CN102134601B - Loop-mediated isothermal amplification detection primer pair of Pseudomonas aeruginosa, detection method and detection kit - Google Patents
Loop-mediated isothermal amplification detection primer pair of Pseudomonas aeruginosa, detection method and detection kit Download PDFInfo
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Abstract
The invention discloses a loop-mediated isothermal amplification (LAMP) detection primer pair of Pseudomonas aeruginosa, a detection method and a detection kit. Aiming at the ecfX gene of Pseudomonas aeruginosa, the invention designs and screens a set of specific detection primer pair, a detection kit containing the detection primer pair and an LAMP detection method utilizing the detection kit. The detection method is adopted to detect a sample to be detected, specially for drinking water to confirm whether the sample contains the special gene segment of Pseudomonas aeruginosa and further confirm whether the sample contains Pseudomonas aeruginosa. The detection kit and detection method disclosed by the invention have high sensitivity and specificity and short detection time, and the whole process from sample processing to result reporting is only 24h; and the polymerase chain reaction (PCR) amplifier and electrophoresis meter are not adopted, the operation process is simple and the method and kit are especially suitable for the self-checking of grassroots detection institutions and drinking water processing enterprises.
Description
Technical field:
The present invention relates to a kind of based on ring mediated isothermal amplification (loop-mediated isothermalamplification, LAMP) the pathogenic bacteria Fast Detection Technique of technology, the fragment that is particularly related to Pseudomonas aeruginosa (Pseudomonas aeruginosa) specific gene ecfX gene has specific detection primer sets, described detection primer sets is detected detection method and the detection kit of Pseudomonas aeruginosa with ring mediated isothermal amplification method LAMP.
Background technology:
Pseudomonas aeruginosa (Pseudomonas aeruginosa) claim Pseudomonas aeruginosa again, it is a kind of single flagellum of giving birth to, motion is active, no brood cell, obligate aerobic gram negative bacillus, extensively be present in water, soil, air and humans and animals body skin and the enteron aisle, be one of primary pollution source in the tap water.Pseudomonas aeruginosa can produce multiple interior extracellular toxin, causes acute intestinal disorders and skin inflammation, in addition, because it has the multidrug resistant ability, under given conditions, this bacterium also can cause secondary infection or polyinfection, cause pneumonia, meningitis, septicemia etc., can cause death when serious.
In recent years, the Pseudomonas aeruginosa pollution condition day by day comes into one's own in the natural mineral water, the investigator pollutes Pseudomonas aeruginosa in the mineral water finished product water and investigates both at home and abroad, find in the finished product water Pseudomonas aeruginosa recall rate between 1.2%～23.6%, and think that processing link is the major cause that causes polluting generation.Pollute the present situation that report obviously increases at present tap water (comprising Drinking Water, natural mineral water, pure water etc.) Pseudomonas aeruginosa, the hygienic quality management has all been strengthened in countries in the world, the U.S., Canada, Brazil, Japan, European various countries and the World Health Organization/food and agricultural organization (WHO/FAO) etc. all limit among bottled drinking water (comprising the natural mineral water water source) Pseudomonas aeruginosa MPN＜3/L or the every 250mL and must not detect." the GB8537-2008 natural mineral water " of the new revision of China is also in formal enforcement on October 1st, 2009, wherein clearly these three kinds of pathogenic bacterium of regulation streptococcus faecium, Pseudomonas aeruginosa and clostridium perfringens must not detect, and require carrying out the Pseudomonas aeruginosa detection before every batch of product export.
At present, aspect the detection technique of Pseudomonas aeruginosa, still continue to use traditional physics and chemistry detection method in the standard both at home and abroad, comprise coating method, many test tubes MPN method, filter membrane method etc., need a plurality of steps such as pure culture and biochemical identification, whole sense cycle 3-5 days, sensitivity was low, and can not satisfy the needs of field quick detection, particularly the rapid detection of water sample in enormous quantities.
The LAMP method is a kind of novel nucleic acids amplification technique of exploitation such as Notomi in 2000,6 zone design 4-6 bar Auele Specific Primers at target gene to be measured, under the effect of BstDNA polysaccharase, in 65 ℃ of left and right sides isothermal condition 1h, can carry out differential high efficient nucleic acid amplification fast, reach 10
9Copy, but amplification direct viewing magnesium pyrophosphate precipitation or adding developer are judged.The LAMP method is compared with round pcr, and detection time is short, has higher susceptibility, and simple to operate, does not need expensive instrument, only needs the ortho-water bath just can finish detection.The LAMP method has been widely used in aspects such as disease prevention and cause of disease detection in recent years.
Summary of the invention:
First purpose of the present invention is to provide a kind of detection primer sets that ring mediated isothermal amplification detects Pseudomonas aeruginosa of using, and this detection primer sets has specificity to the ecfX gene of Pseudomonas aeruginosa.
This detection primer sets that detects Pseudomonas aeruginosa is made up of following primer:
Upstream outer primer F3 (5 '-3 '): GGATGAGCGCTTCCGTG;
Downstream outer primer B3 (5 '-3 '): AAGTTGCGGGCGATCTG;
Upstream inner primer FIP (5 '-3 '): CTTGCGCAGGAAGCGCAGC-GTTCCGTCTCGCATGCCTA;
Downstream inner primer BIP (5 '-3 '): GCCGACCTCGCCCAGGATA-GCTCGACCGATTGCCG.
Second purpose of the present invention provides the detection method of a kind of Pseudomonas aeruginosa, it is characterized in that, ecfX gene with Pseudomonas aeruginosa is target gene, with the specific fragment of above-mentioned detection primer sets to the described target gene ecfX of DNA selective amplification of the thalline in the sample, confirm whether to have amplified production by the LAMP method.
Described sample is preferably tap water.
Described detection method specifically is preferably:
(1) extraction of the preparation of sample and template: sample to be checked is increased bacterium with the enrichment liquid of Pseudomonas aeruginosa cultivate, extract the DNA of thalline in the nutrient solution as template;
(2) ring mediated isothermal amplification LAMP: amplification reaction system is hatched 45～60min, 80 ℃ of termination reaction 1～2min in 65 ℃;
Described amplification reaction system comprises: 1 * Thermopol reaction buffer, 1.4-1.8mmol/L dNTP s, 4-8mmol/L sal epsom (MgSO
4), 0.15-0.25 μ mol/L upstream outer primer F3,0.15-0.25 μ mol/L downstream outer primer B3,1.2-2 the template DNA that the Bst archaeal dna polymerase of μ mol/L upstream inner primer FIP, 1.2-2 μ mol/L downstream inner primer BIP, 0.8-1mol/L trimethyl-glycine, 0.24-0.32U/ μ L and an amount of step (1) obtain, surplus is water;
(3) detection of LAMP reaction product:
Utilize electrophoresis detection, fluoroscopic examination or turbidity to detect the LAMP reaction product and whether have amplified production.
Described fluoroscopic examination is to add SYBR Green I developer in reaction tubes, and observations behind 1～5min is orange as color, detected sample Pseudomonas aeruginosa feminine gender then, be green as color, then the detected sample Pseudomonas aeruginosa positive contains Pseudomonas aeruginosa.
Described electrophoresis detection is through agarose electrophoresis with the LAMP reaction product, present the stepped band of LAMP characteristic as electrophorogram, then the detected sample Pseudomonas aeruginosa positive contains Pseudomonas aeruginosa, and then detected sample is negative not have any band as electrophorogram.
It is to detect by an unaided eye or turbidimeter detects turbidity under 400nm light that described turbidity detects, and shows relatively that with the negative control pipe it is then positive that obvious muddiness appears in detector tube, contains Pseudomonas aeruginosa, as does not see that muddiness is then negative.
10 * Thermopol damping fluid is material of the prior art, can buy from reagent company, and it contains trihydroxy methyl aminomethane-hydrochloric acid (Tris-HCl), 100mmol/L Repone K (KCl), the 100mmol/L ammonium sulfate (NH of 200mmol/LpH8.8
4) 28O
4, 20mmol/L sal epsom (MgSO
4) and 1% triton x-100 (TtitonX-100).
The preparation of the sample of described step (1) and the extraction of template specifically are preferably: described sample is the tap water water sample, water sample is crossed the filter membrane of holding back bacterium, filter membrane is transferred to selective enrichment liquid, cultivate 20h for 36 ± 1 ℃, extract the DNA of thalline in the nutrient solution again as template, described selective enrichment liquid is that every 1000ml LB substratum contains 0.2g cetrimonium bronmide and 15mg nalidixic acid.The Pseudomonas aeruginosa to drinking in the water sample that utilizes this selective enrichment liquid to select increases bacterium, abandons other assorted bacterium.More be conducive to detect drinking water sample.
The described filter membrane of holding back bacterium is preferably the filter membrane that the aperture is 0.45 μ m.
The 3rd purpose of the present invention provides the detection kit of a kind of Pseudomonas aeruginosa, it is characterized in that described detection kit comprises loop-mediated isothermal amplification liquid and the Bst archaeal dna polymerase of the detection primer sets that contains above-mentioned upstream outer primer F3, downstream outer primer B3, upstream inner primer FIP and downstream inner primer BIP at least.
The detection kit of described Pseudomonas aeruginosa preferably also comprises developer: SYBR Green I.
The detection kit of described Pseudomonas aeruginosa preferably also comprises selective enrichment liquid, and this selective enrichment liquid is that every 1000ml LB substratum contains 0.2g cetrimonium bronmide and 15mg nalidixic acid.
The ecfX gene is the specific target gene relevant with the Pseudomonas aeruginosa virulence factor, find after deliberation, this gene has higher specificity than other as outer membrane protein gene oprI, oprL and 16srRNA, therefore be applicable to the detection of Pseudomonas aeruginosa, be specially adapted to the detection of the Pseudomonas aeruginosa in the water surrounding.The present invention is directed to the ecfX gene of Pseudomonas aeruginosa, design and the detection kit of having screened a cover specific detection primer group and having contained this detections primer sets with utilize this detection kit by the detection method of LAMP, detected sample is detected, tap water particularly, confirm whether to exist the specific gene fragment of Pseudomonas aeruginosa, and then whether definite detected sample exists the detection method of Pseudomonas aeruginosa.That detection kit of the present invention and detection method have is highly sensitive, high specificity, detection time are short, only need 24h from sample preparation to the whole process of reporting the result, do not need PCR instrument and electrophoresis apparatus, operating process is simple, is particularly suitable for feeler mechanism of basic unit and the self check use of tap water processing enterprise.
Embodiment:
Below be to further specify of the present invention, rather than limitation of the present invention.
The preparation of selective enrichment liquid:
Selective enrichment liquid: every 1000ml LB substratum contains 0.2g cetrimonium bronmide and 15mg nalidixic acid.
Compound method:
1. nalidixic acid fungistat preparation: add the nalidixic acid dry powder of 15mg in every 10ml water, stir the vibration dissolving, after the film of 0.22 μ m, filtration sterilization obtains the nalidixic acid fungistat.
2. the preparation of selective enrichment liquid: every 1000ml selective enrichment liquid is preparation like this, take by weighing 21g LB culture medium dry powder and 0.2g cetrimonium bronmide, dissolved in distilled water with 1000ml, stirring heating is boiled abundant dissolving, and 121 ℃ of autoclaving 15min are cooled to 40 ℃, under the aseptic condition, add 10ml nalidixic acid fungistat again, mix, and make selective enrichment liquid.
Embodiment 1: the detection of Pseudomonas aeruginosa in the bottled water
1, water sample filters
Get certain bottled water water sample (1) 250mL, aseptic technique filters the 0.45um filter membrane, filter membrane is transferred in the 100ml selective enrichment liquid cultivated, and cultivates 20h, obtains bacterial cultures for 36 ℃ ± 1 ℃.
2, the extraction of DNA of bacteria
Get the 1mL bacterial cultures in the centrifugal 5min of 12000r/min, abandon supernatant, collect thalline, add the 50 μ L TE solution mixing that fully suspends, in 100 ℃ of water-bath 10min, ice bath 3min, the centrifugal 5min of 12000r/min, it is standby to get supernatant liquor, and this supernatant liquor contains the DNA of bacterium, as template DNA.
3, LAMP amplification
Add 1 μ L Bst archaeal dna polymerase (8U/ μ L) and 1 μ L template DNA in the reaction tubes that 23 μ L ring mediated isothermal amplification (LAMP) reaction solutions are housed, hatch 60min for 65 ℃ on thermostat water bath, 80 ℃ of termination reaction 1min take out reaction tubes.
Described 23 μ L ring mediated isothermal amplification (LAMP) reaction solutions consist of: 2.5 μ L, 10 * Thermopol reaction buffer, 4 μ L 10mmol/LdNTPs, 0.5 μ L, 10 μ mol/L upstream outer primer F3,0.5 μ L, 10 μ mol/L downstream outer primer B3,1 μ L40 μ mol/L upstream inner primer FIP, 1 μ L, 40 μ mol/L downstream inner primer BIP, 1.5 μ L 100mmol/L MgSO
4, 9 μ L2.5mol/L trimethyl-glycines, 3 μ L sterilization distilled water ddH
2O.
4, developer is observed
In reaction tubes, add 1 μ L developer (massfraction is 10% fluorescence dye SYBR GREEN I), the colour-change that directly detects by an unaided eye, the reaction tubes color becomes green, contains Pseudomonas aeruginosa in the interpret sample.
Use traditional biochemical identification method (GB8538-2008) this sample is detected, detected result is positive, contains Pseudomonas aeruginosa in the sample, and detected result is coincide, and proves that this method data are reliable.
Embodiment 2: Pseudomonas aeruginosa detects in the bottled water
Present embodiment is substantially the same manner as Example 1, and just water sample is for getting certain bottled water water sample (2) 250mL, detects according to the operation of embodiment 1.The reaction tubes color becomes orangely after testing, and interpret sample is the Pseudomonas aeruginosa feminine gender, does not contain the verdigris pseudomonas.
Use traditional biochemical identification method (GB8538-2008) this sample is detected, detected result is negative, does not contain Pseudomonas aeruginosa in the sample, and detected result is coincide, and proves that this method data are reliable.
Embodiment 3: the detection of Pseudomonas aeruginosa in the bottled water
Present embodiment is substantially the same manner as Example 1, water sample also is certain bottled water water sample (1), just in the LAMP amplification in the step (3), 23 μ L ring mediated isothermal amplification (LAMP) reaction solutions consist of: 2.5 μ L, 10 * Thermopol reaction buffer, 3.5 μ L 10mmol/LdNTPs, 0.375 μ L, 10 μ mol/L upstream outer primer F3,0.375 μ L, 10 μ mol/L downstream outer primer B3,0.75 μ L, 40 μ mol/L upstream inner primer FIP, 0.75 μ L, 40 μ mol/L downstream inner primer BIP, 1 μ L 100mmol/LMgSO
4, 8 μ L 2.5mol/L trimethyl-glycines, 5.75 μ L sterilization distilled water ddH
2O.
The reaction tubes color becomes green after testing, contains Pseudomonas aeruginosa in the interpret sample.Coincide with the detected result of embodiment 1.
Embodiment 4: Pseudomonas aeruginosa detects in the bottled water
Present embodiment is substantially the same manner as Example 2, water sample also is certain bottled water water sample (2), just in the LAMP amplification in the step (3), 23 μ L ring mediated isothermal amplification (LAMP) reaction solutions consist of: 2.5 μ L, 10 * Thermopol reaction buffer, 4.5 μ L 10mmol/LdNTPs, 0.625 μ L, 10 μ mol/L upstream outer primer F3,0.625 μ L, 10 μ mol/L downstream outer primer B3,1.25 μ L, 40 μ mol/L upstream inner primer FIP, 1.25 μ L, 40 μ mol/L downstream inner primer BIP, 2 μ L 100mmol/L MgSO
4, 10 μ L 2.5mol/L trimethyl-glycines, 0.25 μ L sterilization distilled water ddH
2O.
And add 0.75 μ L Bst archaeal dna polymerase (8U/ μ L) and 1.25 μ L template DNAs in the reaction tubes of above-mentioned 23 μ L ring mediated isothermal amplification (LAMP) reaction solutions, hatch 45min for 65 ℃ on thermostat water bath, 80 ℃ of termination reaction 2min take out reaction tubes.Other steps are with embodiment 2.
The reaction tubes color becomes orangely after testing, and interpret sample is the Pseudomonas aeruginosa feminine gender, does not contain the verdigris pseudomonas.Coincide with the detected result of embodiment 2.
Two, specificity and sensitivity detect
1, specificity
Collect Pseudomonas aeruginosa 20 strains and 23 strains of non-Pseudomonas aeruginosa, with these bacterial strains respectively after 37 ℃ of nutrient broths (Vibrio parahemolyticus is at 3.5% sodium-chlor nutrient broth) are cultivated 24h, get 1mL bacterium liquid, extract the DNA of each bacterium according to the method for embodiment 1, and carry out the LAMP amplification respectively and add developer observing.
Its detected result is as shown in table 1.
Table 1: test bacterial strain uses therefor and detected result
Annotate :+: positive findings ,-: negative findings; ATCC: US mode culture collection warehousing (American Type Culture Collection), CMCC: Chinese common micro-organisms culture presevation administrative center (China Microbiological Culture Collection), GIM: Guangdong institute of microbiology (Guangdong Institute ofMicrobiology), NCTC: Britain typical case DSMZ (National Collection of Type Cultures), A.S: the Chinese Academy of Sciences (Academy of Science)
As can be seen from Table 1, detection kit of the present invention and detection method have specificity preferably, have only the pseudomonas aeruginosa strains amplification positive, and other non-pseudomonas aeruginosa strains are negative.
2, sensitivity
Be reference strain with Pseudomonas aeruginosa Pseudomonas aeruginosa ATCC 27853, with its in the LB substratum 37 ℃ cultivate 24h after, get 1mL bacterium liquid, carry out 10 times of gradient dilutions with sterile saline, choose 3 suitable extent of dilution and carry out plate count.The plate count bacterial count of learning from else's experience is respectively 1.57 * 10
0Cfu/mL, 1.57 * 10
1Cfu/mL, 1.57 * 10
2Cfu/mL, 1.57 * 10
3Each 1mL of bacterium liquid of cfu/mL concentration, the method for pressing among the embodiment 1 is extracted DNA of bacteria, carries out the LAMP amplification, adds chromogenic reagent and observes, and verifies sensitivity of the present invention.Through 3 repeated experiments, wherein 2 experimental result sensitivity is 1.57 * 10
1Cfu/mL, 1 experimental result sensitivity is 1.57 * 10
2Therefore cfu/mL determines that the lowest detection of this detection method of the present invention is limited to 1.57 * 10
1Cfu/mL～1.57 * 10
2Cfu/mL has higher sensitivity.
Claims (8)
1. use the detection primer sets that ring mediated isothermal amplification detects Pseudomonas aeruginosa (Pseudomonas aeruginosa) for one kind,
It is characterized in that described detection primer sets is made up of following primer:
Upstream outer primer F3(5 '-3 '): GGATGAGCGCTTCCGTG;
Downstream outer primer B3(5 '-3 '): AAGTTGCGGGCGATCTG;
Upstream inner primer FIP(5 '-3 '): CTTGCGCAGGAAGCGCAGC-GTTCCGTCTCGCATGCCTA;
Downstream inner primer BIP(5 '-3 '): GCCGACCTCGCCCAGGATA-GCTCGACCGATTGCCG.
2. the detection method of a Pseudomonas aeruginosa, it is characterized in that, ecfX gene with Pseudomonas aeruginosa is target gene, use the described detection primer sets of claim 1 to the specific fragment of the described target gene ecfX of DNA selective amplification of the thalline in the sample by the LAMP method, confirm whether have amplified production, described sample is tap water.
3. the detection method of Pseudomonas aeruginosa according to claim 2 is characterized in that, the detection method of described Pseudomonas aeruginosa is specially:
(1) extraction of the preparation of sample and template: sample to be checked is increased bacterium with the enrichment liquid of Pseudomonas aeruginosa cultivate, extract the DNA of thalline in the nutrient solution as template;
(2) ring mediated isothermal amplification LAMP: amplification reaction system is hatched 45 ~ 60min, 80 ℃ of termination reaction 1 ~ 2min in 65 ℃;
Described amplification reaction system comprises: 1 * Thermopol reaction buffer, 1.4-1.8mmol/L dNTP s, 4-8mmol/L sal epsom (MgSO
4), 0.15-0.25 μ mol/L upstream outer primer F3,0.15-0.25 μ mol/L downstream outer primer B3,1.2-2 the template DNA that the Bst archaeal dna polymerase of μ mol/L upstream inner primer FIP, 1.2-2 μ mol/L downstream inner primer BIP, 0.8-1mol/L trimethyl-glycine, 0.24-0.32U/ μ L and an amount of step (1) obtain, surplus is water;
(3) detection of LAMP reaction product:
Utilize electrophoresis detection, fluoroscopic examination or turbidity to detect the LAMP reaction product and whether have amplified production.
4. the detection method of Pseudomonas aeruginosa according to claim 3, it is characterized in that, the preparation of the sample of described step (1) and the extraction of template are specially: water sample is crossed the filter membrane of holding back bacterium, filter membrane is transferred to selective enrichment liquid, cultivate 20h for 36 ± 1 ℃, extract the DNA of thalline in the nutrient solution again as template, described selective enrichment liquid is that every 1000mlLB substratum contains 0.2g cetrimonium bronmide and 15mg nalidixic acid.
5. the detection method of Pseudomonas aeruginosa according to claim 4 is characterized in that, the described filter membrane of holding back bacterium is that the aperture is the filter membrane of 0.45 μ m.
6. the detection kit of a Pseudomonas aeruginosa, it is characterized in that described detection kit comprises loop-mediated isothermal amplification liquid and the Bst archaeal dna polymerase of the detection primer sets that contains the described upstream of claim 1 outer primer F3, downstream outer primer B3, upstream inner primer FIP and downstream inner primer BIP at least.
7. the detection kit of Pseudomonas aeruginosa according to claim 6 is characterized in that, the detection kit of described Pseudomonas aeruginosa also comprises developer: SYBR Green I.
8. according to the detection kit of claim 6 or 7 described Pseudomonas aeruginosas, it is characterized in that, the detection kit of described Pseudomonas aeruginosa also comprises selective enrichment liquid, and this selective enrichment liquid is that every 1000ml LB substratum contains 0.2g cetrimonium bronmide and 15mg nalidixic acid.
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| CN102382879B (en) * | 2011-10-21 | 2013-09-04 | 宁波大学 | Pseudomonas fluorescens LAMP (loop-mediated isothermal amplification) detection agent and kit |
| CN103966200A (en) * | 2013-07-10 | 2014-08-06 | 哈尔滨德歌生物科技有限公司 | Saltatory replication gene amplification method based on nucleic acid isothermal amplification |
| CN103911459A (en) * | 2014-04-29 | 2014-07-09 | 西南大学 | LAMP rapid detection kit for toxin genes of pseudomonas aeruginosa type III secretion system and application and method thereof |
| CN104388566A (en) * | 2014-11-24 | 2015-03-04 | 武汉明曼基因工程有限公司 | Kit for rapidly detecting pseudomonas aeruginosa and application thereof |
| CN107164458A (en) * | 2016-03-08 | 2017-09-15 | 北京福德安科技有限公司 | A kind of multiple detection method of the malicious pseudomonas aeruginosa of field screening production |
| CN107604081A (en) * | 2016-12-14 | 2018-01-19 | 深圳市埃克特生物科技有限公司 | Visualize primer sets, kit and the method for LAMP detection pseudomonas aeruginosas |
| CN106544443B (en) * | 2016-12-30 | 2020-05-05 | 广东环凯生物科技有限公司 | Pseudomonas aeruginosa dry efflorescence LAMP (loop-mediated isothermal amplification) rapid detection kit and use method thereof |
| CN106591483B (en) * | 2017-02-06 | 2020-06-23 | 郑州海关技术中心 | Method for rapidly detecting pseudomonas aeruginosa in textile |
| CN110295243A (en) * | 2019-08-16 | 2019-10-01 | 贵州金玖生物技术有限公司 | A kind of primer sets for detecting pseudomonas aeruginosa, kit and method |
| CN111154900B (en) * | 2020-01-19 | 2022-05-20 | 广东省微生物研究所（广东省微生物分析检测中心） | Pseudomonas aeruginosa specific new molecular target and rapid detection method thereof |
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| CN101429539A (en) * | 2007-11-09 | 2009-05-13 | 中山大学达安基因股份有限公司 | Reagent kit for real time fluorescence quantitative PCR detection of bacillus pyocyaneus |
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| CP03 | Change of name, title or address | ||
| CP03 | Change of name, title or address |
Address after: Guangzhou City, Guangdong province 510070 martyrs Road No. 100 building No. 56 Co-patentee after: Huankai Microbes Tech Co., Ltd., Guangdong Patentee after: Guangdong Institute of Microbiology (Guangdong province microbiological analysis and testing center) Address before: Guangzhou City, Guangdong province 510070 martyrs Road No. 100 Co-patentee before: Huankai Microbes Tech Co., Ltd., Guangdong Patentee before: Guangdong Institute of Microbiology |