CN102242185A - Simple and quick pretreatment method suitable for bacterial PCR (polymerase chain reaction) detection samples - Google Patents
Simple and quick pretreatment method suitable for bacterial PCR (polymerase chain reaction) detection samples Download PDFInfo
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- CN102242185A CN102242185A CN2010101715906A CN201010171590A CN102242185A CN 102242185 A CN102242185 A CN 102242185A CN 2010101715906 A CN2010101715906 A CN 2010101715906A CN 201010171590 A CN201010171590 A CN 201010171590A CN 102242185 A CN102242185 A CN 102242185A
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Abstract
The invention provides a simple and quick pretreatment method suitable for bacterial PCR (polymerase chain reaction) detection samples. The method comprises the following steps of: taking a required amount of the clinical sample of lesion tissues, secretion or feces by aseptic operation, inoculating into an enrichment culture fluid, culturing at constant temperature of 37 DEG C for 12-16 hours; taking a required amount of the culture fluid under an aseptic condition, inoculating into the same kind of enrichment culture liquid, shaking for culturing for 4-6 hours; taking 1-2ml of bacterial liquid, centrifuging at the rotation speed of 5000r/minute for 5-6 minutes to obtain bacterial precipitate; washing the bacteria by double distilled water for 2-3 times, adding 100mu l of aseptic double distilled water for suspension; and boiling at 100 DEG C for about 10-12 minutes, centrifuging at the rotation speed of 12000r/minute for 3-4 minutes to obtain the supernatant, namely the bacterial crude DNA (deoxyribonucleic acid), and performing subsequent PCR typing and detection. In the experimental detection, the standard (or reference) strains used as positive controls are all bought from the China Center for Culture Collection.
Description
Technical field
The present invention relates to the PCR of bacterium is detected, be particularly useful for the pre-treatment that PCR detects, its method is not only easy but also quick.
Background technology
Along with development of molecular biology, PCR method is used to clinical detection and compares with the evaluation of traditional microbial morphology, biochemical reaction and serological method, its method is simply quick, special, responsive, and under appropriate condition, often several bacterium just can be detected.But owing to contain the complicated ingredient of a large amount of inhibition PCR reactions in the clinical sample, limited its method widespread usage in actual applications.General trace routine is at first to adopt the infection sample to increase bacterium to cultivate usually mostly, drawing the plate separation then obtains carrying out preliminary evaluation after the purifying bacterium is cultivated again, comprising morphology such as evaluations such as colonial morphology, dyeing, sometimes also need biochemistry and serological identification to match, complex steps, waste time and energy, thereby limited the practical application that subsequent P CR detects greatly.
The research report is also arranged at present, have approach to expand the application problem of PCR.For example: China Agricultural University's Food science and nutrition engineering college are permitted Wen Tao etc. and have been disclosed 3 kinds of methods of removing these inhibitory substance in " 3 kinds of removal methods of seed rice PCR supressor " paper in 2009 17 (4) " Journal of Agricultural Biotechnology " being entitled as of delivering, be DNA diluted sample method, silicon fiml purified genes group method and low melting-point agarose method of purification; It is the most simple and efficient etc. that wherein DNA diluted sample method is removed supressor.Also have the Zhao Hong of transmissible disease control center of Beijing Military Medical Academy prevention and control of diseases institute celebrating to wait be entitled as " application of multiple PCR technique in cause of disease detects " delivered 2007 18 (5) " biotechnology communication " to summarize the detection of multiplex PCR in addition at bacterial pathogens, the detection of viral pathogen, mycoplasma chlamydozoan and parasitic detection, the application of aspects such as microorganism resistance detection.And for example in pattern detection, adopt test kit can from stool sample, extract DNA and effective inhibition of removing, QIAamp DNA Stool Mini Kit as sky root company, this test kit uses pellosil technology, can have purify DNA the sample of high density PCR inhibition from fresh or freezing human faecal mass and other.But need to buy test kit after all, cost is increased greatly, thereby also limited the practical application that PCR detects.Especially outer more to the treatment process of fecal sample, buoyant density centrifugation filtration method, two step filtration methods, nucleic acid absorption matrix method, metal hydroxides absorption method, phenol extraction process etc. are arranged, all be relatively loaded down with trivial details treatment process.In addition the wide strong people of grade of milk cow research centre, academy of agricultural sciences, Shandong Province horse 2006 22 phases (2) 153 pages " Chinese Amphixenosis's magazine " deliver " directly from ight soil, detect the foundation and the application of the dual-PCR method that causes calf diarrhoea enterotoxigenic escherichia coli " disclose the toxin gene sequence that produces according to enterotoxic Escherichia coli; designed two kinds of primers, set up multiple PCR method.The present invention in contrast, purpose all is to remove inhibition, but blind bacterium is cultivated with cultivating at the expansion bacterium of primer and has one's own knack.Pre-treating process can effectively be removed the PCR inhibition in the sample simply fast, eliminates the restraining effect of clinical samples to the PCR reaction, avoids false negative result to occur.To produce extremely strong practical function for expanding the application that PCR detects.
Summary of the invention
The objective of the invention is to: the pre-treating process of the sample that is applicable to that PCR detects is provided, promptly easily goes simply again, can make the PCR detection method not only easy but also quick in the application of clinical sample, expand the practical value of its method.
The object of the present invention is achieved like this: a kind of pre-treating process that simply is applicable to the bacteria PCR test sample fast, under aseptic technique, get the clinical sample of pathological tissues, secretory product or the ight soil of requirement, be inoculated in the enrichment culture medium, at 37 ℃ of following constant temperature culture 12-16 hours; Get the nutrient solution of requirement under the aseptic condition, be inoculated in the enrichment liquid of the same race shaking culture 4-6 hour once more; It is centrifugal to get 1-2ml bacterium liquid, and rotating speed is 5000 commentaries on classics/min, obtains bacterial sediment in 5-6 minute; With distilled water washing thalline 2-3 all over after, add the 100ul aseptic double-distilled water and suspend, under 100 ℃, boil about 10-12 minute, centrifugal, rotating speed is 12000 commentariess on classics/min, gets supernatant liquor in 3-4 minute, is the rough DNA of bacterium, carries out the detection of subsequent P CR somatotype again and gets final product.
The pre-treating process that described PCR detects, during experiment detects as the standard of positive control (or with reference to) bacterial strain all available from Chinese DSMZ.
The pre-treating process that described PCR detects, the experiment material of selecting for use is the commercially available prod.
The pre-treating process key of the sample of the present invention's design is to have removed the inhibition composition of the follow-up PCR reaction of influence of bringing into when clinical sample increases the bacterium cultivation when bacterium template copy number to be checked is further increased.But, avoid the uncertain and false-negative result's generation of inhibition interferential in the sample because the existence of PCR inhibition in the sample must be groped the condition that DNA concentration can detect in relative extent of dilution.
Present method is got a little clinical sample pre-culturing bacterium in enrichment culture medium by aseptic technique, reach the purpose that increases bacterium, get a little enrichment liquid after cultivating then and inoculate again repeating and cultivate, reach the purpose that enrichment of bacterial is removed other compositions in the sample simultaneously.Thalline is collected in operation routinely at last, carries out subsequent P CR and detects, and the result shows that the thallus DNA that obtains is enough to reach the pcr amplification requirement, promptly obtains amplification, thereby reaches the purpose of detection.
The present invention comprises tissue, blood, secretory product and stool sample thereof etc. with the direct applying clinical sample of this method, wherein contains the multiple composition that can suppress pcr amplification in the stool sample, is the sample of the most difficult extraction nucleic acid and amplification in the clinical samples.For fecal sample, inhibition how to remove PCR reaction in the ight soil is a step that effectively prepares the dna profiling most critical.No matter this invention is theoretical analysis and experimental results show that, this clinical sample treatment process can improve clinical sample greatly and be used for specificity and the susceptibility that PCR detects, avoided false-negative result, the live body for early infection or recessive carrier detects also with helpful simultaneously.
The solution of the inventive method success PCR detect loaded down with trivial details, the restriction bottleneck that the time is long of pre-treatment, by the enrichment to the sample pathogenic bacteria, nucleic acid obtains the removal of PCR inhibition in a large number, specificity and susceptibility that its follow-up PCR is detected increase, and show technical progress.
Description of drawings
Below in conjunction with accompanying drawing invention is described further.
The gel electrophoresis analysis of the multiplex PCR amplified production of accompanying drawing 1-1 pasteurellosis bacillus different shaped type strain is with reference to figure, and from left to right 1, DL2000,2-4 is followed successively by pasteurellosis bacillus type strain A, B, E type, 5 negative contrasts.
The die of illness gel electrophoresis analysis figure of the multiple PCR products of ox different tissues sample after pre-treatment of accompanying drawing 1-2, the multiplex PCR detected result from left to right 1, DL2000,2, pasteurellosis bacillus type strain A type type strain positive control, 3-5 is followed successively by lung, liver, spleen, and 6, negative control.
The gel electrophoresis analysis of accompanying drawing 2-1 clostridium perfringens different shaped type strain multiplex PCR amplified production is with reference to figure, and from left to right 1, DL2000,2-4 is followed successively by clostridium perfringens type strain A, B, C, D type, 5 negative contrasts.
The die of illness gel electrophoresis analysis figure of the multiple PCR products of sheep different tissues sample after pre-treatment of accompanying drawing 2-2, the multiplex PCR detected result from left to right 1, DL2000,2, clostridium perfringens type strain D strain positive control, 3-7 is followed successively by lung, liver, spleen, intestines inner membrance, ight soil, and 8, negative control.
Embodiment
Embodiment
The pre-treating process of bacteria PCR test sample is applied to the detection that pasteurella infection sample and clostridium perfringens infect sample:
One, the multiplex PCR of pasteurella infection sample detects;
Get sheep pneumonia disease material package and draw together lung, liver and spleen tissue;
Aseptic technique: a little pathological tissues (lung, liver, spleen) respectively is inoculated in contains in TSB (soybean Tryptones) nutrient solution 37 ℃ of constant temperature culture, 12 hours;
Aseptic technique: dip in transfering loop and to get a little nutrient solution and be linked in the TSB nutrient solution 37 ℃ of shaking culture 5h;
Get 1ml bacterium liquid, centrifugal 5000 commentaries on classics/min, 5min;
Abandon supernatant and keep the bacterium precipitation, wash 2 times with aseptic double-distilled water;
Adding the 100ul aseptic double-distilled water suspends.100 ℃ were boiled about about 10 minutes;
Centrifugal 12000 commentaries on classics/min, 3min, supernatant are the rough DNA of bacterium.
The PCR somatotype detects and comprises the PCR reaction solution, pasteurellosis bacillus serotype A, three pairs of special primer combinations of B, E, the preparation of bacterium dna profiling;
Wherein the annealing temperature of the reaction of pasteurellosis bacillus is 55 ℃; Amplification system is: 10 times of buffer (containing Mg2+) 5.0ul, and dNTP 1.5ul (10mmol/l), 3 couples of special each 1.0ul of upstream and downstream primer (10umol/l), Taq archaeal dna polymerase 2.5U, template 10ul complements to 50ul with distilled water; The pcr amplification reaction condition is: 95 ℃ of pre-sex change in 5 minutes; Then 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 45 seconds, after totally 30 circulations again 72 ℃ extended in 10 minutes;
The gel electrophoresis analysis of pasteurellosis bacillus PCR product wherein: behind the amplified production electrophoresis, the nucleic acid samples of 3 reference cultures all can amplify the purpose band that conforms to the test design size; C403-A strain (pasteurellosis bacillus A type) sample amplification has gone out 1044bp and 460bp two bands, C405-B strain (pasteurellosis bacillus Type B) has amplified 760bp and 460bp two bands, C393-E strain (pasteurellosis bacillus E type) has amplified 511bp and 460bp two bands, Fig. 1-1 be pasteurellosis bacillus A B the PCR electrophoresis result of E three strain reference cultures as reference.Fig. 1-2 is a PCR detected result after the pre-treatment of sheep pneumonia clinical tissue sample process, defines pasteurellosis bacillus A type according to the amplified fragments size and infects.
Two, clostridium perfringens infects sample detection;
Get the lamb sudden death and cut open the sample product, comprise liver, spleen, lung, intestines and ight soil;
Aseptic technique is inoculated in sample respectively in the liver soup of being sick of, and 37 ℃ of constant temperature anaerobism are cultivated 12 hours;
Aseptic technique: dip in transfering loop and to get a little nutrient solution and be linked in the liver soup of being sick of, 37 ℃ of constant temperature anaerobism are cultivated 12 hours;
Get 1ml bacterium liquid, centrifugal 5000 commentaries on classics/min, 5min;
Abandon supernatant and keep the bacterium precipitation, wash 2 times with aseptic double-distilled water;
Adding the 100ul aseptic double-distilled water suspends.100 ℃ were boiled about about 10 minutes;
Centrifugal 12000 commentaries on classics/min, 3min, supernatant are rough bacterium DNA.
The PCR somatotype detects and comprises the PCR reaction solution, pasteurellosis bacillus serotype A, three pairs of special primer combinations of B, E, the preparation of bacterium dna profiling;
Wherein the annealing temperature of the reaction of pasteurellosis bacillus is 55 ℃; Amplification system is: 10 times of buffer (containing Mg2+) 5.0ul, and dNTP 1.5ul (10mmol/l), 3 couples of special each 1.0ul of upstream and downstream primer (10umol/l), Taq archaeal dna polymerase 2.5U, template 10ul complements to 50ul with distilled water; The pcr amplification reaction condition is: 95 ℃ of pre-sex change in 5 minutes; Then 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 45 seconds, after totally 30 circulations again 72 ℃ extended in 10 minutes;
The gel electrophoresis analysis of pasteurellosis bacillus PCR product wherein: behind the amplified production electrophoresis, the nucleic acid samples of 3 reference cultures all can amplify the purpose band that conforms to the test design size; C403-A strain (pasteurellosis bacillus A type) sample amplification has gone out 1044bp and 460bp two bands, C405-B strain (pasteurellosis bacillus Type B) has amplified 760bp and 460bp two bands, C393-E strain (pasteurellosis bacillus E type) has amplified 511bp and 460bp two bands, Fig. 2-1 be pasteurellosis bacillus A B the PCR electrophoresis result of E three strain reference cultures as reference.Fig. 2-2 for the clinical tissue sample through PCR detected result after the pre-treatment, define pasteurellosis bacillus A type according to the amplified fragments size and infect.
The reference culture of the positive control that is used as is all available from Chinese DSMZ in more than detecting.
Claims (3)
1. a pre-treating process that simply is applicable to the bacteria PCR test sample fast is characterized in that: get the clinical sample of pathological tissues, secretory product or the ight soil of requirement under aseptic technique, be inoculated in the enrichment culture medium, at 37 ℃ of following constant temperature culture 12-16 hours; Get the nutrient solution of requirement under the aseptic condition, be inoculated in the enrichment liquid of the same race shaking culture 4-6 hour once more; It is centrifugal to get 1-2ml bacterium liquid, and rotating speed is 5000 commentaries on classics/min, obtains bacterial sediment in 5-6 minute; With distilled water washing thalline 2-3 all over after, add the 100ul aseptic double-distilled water and suspend, under 100 ℃, boil about 10-12 minute, centrifugal, rotating speed is 12000 commentariess on classics/min, gets supernatant liquor in 3-4 minute, is the rough DNA of bacterium, carries out the detection of subsequent P CR somatotype again and gets final product.
2. the pre-treating process that detects according to the described PCR of claim 1 is characterized in that: during experiment detects as the standard of positive control (or with reference to) bacterial strain all available from Chinese DSMZ.
3. according to the pre-treating process of the described PCR detection of claim 1, it is characterized in that: the experiment material of selecting for use is the commercially available prod.
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| CN104480104A (en) * | 2014-12-25 | 2015-04-01 | 遵义医学院 | Method for extracting genome DNA (deoxyribonucleic acid) of rhinopithecus brelichi through feces sampling |
| CN108467896A (en) * | 2018-03-02 | 2018-08-31 | 中山出入境检验检疫局检验检疫技术中心 | Detect the primer of C.perfringens and etx genes, kit and method in food |
| CN108977497A (en) * | 2018-06-12 | 2018-12-11 | 深圳市领治医学科技有限公司 | A method of acquisition and depositary's trace fecal sample |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104480104A (en) * | 2014-12-25 | 2015-04-01 | 遵义医学院 | Method for extracting genome DNA (deoxyribonucleic acid) of rhinopithecus brelichi through feces sampling |
| CN108467896A (en) * | 2018-03-02 | 2018-08-31 | 中山出入境检验检疫局检验检疫技术中心 | Detect the primer of C.perfringens and etx genes, kit and method in food |
| CN108977497A (en) * | 2018-06-12 | 2018-12-11 | 深圳市领治医学科技有限公司 | A method of acquisition and depositary's trace fecal sample |
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Application publication date: 20111116 |