clinical diagnosis field, but do not used as yet in dairy products food safety field.

Since SARS, the outburst of bird flu epidemic situation big area, the PCR detection technique becomes or is becoming one of main detection technique platform in disease prevention and control centers at different levels rapidly.The early diagnosis of bovine tuberculosis has important public health meaning, and it can be strengthened food safety, reduce human tuberculosis's new cases quantity to greatest extent, improves economic results in society.

Bovine tuberculosis can adopt the diverse ways diagnosis according to the particular case of cows.At present, sick cows are based on clinical and laboratory diagnosis, and suspicious cows and healthy cows verify as the master with tuberculin, the fit applications laboratory diagnosis.Be used at present the detection method of diagnosing ox tuberculosis both at home and abroad, can be divided into three major types substantially.The first kind is the bacteriology checking method, as smear for microscopic examination, microbial culture etc.The incubation time that tubercule bacillus needs is long, and sensitivity is low, can not satisfy the needs of modern clinical diagnosis.Second class is a molecular biology method, and as PCR, based on methods such as the real-time fluorescence PCR of TaqMan technology, DNA collection of illustrative plates, these class methods are highly sensitive, and specificity is good, but present conventional PCR method generally can only detect the pcr amplification product of 100mg.(1998) such as Fabrizio Vitale have set up the PCR detection technique of cattle on the hoof tuberculosis diagnosis, detect the negative and intradermoreaction male animal of intradermoreaction.The nest-type PRC amplification obtains the target sequence of the 182bp in the IS6110 insertion sequence, this fragment cloning and order-checking, and mark is used for the dot hybridization analysis later on.From the different specimens source (comprising lymphoglandula aspirate, breast and nose swab) of 100 cattles on the hoof, extract mycobacterium tuberculosis complex DNA and PCR detection; Check later on from 60 scalp internal reaction male animal slaughterings, and from lymphoglandula, lung and breast tissue sample, extract DNA and make PCR detection, detected result total positives.The tissue sample that detects slaughtered animals with PCR is as standard, and PCR detection sensitivity, specificity, positive predictive value and the negative predictive value to emulsion, lymphoglandula sample of calculating all are 100%, and the nose swab sample is respectively 58,100,, 00, and 28%.All PCR detection sensitivity, specificity, positive predictive value and negative predictive values that detect sample are respectively 74,100,100, and35%.PCR check and analysis to lymphoglandula aspirate, breast and the nose swab of the negative animal of intradermoreaction show that 52% intradermoreaction negative findings is a false negative.

(2000) such as Srinand Sreevatsan have developed multiplex PCR augmentation detection platform, detect Mycobacterium bovis and Bacillus abortus in cow's milk and the nasal discharge sample simultaneously.This system comprises the specific target sequence of dual amplification kind (the tumor-necrosis factor glycoproteins district of Bacillus abortus BCSP31K gene regions and Mycobacterium bovis hsp65 gene), catches the method detection of hybridization amplified production then by the solid phase probe.In the test of the normal newborn sample of stab inoculation, detection sensitivity is 800CFU equivalent/ml to Bacillus abortus, is 4CFU equivalent/ml to Mycobacterium bovis.

Malcolm James Taylor etc. calendar year 2001 reported first use the lightcycler real-time fluorescence PCR to detect the technology of Mycobacterium bovis in the tissue.Detected 38 sick oxen that the tuberculosis sign is arranged, the method for extracting that adopts nucleotide sequence to catch extracts Mycobacterium bovis DNA from the lymphoglandula sample, and product detects and uses real-time fluorescence PCR and conventional PCR, result's contrast of result and cultivation and histopathological analysis.Conventional PCR method was finished in about 9 hours, cultivated in the sick ox of male at 28 and can detect 26 (recall rates 93%), and the method for real-time fluorescence PCR is finished in 30min, can detect 20 (recall rates 71%) in 28.The specificity that real-time fluorescence PCR detects the compound group of mycobacterium guarantees by using SYBR Green 1 and the special Cy5 mark fluorescent resonance energy of mycobacterium tuberculosis complex to shift probe.

(2002) reported first such as Janin Stratmann the application of phage display technology in microorganism diagnosis.The immunomagnetic beads of phage display technology and polypeptide media separated combining, developed the technology that selective separation mycobacterium paratuberculosis the newborn samples of a kind of milk cows from the natural infection johne's disease belongs to the mycobacterium avium kind.From the commercialization phage polypeptide library of the 12-mer polypeptide at random of encoding, isolate the recombinant phage that 9 specificitys belong in conjunction with mycobacterium paratuberculosis.From these 9 kinds of phages, filter out again mycobacterium paratuberculosis is belonged to the phage Fmp3 that binding ability is strong, specificity is high, the order-checking of Fmp3 nucleosides, the peptide sequence of being derived by nucleotide sequences is NYVIHDVPRHPA, chemically synthesized polypeptide, its aminoterminal connects biotin residue by 6 carbon amino acid moleculars, and this peptide sequence is designated as AMP3.Can catch the mycobacterium paratuberculosis molecule after immunomagnetic beads surface coverage FMP3 or the AMP3 from emulsion, be that the PCR result of amplified target sequence shows that this method detection sensitivity in the artificial clinical newborn sample of acupuncture mimic can reach 10 bacterium/ml with insertion sequence ISMav2.

The 3rd class is an immunological method, as methods such as intracutaneous transformation reactions, ELISA.Up to the present, the diagnosing bovine tuberculosis method of OIE (OIE) recommendation has only tuberculin (PPD) transformation reactions.But there are a lot of drawbacks in this method, except that the factor of physics and chemistry, more is the interference of atypia mycobacterium, often causes the generation of nonspecific reaction.Though this is that it is actually the mixture that includes multiple antigenic component because PPD is called purified protein derivative, can not differentiate that TB infects, the DTH that mycobacterium avium sensitization and BCG immunity produce replys.

Summary of the invention:

Technical problem to be solved by this invention is to overcome above-mentioned weak point, provides a kind of sensitivity higher, is convenient to the method for mycobacterium tuberculosis in the early detection dairy products.

The invention provides the real-time fluorescence PCR detection method of mycobacterium tuberculosis complex in a kind of dairy products.The foundation of the inventive method detects based on the real-time fluorescence PCR of TaqMan technology, the target sequence that detects is peculiar by causing humans and animals mycobacterium tuberculosis complex lungy (comprising four kinds of mycobacterium tuberculosis), Auele Specific Primer and specific probe to comprise the soil, do not produce look, more than 20 kind of non-virulent mycobacterium of Amur, mycobacterium triviale and comprise in fowl type, the born of the same parents, the pathogenic mycobacterium of kind more than ten of toad, Kansas do not have S type real-time fluorescence PCR amplification curve, its amplification curve is a sea line.Real-time fluorescence (real-time) PCR based on the TaqMan technology detects, key is to have increased a specific fluorescence double-tagging probe on a pair of Auele Specific Primer basis of regular-PCR, this probe combines with the pathogen nucleic acid specificity, and combining site is positioned at the centre in PBR territory equally.Because fluorescent signal detection system that fluorescent PCR itself is advanced and powerful information processing capability, the more conventional PCR electrophoretic detection of detection sensitivity has breakthrough raising, conventional PCR method generally can detect the pcr amplification product of 100ng, and fluorescent PCR can detect the pcr amplification product of 0.1ng; And the standard substance of fluorescent PCR by concentration known are as the template of pcr amplification reaction, and standard of comparison product and the Ct value that detects sample can be realized quantitative to pathogen nucleic acid.

The real-time fluorescence PCR detection method of mycobacterium tuberculosis complex is as follows through the overtesting detected result in the dairy products of the present invention:

1, material and method

1.1 bacterial strain

The lyophilized powder bacille Calmette-Guerin vaccine is provided by Shanghai Disease Prevention and Control Centre,-20 ℃ of preservations, stipulate by national Freeze Dried Bacillus Calmette-Guerin Vaccine manufacturing and vertification regulation: freeze-drying vaccine bacterium number should be more than 1,000,000/mg, bacille Calmette-Guerin vaccine per ampoule bottle 5 person-portions that the present invention uses, contain bacill calmette-guerin 0.2-0.3mg, the bacterium number should be more than 200,000.Bacille Calmette-Guerin vaccine adds the dissolving of 1ml distilled water, dissolves later bacterium liquid and does 1: 10,1: 100,1: 1000,1: 10000,1: 100000 dilution.

1.2 simulate clinical newborn sample

Adopt the dilution bacterium liquid of 50ul gradient concentration to sneak in the 450ul emulsion for detecting sample, emulsion is used commercially available ultra high temperature sterilization (UHTS) pure milk, each detects sample: solution A: the bcg bacteria liquid that comprises the dilution in 1: 10 of 50ul in the 500ul emulsion, it is several more than 1000 that 50ul bacterium liquid contains bacterium, 1000 of final concentrations are more than bacterium/0.5ml, solution B: 500ul emulsion, 100 of bacille Calmette-Guerin vaccine final concentrations are more than bacterium/0.5ml, solution C: 500ul emulsion, 10 of bacille Calmette-Guerin vaccine final concentrations are more than bacterium/0.5ml, solution D: the 500ul emulsion, 1 of bacille Calmette-Guerin vaccine final concentration is more than bacterium/0.5ml.

1.3 primer, probe design

According to ITS (internaltranscribed spacer) sequence between mycobacterium coding 16sRNA gene and the 23sRNA gene among the GenBank, use OMIGA software do sequence relatively after, use OLIGO6.0, PRIMER5.0, a pair of Auele Specific Primer of BEACON software design and specific probe, pcr amplification product is peculiar by mycobacterium tuberculosis complex.Primer, probe are given birth to worker company by Shanghai and are synthesized.

1.4 bacterial strain DNA extraction

For the bacille Calmette-Guerin vaccine of different gradient concentrations dilution bacterium liquid, 50ul dilution bacterium liquid directly adds mycobacterium tuberculosis (TB) the DNA extraction liquid that the inventor produces, 100 ℃ 15 minutes, 13000rpm 2 minutes can be used for pcr amplification.Because dilution bacterium liquid composition is simple,, can reflect quite truly to contain bacterium quantity in the dilution bacterium liquid directly with TB extracting solution cracking TB cell and results.

To the DNA extraction of bacille Calmette-Guerin vaccine in the milk (Mycobacterium bovis), the solid phase column method of selecting for use the inventor to develop is extracted reagent, and its structure includes the Silica material and is fixed on the interior preparation pipe at the pipe end and is enclosed within the interior outer centrifuge tube for preparing on the pipe.Because cow's milk comprise multiple complicated ingredients such as butterfat, milk-protein, common extraction reagent is unsuitable for the processing of this complex samples.Cardinal principle based on the solid phase extractions technology of Silica material is that sample cracking in high exsolution liquid discharges nucleic acid, be attached to then on the Silica material, remove cell debris, albumen or protoheme impurity such as (in the clinical samples) through cleaning, under low ionic strength nucleic acid dissociates from the Silica material and is discharged into the elutriant then, and the concrete operations step is:

(1) in 1980ul LB lysis buffer, adds 20 μ l Poly (A) poly VITAMIN B4, use behind the mixing.

(2) in the 1.5ml centrifuge tube that 500 μ l samples are housed, add the LB that 500 μ l are mixed with Poly (A) in advance, vibration mixing, 72 ℃ of insulation cracking 25min.

(3) add 200 μ l Virahols in the solution after above-mentioned insulation, shift 600ul solution in pillar preparation/centrifuge tube behind the vibration mixing, 10, abandon filtrate behind the centrifugal 1min of 000rpm.

(4) remaining 600ul solution is transferred in the pillar preparation/centrifuge tube, 10, abandon filtrate behind the centrifugal 1min of 000rpm.

(5) in the preparation pipe, add WB elution buffer 500 μ l, 10, pipe abandon bottom centrifugal filtrate behind the centrifugal 1min of 000rpm.

The centrifugal 1min of (6) 13,000rpm removes remaining WB elution buffer with thorough, will prepare pipe then and be placed in the new clean centrifuge tube, adds to be preheating to 72 ℃ EB 50 μ l in advance, room temperature placement 2min.

Centrifugal filtrate at the bottom of the centrifugal 1min collection tube of (7) 10,000rpm is isolating nucleic acid elute soln from sample, then is kept at-70 ℃ of refrigerators if DNA need be kept at-20 ℃, RNA.

1.5 real-time fluorescence PCR reaction

In the reaction system of 50ul, add 7ul extraction product and make template, add the real-time fluorescence PCR reaction buffer 30ul that is mixed with primer, add MgCl ₂5ul adds fluorescent probe 5ul, adds the Taq enzyme 3ul that is mixed with the UNG enzyme, and the UNG enzyme can effectively be eliminated the influence of pcr amplification product pollution to experiment.Reaction conditions at the icycler of BioRad company fluorescent PCR amplification instrument is: 50 ℃ of 2min; 94 ℃ of 5min; 93 ℃ of 30sec, 60 ℃ of 1min, 40 circulations.

Described in the methods of the invention lysis buffer and elution buffer all are conventional damping fluids that use, and can commercially availablely obtain.

2, result

2.1 the fluorescent PCR detected result of bacille Calmette-Guerin vaccine dilution bacterium liquid

5 person-portion bacille Calmette-Guerin vaccines add the dissolving of 1ml distilled water, dissolve later bacterium liquid and remake 1: 10,1: 100,1: 1000,1: 10000 dilution; Each gradient concentration is got 3 of 50ul bacterium liquid at random, is designated as sample 1, sample 2 and sample 3, adds 50ulTB DNA extraction liquid and extracts, and reacts on the icycler of BioRad company fluorescent PCR amplification instrument.Real-time fluorescence PCR detected result (Ct value) sees Table 1, fluorescence threshold 25.

Table 1 bacille Calmette-Guerin vaccine dilution bacterium liquid fluorescent PCR detected result (Ct value)

	Sample 1	Sample 2	Sample 3
				Dilution in 1: 10	22.49	22.13	22.10
Dilution in 1: 100	26.86	26.54	26.51
				Dilution in 1: 1000	30.50	31.38	31.88
Dilution in 1: 10000	35.59	30.49	34.98

2.2 simulate the fluorescent PCR detected result of clinical newborn sample

Simulate clinical newborn sample and prepare, comprise solution A by preceding method, solution B, the sample of solution C and four kinds of bcg bacteria gradient concentrations of solution D, each concentration samples is all done 3 parts of detections, is designated as sample 1,2,3.Use solid phase column method reagent to extract mycobacterium DNA, on the icycler of BioRad company fluorescent PCR amplification instrument, react.Real-time fluorescence PCR amplification (Ct value) sees Table 2, and bacille Calmette-Guerin vaccine dilution bacterium liquid is once finished fluorescence threshold 25 in the test together with the real-time fluorescence PCR detection of the clinical newborn sample of simulation.

Table 2 simulation clinical newborn sample fluorescent PCR detected result (Ct value)

	Sample 1	Sample 2	Sample 3
				Solution A	25.30	26.72	25.90
Solution B	28.92	28.94	29.82

Solution C	32.07	32.90	33.58
				Solution D	35.54	37.29	-

As seen from Table 2, even solution D (comprise the bcg bacteria liquid of dilution in 1: 10000 of 50ul in the 500ul emulsion, it is several more than 1 that 50ul bacterium liquid contains bacterium, final concentration 1 more than bacterium/0.5ml) after the solid phase column method is extracted, only has one not detect in three samples.After, in order to verify this experimental result repeatedly, in the different time repeated experiments.The bcg bacteria liquid of 1: 10,1: 100,1: 1000 and 1: 10000 dilution, bacteria containing amount with solution A, solution B, solution C, solution D is identical respectively in theory, but in fact both detect the sample difference, simply and not there is inhibition pcr amplification composition in the bacterium liquid that bcg bacteria adds the distilled water dilution, simulates the complicated also existence of clinical newborn sample and suppresses the pcr amplification composition; Detection method is also different, be used for handling the process of the solid phase column method reagent of clinical newborn sample in use, since repeatedly the DNA of the DNA loss that causes of wash-out and silicon matrix absorption fully the loss that causes of wash-out reduced final DNA and gathered in the crops efficient, therefore the concentration ratio that is reflected in both is gone up, and the Ct value of simulating clinical newborn sample will lag behind the Ct value of bacille Calmette-Guerin vaccine dilution bacterium liquid.

2.3 simulate the revision test of clinical newborn sample

Finish repeated experiments twice.For the first time, to the low solution C of bacteria concentration, solution D repeated experiments, fluorescent PCR preceding 5 amplification systems that sample is a solution C that increase, the Ct value is respectively 31.0,31.2,31.5,31.2,31.7,5 amplification systems that sample is a solution D in back, the Ct value is respectively 34.5,33.7,34.0,34.1,35.0.

For the second time, to solution A, solution B, solution C and solution D repeated experiments, each concentration is done 4 and is detected sample, is designated as sample 1,2,3,4 respectively, and each sample is made 1 multiple pipe when fluorescent PCR increases, and the real-time fluorescence PCR detected result sees Table 3.As known from Table 3, the detection sample of 8 solution D all detects, and sensitivity reaches 100%.

Table 3 is the fluorescent PCR amplification (Ct value) of revision test for the second time

	Solution A	Solution B	Solution C	Solution D
					Sample 1	23.2	26.9	30.7	34.3
Multiple pipe	23.1	26.6	30.3	33.9
					Sample 2	23.2	26.7	30.3	37.8
Multiple pipe	23.0	27.1	30.7	34.6

Sample 3	22.9	26.7	30.5	33.4
					Multiple pipe	23.1	26.3	30.8	33.8
Sample 4	23.0	27.0	30.8	34.7
					Multiple pipe	23.3	26.9	31.1	34.2

Analytical table 3 results, we find solution A, B, C at higher concentration, each experimental result repeatability is fine, the Ct value is approaching, and in the solution D of lower concentration very, because bacteria containing amount own seldom, certain difference is arranged between the Ct value, last table result and experimental result comparison before, the Ct value is approaching between the sample of same concentrations, and illustrative experiment is good reproducibility as a result.

The above results shows the inventive method after clinical application, can not influence under the prerequisite of cows ordinary production at all, reaches the purpose of bovine tuberculosis monitoring and early detection, meets the milk cow of modern dairy industry and the needs of milk preparation safety in production.

The inventive method is highly sensitive, and specificity is good, also has following characteristics: the first, and simple to operate, quick, mycobacterium DNA extraction and real-time fluorescence PCR process were finished in 3-4 hour, met the needs of modern clinical diagnosis technology.The second, sampling is convenient, gets the milk cow emulsion and detects, and milk cow is not caused invasive injury or stress reaction.The 3rd, be widely used, except that being used for diagnosing bovine tuberculosis, detecting the nodule in the breast mycobacterium and have important food safety meaning, owing to possess early diagnosis, application value is preferably arranged.

Embodiment:

The fluorescent PCR detected result of example 1 clinical cow's milk sample

Adopt solid phase column method reagent to extract mycobacterium DNA 10 routine clinical cow's milk tuberculosis positive samples, on the icycler of BioRad company fluorescent PCR amplification instrument, react.The real-time fluorescence PCR amplification sees table 4 for details, and fluorescence threshold is 25.

Table 4 clinical sample fluorescent PCR amplification (Ct value)

Sample	1	2	3	4	5	6	7	8	9	10
											The Ct value	35.03	37.83	35.95	38.04	36.05	30.90	30.50	42.50	45.26.	N/A

As seen from Table 4, there is 1 example not to be detected in the 10 routine clinical positive sample, but it may be because the bacterium number is fewer in the sample that this 1 example does not detect sample, as the clinical newborn sample lower concentration of simulation, the solid phase column method reagent of handling clinical newborn sample is in the process of using, because repeatedly the DNA loss that causes of wash-out and the DNA of silicon matrix absorption, can not finish the loss that wash-out causes and reduce final DNA results efficient, thereby cause false negative result.

The fluorescent PCR detection specificity experimental result of example 2 clinical cow's milk positive sample

Adopt hepatitis B virus DNA, Salmonellas DNA, Shigellae DNA, various clinical positive sample extracts such as e. coli dna and human papillomavirus HPV DNA carry out specificity experiment (concentration is all greater than 104copies/ μ L), all there is not amplified signal, come to the same thing with negative control, only clinical cow's milk positive sample DNA has amplified signal.

The specificity experimental result of the clinical positive sample of table 5.

Title	Negative control	Human papillomavirus HPV DNA	Staphylococcus aureus DNA	Hepatitis B virus DNA	Salmonellas DNA	E. coli dna	Cow's milk tuberculosis viral DNA
								The Ct value	N/A	N/A	N/A	N/A	N/A	N/A	33.5

The result that example 3 clinical cow's milk tuberculosis positive sample adopt different pcr amplification instrument to detect

Adopt clinical cow's milk tuberculosis positive sample by this detection technique being optimized (comprising the optimization of reaction system and response procedures), and in upward amplification of different fluorescent PCR amplification instrument (fluorescent PCR amplification instrument such as PE5700, PE7000, Lightcyler, iCycle, SLAN), the Ct value of amplification sees Table 6.

The different pcr amplification instrument experimental results (Ct value) of the clinical positive sample of table 6.

	1	2	3	4	5	6	7	8	9	10
											PE7000	35.22	37.54	36.05	37.84	35.95	31.56	32.20	45.50	42.06.	N/A
PE5700	35.85	37.83	35.95	38.53	36.32	32.07	31.89	N/A	41.98.	N/A
											Lightclyer	34.52	38.03	36.42	37.51	34.86	29.87	30.23	44.64	39.26.	48.09
iCycle	35.03	37.83	35.95	38.04	36.05	30.90	30.50	42.50	45.26.	N/A
											Slan	36.10	36.97	36.87	38.34	35.23	31.42	31.58	47.04	43.55.	N/A

。

Claims (2)

1, the real-time fluorescence PCR detection method of mycobacterium tuberculosis complex in a kind of dairy products: it is characterized in that this method comprises the following steps:

(1) material and method

Bacterial strain

The lyophilized powder bacille Calmette-Guerin vaccine is provided by Shanghai Disease Prevention and Control Centre,-20 ℃ of preservations, stipulate by national Freeze Dried Bacillus Calmette-Guerin Vaccine manufacturing and vertification regulation: freeze-drying vaccine bacterium number should be more than 1,000,000/mg, bacille Calmette-Guerin vaccine per ampoule bottle 5 person-portions that the present invention uses, contain bacill calmette-guerin 0.2-0.3mg, the bacterium number should be more than 200,000, and bacille Calmette-Guerin vaccine adds the dissolving of 1ml distilled water, dissolve later bacterium liquid and do 1: 10,1: 100,1: 1000,1: 10000,1: 100000 dilution;

Simulate clinical newborn sample

Adopt the dilution bacterium liquid of 50ul gradient concentration to sneak in the 450ul emulsion for detecting sample, emulsion is used commercially available ultra high temperature sterilization (UHTS) pure milk, each detects sample: solution A: the bcg bacteria liquid that comprises the dilution in 1: 10 of 50ul in the 500ul emulsion, it is several more than 1000 that 50ul bacterium liquid contains bacterium, 1000 of final concentrations are more than bacterium/0.5ml, solution B: 500ul emulsion, 100 of bacille Calmette-Guerin vaccine final concentrations are more than bacterium/0.5ml, solution C: 500ul emulsion, 10 of bacille Calmette-Guerin vaccine final concentrations are more than bacterium/0.5ml, solution D: the 500ul emulsion, 1 of bacille Calmette-Guerin vaccine final concentration is more than bacterium/0.5ml;

Primer, probe design

According to ITS (internaltranscribed spacer) sequence between mycobacterium coding 16sRNA gene and the 23sRNA gene among the GenBank, use OMIGA software do sequence relatively after, use OLIGO6.0, PRIMER5.0, a pair of Auele Specific Primer of BEACON software design and specific probe, pcr amplification product is peculiar by mycobacterium tuberculosis complex, and primer, probe are given birth to worker company by Shanghai and synthesized;

The bacterial strain DNA extraction

For the bacille Calmette-Guerin vaccine of different gradient concentrations dilution bacterium liquid, 50ul dilution bacterium liquid directly adds self-produced mycobacterium tuberculosis DNA extraction liquid, 100 ℃ 15 minutes, 13000rpm 2 minutes can be used for pcr amplification;

The real-time fluorescence PCR reaction

In the reaction system of 50ul, add 7ul extraction product and make template, add the real-time fluorescence PCR reaction buffer 30ul that is mixed with primer, add MgCl ₂5ul adds fluorescent probe 5ul, adds the Taq enzyme 3ul that is mixed with the UNG enzyme, and the UNG enzyme can effectively be eliminated pcr amplification product and pollute influence to experiment, at the reaction conditions of the icycler of BioRad company fluorescent PCR amplification instrument is: 50 ℃ of 2min; 94 ℃ of 5min; 93 ℃ of 30sec, 60 ℃ of 1min, 40 circulations.

2, the real-time fluorescence PCR detection method of mycobacterium tuberculosis complex in a kind of dairy products according to claim 1 is characterized in that the bacterial strain extracting method in the wherein said step (1) comprises the following steps:

(1) in the 1980ul lysis buffer, adds 20 μ l poly VITAMIN B4, use behind the mixing;

(2) in the 1.5ml centrifuge tube that 500 μ l samples are housed, add the lysis buffer that 500 μ l are mixed with the poly VITAMIN B4 in advance, vibration mixing, 72 ℃ of insulation cracking 25min;

(3) add 200 μ l Virahols in the solution after above-mentioned insulation, shift 600ul solution in pillar preparation/centrifuge tube behind the vibration mixing, 10, abandon filtrate behind the centrifugal 1min of 000rpm;

(4) remaining 600ul solution is transferred in the pillar preparation/centrifuge tube, 10, abandon filtrate behind the centrifugal 1min of 000rpm;

(5) in the preparation pipe, add elution buffer 500 μ l, 10, pipe abandon bottom centrifugal filtrate behind the centrifugal 1min of 000rpm;

The centrifugal 1min of (6) 13,000rpm removes remaining elution buffer with thorough, will prepare pipe then and be placed in the new clean centrifuge tube, add be preheating in advance 72 ℃ washing towards damping fluid 50 μ l room temperature placement 2min;

Centrifugal filtrate at the bottom of the centrifugal 1min collection tube of (7) 10,000rpm is isolating nucleic acid elute soln from sample, then is kept at-70 ℃ of refrigerators if DNA need be kept at-20 ℃, RNA.