CN112760212A - Fast live bacteria DNA screening instrument, and use method and application thereof - Google Patents
Fast live bacteria DNA screening instrument, and use method and application thereof Download PDFInfo
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Abstract
The invention discloses a fast screening instrument for live bacteria DNA, a use method and application thereof, the screening instrument comprises a PMA top cover, an LED blue light source, a cuboid box type structure which is formed by fixedly connecting an upper box body with a groove structure and a lower box body internally provided with a motor into a whole, and the fast screening instrument also comprises a digital display type timer, a digital display type rotary regulator and a rotary mixer which are arranged in the upper box body, wherein the rotary mixer is parallelly fixed at the inner side of a groove of the upper box body and can rotate along the central axis of the groove, a plurality of sample placing holes are uniformly distributed on the rotary mixer, the LED blue light source is a plurality of L-shaped flat plate structures which are uniformly distributed in the upper box body, the PMA top cover is made of opaque materials and are sealed by rubber, and the PMA top cover can be freely opened and closed along the other. The invention combines dark treatment and photo-crosslinking into a whole, realizes the integration and full automation of the PMA pretreatment process, and can be used for quantitatively detecting the live bacteria of vibrio parahaemolyticus and vibrio cholerae in aquatic products.
Description
Technical Field
The invention relates to the technical field of microbiological analysis equipment, in particular to a live bacterium DNA rapid screening instrument, a using method thereof and application of the live bacterium DNA rapid screening instrument in quantitative detection of vibrio in aquatic products.
Background
Vibrio (Vibrio) is the most common pathogenic bacteria in aquatic products, Vibrio parahaemolyticus (Vibrio parahaemolyticus) is the main aquatic product pathogenic bacteria in China, and a food poisoning event caused by the Vibrio parahaemolyticus is the first of bacterial food poisoning events and is also regarded as one of the most main factors causing diarrhea diseases worldwide. Vibrio cholerae (Vibrio cholerae) is a main source causing human bodies to generate cholera diseases, is wide in prevalence and belongs to one of virulent infectious diseases, has caused large-area diseases for many times, is mainly manifested by severe vomiting, diarrhea and water loss, can cause death seriously, and has important significance on how to quickly, accurately and efficiently detect pathogenic vibrios in aquatic products.
The traditional method for quantitatively detecting vibrio parahaemolyticus and vibrio cholerae in food usually needs 1 week, and a qPCR quantitative technology based on DNA is simple, convenient and quick, but can not distinguish dead bacteria and live bacteria in a sample, so that a false positive phenomenon is easily caused. The photosensitive nucleic acid dye azide propidium bromide (PMA) is combined with a qPCR technology, can selectively identify live bacteria DNA in aquatic products, and can be used for carrying out live bacteria quantitative detection. However, the pre-treatment of the PMA includes two key steps of dark treatment and photo-crosslinking, the traditional pre-treatment method of the PMA usually carries out the dark treatment in a closed environment, and the sodium tungsten lamp which is about 600W is held by hand to carry out the photo-crosslinking (as shown in figure 1), so that the process is complicated, and the whole process is time-consuming and labor-consuming in manual operation; although a light cross-linking instrument (from PMA company) based on a blue light LED tube can greatly simplify the pre-treatment process of PMA, the light cross-linking instrument separates two steps of dark treatment and light cross-linking, and still needs complicated manual transfer operation.
Disclosure of Invention
The invention mainly aims to provide a quick screening instrument for live bacteria DNA, which can combine two core structures of dark treatment and photo-crosslinking into a whole, realize the integration and full automation of the pretreatment process of PMA and can quickly, accurately and efficiently detect pathogenic vibrios in aquatic products.
The invention further aims to provide a using method of the living bacterium DNA rapid screening instrument.
The invention further aims to provide the application of the live bacterium DNA rapid screening instrument in quantitative detection of vibrio in aquatic products.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a quick screening instrument for viable bacteria DNA, which comprises a PMA top cover, an LED blue light lamp light source and a cuboid box structure, wherein the cuboid box structure is formed by fixedly connecting an upper box body with a groove structure and a lower box body internally provided with a motor into a whole;
the device also comprises a digital display type timer, a digital display type rotation regulator and a rotation mixer which are arranged in the upper box body, wherein the rotation mixer is fixed on the inner side of the groove of the upper box body in parallel and can rotate along the central axis of the groove, and a plurality of sample placing holes are uniformly distributed on the rotation mixer;
the LED blue light lamp light sources are multiple and uniformly distributed in the upper box body;
the PMA top cap is of an L-shaped flat plate structure made of opaque materials and provided with a rubber sealing edge, and is located above the rotary blending machine, one side of the PMA top cap is fixed on one side of the opening of the groove of the upper box body, the other side of the PMA top cap can be freely opened and closed along the other side of the opening of the groove of the upper box body, and the PMA top cap is completely sealed with the opening of the groove of the upper.
Preferably, the exterior side of the PMA top cover is provided with a waterproof insulated door handle.
Preferably, the number of the LED blue light lamp light sources is 24, the power is 1-3w, the voltage parameter is 3.0-3.7V, the current is 350-.
Preferably, the rotating mixer is made of 304 stainless steel.
Preferably, the size of the living bacteria DNA rapid screening instrument is 25cm multiplied by 20 cm.
Preferably, the control panels of the digital display type timer and the digital display type rotation adjuster are located on the upper surface of the upper case.
The invention also provides a use method of the live bacteria DNA rapid screening instrument, the live bacteria DNA rapid screening instrument is adopted to carry out live bacteria DNA detection on a sample to be detected, and the method comprises the following steps:
(1) adding PMA into a sample to be detected, placing the sample into a sample placing hole of a rotary mixer, starting the viable bacteria DNA rapid screening instrument, setting parameters of dark treatment time, photo-crosslinking time and mixing speed, starting an LED blue light lamp light source, closing a PMA top cover, and forming an environment for performing dark treatment and photo-crosslinking reaction simultaneously;
(2) and (3) simultaneously carrying out dark treatment and photo-crosslinking reaction according to the set parameters: under the dark condition, PMA selectively enters dead bacteria cells with damaged cell membranes in a detection sample, and an azide group of the PMA is decomposed into a nitrene compound under the activation of an excitation light source and generates a photocrosslinking reaction with a hydrocarbon compound of double-stranded DNA to obtain a covalent crosslinking precipitate so as to inhibit the amplification of the dead bacteria DNA.
The invention also provides application of the live bacterium DNA rapid screening instrument in quantitative detection of vibrio in aquatic products.
Preferably, the Vibrio is selected from Vibrio parahaemolyticus ATCC33847 or Vibrio cholerae GIM 1.449.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention integrates the design of two core steps of dark treatment and photo-crosslinking into a whole, realizes the integration and full automation of the PMA pretreatment process, fundamentally simplifies the pretreatment process of PMA-qPCR and improves the treatment efficiency of PMA.
2. The invention can set the dark treatment time, the photocrosslinking time and the uniform mixing speed according to the self requirement, the PMA pretreatment condition is flexible, the adopted LED blue light lamp light source can keep lower heat energy output under long-time work and can not damage the sample DNA, the defect that the living bacteria DNA in the sample is burnt by a large amount of heat released when the traditional halogen lamp (600 plus 1000W) irradiates is avoided, and meanwhile, the PMA and the dead bacteria DNA are fully crosslinked, thereby being safe and reliable.
3. The detection method disclosed by the invention has no influence on dead bacteria with complete cell membranes, can completely eliminate false positive results of dead bacteria DNA, and is used for detecting pathogenic vibrios (such as vibrio parahaemolyticus ATCC33847 and vibrio cholerae GIM1.449) in aquatic products quickly, accurately and efficiently.
4. The quick screening instrument for the DNA of the viable bacteria has an integrated box-type structure, and is small and portable.
The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the invention without limiting the invention to its proper form. It is obvious that the drawings in the following description are only some embodiments, and that for a person skilled in the art, other drawings can be derived from them without inventive effort. In the drawings:
FIG. 1 is a diagram of a conventional PMA treatment process including halogen lamps and commercial PMA pre-treatment equipment;
FIG. 2 is a schematic structural diagram of a live bacteria DNA rapid screening apparatus according to an embodiment of the present invention;
FIG. 3 is a diagram showing the change of the cavity temperature with time when the living bacterium DNA rapid screening apparatus according to an embodiment of the present invention is in operation;
FIG. 4 is a standard curve of PMA dual qPCR according to an embodiment of the present invention;
the reference numbers are as follows: 1-PMA top cover, 2-excitation light source, 3-digital display type timer, 4-digital display type rotation regulator, 5-rotation mixer, 6-motor, 7-upper box body and 8-lower box body.
Detailed Description
The technical scheme of the invention is further explained by the concrete examples and the attached drawings. It should be understood that the following specific examples are illustrative only and are not limiting upon the present invention. The described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and all other embodiments obtained without any inventive work by those skilled in the art are within the scope of the present invention.
Referring to fig. 2, a fast screening instrument for viable bacteria DNA is exemplarily described, which comprises a PMA top cover 1, an LED blue light lamp light source 2, and a cuboid box structure which is fixedly connected into a whole by an upper box 7 with a groove structure and a lower box 8 with a motor 6 built in;
the device also comprises a digital display type timer 3, a digital display type rotation regulator 4 and a rotation mixer 5 which are arranged in the upper box body 7, wherein the rotation mixer 5 is fixed on the inner side of the groove of the upper box body 7 in parallel and can rotate along the central axis thereof, and a plurality of sample placing holes are uniformly distributed on the rotation mixer 5;
the LED blue light lamp light sources 2 are uniformly distributed in the upper box body 7;
the PMA top cover 1 is an L-shaped flat plate structure which is made of opaque materials and sealed by rubber, is positioned above the rotary mixer 5, one side of the PMA top cover is fixed on one side of a groove opening of the upper box body 7, the other side of the PMA top cover can be freely opened and closed along the other side of the groove opening of the upper box body 7, and when the PMA top cover is closed, the PMA top cover can be completely sealed with the groove opening of the.
In one embodiment, a waterproof insulated door handle is arranged outside the PMA top cover 1.
In one embodiment, the number of the LED blue light lamp light sources 2 is 24, the power is 1-3w, the voltage parameter is 3.0-3.7V, the current is 350-.
In one embodiment, the spin mixer 5 is 304 stainless steel.
In one embodiment, the size of the viable bacteria DNA rapid screening instrument is 25cm × 20cm × 20 cm.
In one embodiment, the control panels of the digital display type timer 3 and the digital display type rotary actuator 4 are located on the upper surface of the upper case 7.
Example 1
The live bacteria DNA rapid screening instrument shown in figure 2 is adopted to quantitatively detect the vibrio in the penaeus vannamei boone, and the steps are as follows:
(1) quick screening instrument for constructing live bacteria DNA
The LED blue light lamps are purchased from CREE of America, the total number of the LED blue light lamps is 24, the power is 1-3w, the voltage parameter is 3.0-3.7V, the current range is 350-; digital display timers were purchased from wazhou dawa instruments ltd; digital display rotary adjusters were purchased from taiwan lapping motors limited (JSCC); the transformer was purchased from taiwan mingwei ltd; the welding instrument uses argon arc welding machine and purchases from Shenzhen Ruilong welding machine, and the product model: TIG 400 GT; stainless steel panels were purchased from tungsten building, 1.5mm, food grade 304.
(2) Vibrio parahaemolyticus and Vibrio cholerae strains
The strains are Vibrio parahaemolyticus ATCC33847 and Vibrio cholerae GIM1.449, and the Vibrio cholerae GIM1.449 is a low-toxicity strain which is not O1 or O139 and is purchased from Guangdong institute of microorganisms. Taking strain liquid in the glycerol tube, streaking and inoculating the strain liquid to a Colma Jia Vibrio chromogenic medium plate, selecting a single colony in 10mL of TSB (1% NaCl, pH 8.0), and carrying out the whole process in a biological safety cabinet. Placing the inoculated bacterial liquid in a bacterial incubator with the culture condition of 37 ℃ and the rotation speed of 200r/min, and culturing overnight for 16-24h to obtain 109CFU/mL Parahemolytic vibrio culture solution, and 107CFU/mL of a culture solution of vibrio cholerae.
(3) Pretreatment of azide propidium bromide
mu.L of PMAxx (Biotium, Hayward, Calif., USA) was dissolved in 900. mu.L of double distilled water to obtain 2mM of the mother liquor, which was stored in a refrigerator at-20 ℃ in the dark. 25 mu L of PMA solution is measured by a pipette gun and added into 975 mu L of sample to obtain PMA-sample mixed solution with the final concentration of 50 mu M, and the sample is treated in the dark for 30min to ensure that PMA penetrates dead cells and is crosslinked with DNA of the dead bacteria.
The dark treated sample is placed in an instrumentDevice PMA-LiteTMIn an LED blue light photocrosslinking device (Biotium, Hayward, Calif., USA), an LED light source is used for exposure for 30min to ensure that PMA and DNA of dead bacteria are completely crosslinked together. The sample solution was centrifuged at high speed at 13,400 Xg for 10min, and the cells were collected for subsequent DNA extraction and PCR analysis.
(4) PMA pretreatment process based on viable bacteria DNA screening instrument
980. mu.L of double distilled water was added to 1mg of PMA to prepare a 2mM PMA stock solution. Adding 12.5 mu L of PMA mother liquor into a sample to be detected to obtain a mixed solution of PMA and the sample with the final concentration of 50 mu M. And (2) placing the sample in the viable bacteria DNA screening instrument constructed in the step (1), setting the dark treatment time to be 30min and the light treatment time to be 30min, and starting the instrument. The method comprises the steps of enabling dead bacteria DNA in a sample to fully react with PMA in an instrument at the rotating speed of 1000rpm to form stable covalent cross-linked precipitates so as to achieve the purpose of screening the viable bacteria DNA, centrifuging the fully reacted sample at high speed of 12000rpm to collect viable bacteria, and extracting the viable bacteria DNA in a sample by using a Tiangen bacteria DNA extraction kit, wherein the living bacteria DNA is shown in figure 3.
(5) Primers, probes and PCR reaction conditions
a. Primers and probes for quantitative detection of Vibrio parahaemolyticus and Vibrio cholerae are shown in Table 1 and are custom made by Yiwei Jie based Invitrogen, subsidiary under the heading of Thermofish, USA.
TABLE 1 primers and probes for PMA-qPCR
Note: VP: vibrio parahaemolyticus; VC Vibrio cholerae.
qPCR reaction conditions
Pre-denaturation temperature: at 95 ℃ for 2 min; denaturation 95 ℃, denaturation time: 15 s; annealing temperature: 60 ℃, annealing time: the 1min denaturation and annealing process took 40 cycles.
c.20. mu.L qPCR reaction:
0.2. mu.L Taq DNA polymerase, 0.5. mu.L forward primer, 0.5. mu.L reverse primer, 0.2mu.L of TaqMan probe, 2. mu.L of PCR reaction solution, and 1.2. mu.L of Mg2+Solution, 0.8. mu.L dNTPs solution, 1. mu.L template, and ddH2O make up to 20. mu.L.
The reagents were obtained from the Acriegat Invitrogen corporation, subsidiary under the Thermofish flag, USA, and the PCR reaction was carried out in a 7500Fast real-time PCR instrument.
(6) Specificity analysis of PMA Dual qPCR technology
The strains used for specific detection of PMA by dual qPCR are shown in Table 2, and include 62 strains of Vibrio parahaemolyticus, 31 strains of Vibrio cholerae, 3 other strains of Vibrio, and 7 strains of non-Vibrio. DNA of different strains is extracted by using a Tiangen bacterium DNA extraction kit, and PMA double qPCR detection is carried out to verify the specificity and specificity of the PMA double qPCR, and each group of experiments are repeated for 3 times.
TABLE 2 strains for specificity analysis
| Species of the Strain | Strain numbering | Number of strains | |
| Genus Vibrio | |||
| Vibrio parahaemolyticus | ATCC 33847 | 1 | |
| ATCC 17802 | 1 | ||
| VPD1-VPD20 (aquatic sample isolate) | 20 | ||
| VPC1-VPC40 (clinical specimen isolate) | 40 | ||
| Vibrio cholerae | GIM1.449 | 1 | |
| VCW1-VCW30 (aquatic sample isolate) | 30 | ||
| Vibrio anguillarum | CICC 10475 | 1 | |
| Vibrio fluvialis | CGMCC 1.1611 | 1 | |
| Vibrio | MCCC 1H0006 | 1 | |
| Other genera of genera | - | - | |
| Listeria monocytogenes | ATCC 19112 | 1 | |
| Listeria engleri | ATCC 33090 | 1 | |
| Listeria Williams | ATCC 43548 | 1 | |
| Salmonella enteritidis | CMCC 50041 | 1 | |
| Salmonella typhimurium | CICC 21484 | 1 | |
| |
ATCC 43889 | 1 | |
| Staphylococcus aureus | CCTCC AB 91093 | 1 |
(7) Construction of PMA dual qPCR technology standard curve
A standard curve is constructed based on actual samples, and fresh south American white prawns are purchased in the aquatic product market in the Pudong new area of Shanghai. Weighing 25g of Penaeus vannamei Boone, and respectively adding 225mL of alkali peptone water(APW), homogenizing in a homogenizer to obtain a homogeneous solution of the penaeus vannamei boone. Get 109CFU/mL Vibrio parahaemolyticus and 107Diluting the CFU/mL vibrio cholerae bacterial solution with Penaeus vannamei homogeneous solution to obtain carrier 101To 108CFU/mL (g) sample of Vibrio parahaemolyticus, and a sample carrying 101To 106CFU/mL (g) samples of Vibrio cholerae.
Performing PMA pretreatment, DNA extraction and PMA dual qPCR quantitative detection, and constructing a standard curve of reaction by using the colony counting result and the CT value of the dual qPCR to judge the lowest limit of detection (LOD) of the PMA dual qPCR. Using the formula E as 10 according to the slope (k) of the standard curve-1/k-1 calculating the amplification efficiency of the PMA twofold qPCR reaction. The results are shown in FIG. 4, in which R of the Vibrio parahaemolyticus standard curve20.980, the slope of the standard curve was-3.16, the amplification efficiency was 107.23%, R of the Vibrio cholerae standard curve20.985, the slope of the standard curve was-3.16, and the amplification efficiency was 94.92%.
(8) Preparation of dead bacteria of pathogenic vibrio
Suction 10mL, 107And (3) placing the CFU/mL of the vibrio parahaemolyticus and vibrio cholerae bacterial liquid in a high-temperature high-pressure sterilization pot at 121 ℃ for treating for 20min to obtain dead bacterial cells of the vibrio parahaemolyticus and the vibrio cholerae. The viability of the bacteria was confirmed using chromogenic culture of vibrio kema and secondary confirmation was performed with Tryptone Soy Broth (TSB) and Tryptone Soy Agar (TSA): culturing at 37 deg.C for 18-24h, and if no bacteria grow, it is proved that the bacteria have completely died.
(9) Application of PMA dual qPCR in penaeus vannamei boone
Fresh south America white shrimps for artificial inoculation are purchased from the aquatic product market in the Pudong new area of Shanghai, 9 groups of south America white shrimp samples are selected to be inoculated with dead and live bacteria in different proportions, and the specific inoculation mode is shown in Table 3. Adding 106CFU/mL (g) dead bacteria are interfered, the capability of distinguishing dead bacteria by using a PMA dual qPCR method is verified, and a traditional plate counting method and a common qPCR method are used as controls.
TABLE 3 ability of PMA Dual qPCR technique to discriminate dead viable bacteria in Penaeus vannamei
Note: VP: vibrio parahaemolyticus; VC Vibrio cholerae.
Example 2
This example is essentially the same as example 1, except that the sample was pacific oysters from the aquaculture market, purchased in the purdong new area of the shanghai. The PMA dual qPCR standard curve is shown in FIG. 4, wherein R of the Vibrio parahaemolyticus standard curve20.999%, the slope of the standard curve is-3.47, the amplification efficiency is 94.17%, and the R of the Vibrio cholerae standard curve2The results are shown in Table 4, with a slope of-3.47 for the standard curve and an amplification efficiency of 93.07%, at 0.975.
Table 4: capability of PMA dual qPCR technology in distinguishing dead bacteria from pacific oysters
Note: VP: vibrio parahaemolyticus; VC Vibrio cholerae.
Example 3
The embodiment is basically the same as the embodiment 1, and the difference is that the application sample is the prawn culture environment water body adopted in Shanghai Shangxian district Jixian shrimp industry. The PMA dual qPCR standard curve is shown in FIG. 4, wherein R of the Vibrio parahaemolyticus standard curve20.988, the slope of the standard curve was-3.49, the amplification efficiency was 93.43%, R of the Vibrio cholerae standard curve2The results are shown in Table 5, in which the standard curve has a slope of-3.49 and an amplification efficiency of 90.94% and 0.994, respectively.
Table 5: capability of PMA dual qPCR technology for distinguishing dead bacteria from live bacteria in prawn culture environment water body
Note: VP: vibrio parahaemolyticus; VC Vibrio cholerae.
Example 4
The present example is substantially the same as example 1, except that the sample is collected from the international center for aquatic products in oriental Shanghai city, and 108 samples of aquatic products including 48 shrimp samples, 23 shellfish samples, 18 freshwater fish samples, and 19 seaweed samples are collected. The results of the screening using PMA twofold qPCR technique and validation using ISO standard method (2007) are shown in table 6.
Table 6: application of PMA (Poly-alpha-amino-acid) dual qPCR (quantitative polymerase chain reaction) technology in actual aquatic product samples
Note: VP: vibrio parahaemolyticus, VC: vibrio cholerae.
The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that are not thought of through the inventive work should be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope defined by the claims.
Claims (9)
1. A quick screening instrument for viable bacteria DNA is characterized by comprising a PMA top cover, an LED blue light source and a cuboid box-type structure, wherein the cuboid box-type structure is formed by fixedly connecting an upper box body with a groove structure and a lower box body internally provided with a motor;
the device also comprises a digital display type timer, a digital display type rotation regulator and a rotation mixer which are arranged in the upper box body, wherein the rotation mixer is fixed on the inner side of the groove of the upper box body in parallel and can rotate along the central axis of the groove, and a plurality of sample placing holes are uniformly distributed on the rotation mixer;
the LED blue light lamp light sources are multiple and uniformly distributed in the upper box body;
the PMA top cap is of an L-shaped flat plate structure made of opaque materials and provided with a rubber sealing edge, and is located above the rotary blending machine, one side of the PMA top cap is fixed on one side of the opening of the groove of the upper box body, the other side of the PMA top cap can be freely opened and closed along the other side of the opening of the groove of the upper box body, and the PMA top cap is completely sealed with the opening of the groove of the upper.
2. The viable bacteria DNA rapid screening instrument according to claim 1, wherein a waterproof insulated door handle is arranged on the outer side of the PMA top cover.
3. The viable bacteria DNA rapid screening instrument of claim 1, wherein the number of the LED blue light lamp light sources is 24, the power is 1-3w, the voltage parameter is 3.0-3.7V, the current is 350-.
4. The viable bacteria DNA rapid screening instrument according to claim 1, wherein the rotary mixer is made of 304 stainless steel.
5. The viable bacteria DNA rapid screening instrument according to claim 1, wherein the size of the viable bacteria DNA rapid screening instrument is 25cm x 20 cm.
6. The viable bacteria DNA rapid screening instrument according to claim 1, wherein the control panels of the digital display type timer and the digital display type rotary regulator are located on the upper surface of the upper case.
7. The use method of the viable bacteria DNA rapid screening instrument according to any one of claims 1 to 6, characterized in that the viable bacteria DNA rapid screening instrument of any one of claims 1 to 6 is adopted to detect viable bacteria DNA of a sample to be detected, and the method comprises the following steps:
(1) adding PMA into a sample to be detected, placing the sample into a sample placing hole of a rotary mixer, starting the viable bacteria DNA rapid screening instrument, setting parameters of dark treatment time, photo-crosslinking time and mixing speed, starting an LED blue light lamp light source, closing a PMA top cover, and forming an environment for performing dark treatment and photo-crosslinking reaction simultaneously;
(2) and (3) simultaneously carrying out dark treatment and photo-crosslinking reaction according to the set parameters: under the dark condition, PMA selectively enters dead bacteria cells with damaged cell membranes in a detection sample, and an azide group of the PMA is decomposed into a nitrene compound under the activation of an excitation light source and generates a photocrosslinking reaction with a hydrocarbon compound of double-stranded DNA to obtain a covalent crosslinking precipitate so as to inhibit the amplification of the dead bacteria DNA.
8. The use of the viable bacteria DNA rapid screening instrument of any one of claims 1 to 6 in quantitative detection of vibrio in aquatic products.
9. Use according to claim 8, wherein the vibrio is selected from vibrio parahaemolyticus ATCC33847 or vibrio cholerae GIM 1.449.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH10248597A (en) * | 1997-03-17 | 1998-09-22 | Nippon Mizushiyori Giken:Kk | Prompt discrimination of viable and dead microoranismic cells and device therefor |
| CN205635610U (en) * | 2016-05-13 | 2016-10-12 | 徐敏 | Little biological detection's preprocessing device |
| CN211497643U (en) * | 2019-12-23 | 2020-09-15 | 中国疾病预防控制中心环境与健康相关产品安全所 | Microbial detection incubation box combined with EMA/PMA |
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2021
- 2021-01-21 CN CN202110082274.XA patent/CN112760212A/en active Pending
- 2021-09-17 ZA ZA2021/06864A patent/ZA202106864B/en unknown
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| JPH10248597A (en) * | 1997-03-17 | 1998-09-22 | Nippon Mizushiyori Giken:Kk | Prompt discrimination of viable and dead microoranismic cells and device therefor |
| CN205635610U (en) * | 2016-05-13 | 2016-10-12 | 徐敏 | Little biological detection's preprocessing device |
| CN211497643U (en) * | 2019-12-23 | 2020-09-15 | 中国疾病预防控制中心环境与健康相关产品安全所 | Microbial detection incubation box combined with EMA/PMA |
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| CN114891903A (en) * | 2022-04-25 | 2022-08-12 | 华南农业大学 | Kit and method for rapidly detecting salmonella live bacteria based on PMAxx-LAMP-NALFA |
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