CN111321235A - Method for detecting number of Listeria monocytogenes in food - Google Patents
Method for detecting number of Listeria monocytogenes in food Download PDFInfo
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- 238000007847 digital PCR Methods 0.000 claims abstract description 23
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Abstract
The invention provides a method for detecting the number of Listeria monocytogenes in food, which comprises the steps of extracting genome nucleic acid from food by using a genome extraction kit, dissolving by using 50ul deionized water, performing primary quantification of a whole genome by using NanoDrop, calculating a possible copy number according to the molecular weight of Listeria monocytogenes, performing gradient dilution on a dissolved whole genome nucleic acid solution, and mixing the dissolved whole genome nucleic acid solution with a PCR reaction mixed solution and a primer probe sequence to form a reaction premixed solution; injecting the reaction premixed solution into a digital PCR reaction chip, scraping the reaction premixed solution into micropores of the reaction chip by using a special scraper, and injecting mineral oil to physically isolate the reaction micropores from the outside; and (3) sealing the digital PCR reaction chip, and then placing the chip injected with the reaction premix into an integrated digital PCR instrument for PCR reaction.
Description
Technical Field
The invention relates to the technical field of food detection of PCR chips, in particular to a method for detecting the number of Listeria monocytogenes in food based on a chip-type digital PCR method.
Background
Food safety is related to physical and mental health and property safety of consumers. Pathogenic microorganisms in food are one of important reasons for causing food safety problems, the total number of bacterial colonies is an important microorganism index for food safety, the detection of the total number of the bacterial colonies in the food is a mark for judging whether a sample is polluted by human and animal excreta and the pollution degree, and the possibility of the pollution of intestinal pathogenic bacteria in the food and the threat of latent food poisoning and epidemic disease can be presumed, so that whether the food has potential danger to human health. Therefore, each country establishes a defined standard for the total number of bacterial colonies in various foods such as meat, eggs, milk, beverages and the like, and performs a spot check and detection on the microbial content of each food according to law. For example, the total number of colonies in soy sauce and pasteurized milk cannot exceed 30000cfu/ml and the total number of colonies in fresh milk cannot exceed 200 ten thousand cfu/ml. Because the food matrix is complex and the number of microorganisms in food is small, the current detection method for the total number of colonies in food depends on bacterial culture and detection is carried out on the basis of the culture, so that the detection time needs to be finished within 1-2 days. If the current national standard culture method needs 48-72 hours to complete, the test piece for the total number of 3M colonies imported from the United states also needs 48 hours to confirm the result. For fresh and live food, the detection time is too long, the food supply is greatly influenced, and the quality of the fresh and live food is also reduced. Therefore, a rapid method for detecting the total number of colonies in food is urgently needed.
Digital PCR (digital PCR, dPCR) is a new absolute quantitative technology which is started in recent years, theoretical single-molecule amplification is realized by extreme dilution, a PCR reaction system containing DNA or RNA is divided into tens of thousands of micropores, after PCR amplification, photographing is carried out, the micropore with a fluorescent signal is judged to be 1, the micropore without the fluorescent signal is judged to be 0, and then the initial copy number of a sample is calculated by using an end-point method PCR, Poisson distribution and the number and proportion of positive micropores. Different from the traditional quantitative PCR technology, the digital PCR does not depend on the Cycle Threshold (CT) of an amplification curve for quantification, is not influenced by the amplification efficiency, does not need to adopt housekeeping genes and a standard curve, has good accuracy and reproducibility, and can realize absolute quantitative analysis. Because a standard curve does not need to be established, the method can be used for carrying out absolute quantification on the copy number of the nucleic acid with high sensitivity and accuracy under the condition of low-concentration nucleic acid content, so that the dPCR technology is widely applied to gene expression analysis, copy number variation analysis, pathogen detection, transgenic food detection and the like, and has a good application prospect.
Listeria monocytogenes is a pathogenic bacterium with high lethality in food-borne infections, and since most cases of Listeria monocytogenes occur after eating contaminated food, Listeria monocytogenes has become a major problem worldwide.
Disclosure of Invention
The present invention aims to provide a method for detecting the number of Listeria monocytogenes in food, so as to solve the technical problems.
In order to achieve the above objects, the present invention provides a method for detecting the number of Listeria monocytogenes in food,
the method comprises the following steps:
step a, extracting whole genome nucleic acid from food by using a genome extraction kit, dissolving by using deionized water, carrying out preliminary quantification of the whole genome, calculating a possible copy number according to the molecular weight of the Listeria monocytogenes, carrying out gradient dilution on a dissolved whole genome nucleic acid solution, and then selecting a proper concentration to mix with a PCR reaction mixed solution and a primer probe sequence to form a reaction premixed solution;
b, injecting the reaction premixed solution into a digital PCR reaction chip, scraping the reaction premixed solution into micropores of the reaction chip by using a special scraper, and injecting mineral oil to physically isolate the reaction micropores from the outside;
step c, sealing the digital PCR reaction chip, and then placing the chip injected with the reaction premix into an integrated digital PCR instrument for PCR reaction;
wherein, the primer probe fragment in the step a is positioned on the inlA gene in the genome of the Listeria monocytogenes, and the size of the amplified fragment is 118 bp;
c, shooting the digital PCR chip by using a CCD camera before carrying out PCR reaction in the step c to calculate the effective hole entering rate of the reaction premix; after the PCR reaction was completed, a photograph was taken with a CCD camera, and then the photograph was processed to convert into a digital signal and the absolute quantification of the number of positive DNA copies was calculated.
Further, in the step a,
the primer sequence is as follows:
an upstream primer InlA-R: 5'-GAGACCGTGTCTGTTACATTCGTT-3', respectively;
the downstream primer InlA-F: 5'-ATGCTCAGGCAGCTACAATTACAC-3' are provided. Further, in the step a, the sequence of the probe InlA-probe is as follows: FAM-CCTATTAAT × CAGATTTTTACAGAYRCAGCTCTAGCGG-BHQ1, wherein, represent ZEN quenching group.
Further, in the above step c, the PCR reaction conditions are as follows:
pre-denaturation: the reaction temperature was 95 ℃ for 5 minutes;
and (3) cyclic reaction: the reaction temperature is 95 ℃ for 30 seconds; the reaction temperature is 55 ℃ and the duration is 30 seconds; the reaction temperature was 72 ℃ for 1 minute and cycled 45 times.
Further, the PCR reaction is carried out in PCR reaction unit, PCR reaction unit includes reaction chip, dichroic mirror, look mirror support, light source and the CCD camera, wherein, the reaction chip is arranged in and is looked mirror support below, and the dichroic mirror sets up on look mirror support, and, the dichroic mirror is 45 degrees angular layout, and the light source setting sets up in one side of dichroic mirror, sets up the CCD camera in the top of dichroic mirror for take a picture to the reaction chip.
Further, in the step c, before the PCR reaction, the CCD camera acquires a first image T1 of the reaction premix in the reaction chip before the reaction premix is injected into the microwell, and acquires the first image area S1 through the first image T1; after the injection is started, acquiring a second image T2 of the reaction premixed liquid in the reaction chip at a first preset time T1, and acquiring a second image area S2 through the second image T2, wherein the second preset time is T2; acquiring a third image T3 of the reaction premixed liquid in the reaction chip at a second preset time, and acquiring a third image area S3 through the third image T3, wherein the third preset time is T3; and acquiring an nth image of the reaction premixed liquid in the reaction chip at nth preset time, and acquiring the area Sn of the nth image through the nth image, wherein the nth preset time is tn.
Further, in the step c, the first flow rate v1 of the reaction premix is obtained through the image,
v1＝(S1-S2)x h/(t2-t1) (1)
wherein, the first image area S1, the second image area S2, the first preset time t1, the second preset time t2, h represents the height of the reaction premixed liquid;
a second flow rate v2 of the reaction premix was obtained,
V2＝(S2-S3)x h/(t3-t2) (2)
wherein, the second image area S2, the third image area S3, the second preset time t2, the third preset time t3, and h represents the height of the reaction premixed solution;
obtaining the n-1 flow velocity v (n-1) of the reaction premixed liquid,
V(n-1)＝(Sn-S(n-1))x h/[tn-t(n-1)](3)
in the formula, the n-1 image area S (n-1), the n-1 image area Sn, the n-1 preset time is t (n-1), the n-1 preset time is tn, and h represents the height of the reaction premix.
8. The method of claim 7, wherein the amount of Listeria monocytogenes in the food product is determined by,
obtaining the average flow speed Vm of the reaction premix liquid as [ V1+ V2+ … … + V (n-1) ]/(n-1),
in the formula, the first flow rate V1 of the reaction premix, the second flow rate V2 of the reaction premix, and the n-1 th flow rate V (n-1) of the reaction premix are set.
Further, the CCD camera acquires the cut-off time t0 when the area of the reaction premixed liquid of the photographed image is 0, calculates and acquires the initial volume Q1 of the reaction premixed liquid,
Q1＝Vm x t0 (4)
in the formula, the reaction premix has an average flow rate Vm and a cut-off time t 0.
Further, the air conditioner is provided with a fan,
the CCD camera respectively obtains the height h1 of the liquid level of the reaction premixed liquid in the first micropore and the surface of the first micropore on the reaction chip, the height h2 of the liquid level of the reaction premixed liquid in the second micropore and the surface of the second micropore, and the height h3 of the liquid level of the reaction premixed liquid in the third micropore and the surface of the third micropore, and then
Obtaining the error amount Q0 of the reaction premix liquid as h1 x S0+ h2 x S0+ h3 x S0 (5)
Wherein the height h1 between the liquid level of the reaction premixed liquid in the first micropore and the surface of the first micropore, the height h2 between the liquid level of the reaction premixed liquid in the second micropore and the surface of the second micropore, the height h3 between the liquid level of the reaction premixed liquid in the third micropore and the surface of the third micropore, and the cross-sectional area of each micropore is S0;
the volume of the finally obtained reaction premix, Q ═ Q1+ Q0 (6)
The initial volume of the reaction premix Q1 and the error amount of the reaction premix Q0.
Compared with the prior art, the method for detecting the number of the Listeria monocytogenes in the food has the technical effects that 50ul of deionized water is used for dissolving after the whole genome nucleic acid is extracted from the food by using the genome extraction kit, then the NanoDrop is used for carrying out the initial quantification of the whole genome, the possible copy number is calculated according to the molecular weight of the Listeria monocytogenes, and then the dissolved whole genome nucleic acid solution is subjected to gradient dilution and then is mixed with the PCR reaction premix and the primer probe sequence to form the reaction premix. The method can overcome the defects of long time consumption, insufficient accuracy, complicated operation and the like of the traditional culture identification and real-time fluorescence quantitative PCR method and the like.
Particularly, the food flora identification method provided by the invention has the advantages of simple and convenient operation, sensitivity, high efficiency, convenience for wide popularization and application and the like, and can complete the quantitative identification of pathogenic microorganism components in food within two hours; the integrated digital PCR equipment can ensure the simplicity and effectiveness of the operation of the digital PCR reaction process; the absolute quantitative property of the digital PCR technology ensures the accuracy of the method for identifying the pathogenic microorganism components in the food.
Particularly, the method can accurately measure the amount of the reaction premixed liquid, photograph the reaction premixed liquid through the CCD camera, and obtain the volume V of the reaction premixed liquid entering the reaction bin so as to calculate the effective hole entering rate of the reaction premixed liquid. The CCD camera obtains a standard image T1 of the reaction premixed liquid in the reaction chip before the reaction premixed liquid is injected into the micropores, obtains a first image area S1 through the first image T1, and obtains a preset value through n times of calculation; meanwhile, the volume of the reaction premixed liquid with micropore errors is also considered, and the amount of the reaction premixed liquid is accurately measured by combining the errors.
Drawings
Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to refer to like parts throughout the drawings.
FIG. 1 is a schematic diagram of a PCR chip detection structure according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a premixed reagent injection pattern according to an embodiment of the present invention;
FIG. 3 is a schematic cross-sectional view illustrating injection of premixed reagent according to an embodiment of the present invention.
Detailed Description
Preferred embodiments of the invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only for explaining the technical principle of the invention, and do not limit the scope of the invention.
It should be noted that in the description of the present invention, the terms of direction or positional relationship indicated by the terms "upper", "lower", "left", "right", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings, which are only for convenience of description, and do not indicate or imply that the device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention.
Furthermore, it should be noted that, in the description of the present invention, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meaning of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
Referring to fig. 1, which is a schematic view of a PCR chip detection structure according to an embodiment of the present invention, a PCR reaction apparatus according to the embodiment of the present invention includes a reaction chip 1, a dichroic mirror 2, a dichroic mirror support 3, a light source 5, and a CCD camera 4, wherein the reaction chip is disposed below the dichroic mirror support, the dichroic mirror is disposed on the dichroic mirror support, the dichroic mirror is disposed at an angle of 45 degrees, the light source is disposed at one side of the dichroic mirror, and the CCD camera is disposed above the dichroic mirror for taking a picture of the reaction chip.
In the embodiment, a specific primer probe sequence is designed based on public pathogenic microorganism genome data (such as GeneBank and the like) for carrying out digital PCR amplification reaction, a primer probe fragment related to the embodiment of the invention is positioned on an inl A gene in a Listeria monocytogenes genome, the size of an amplified fragment is 118bp, the probe sequence is designed by adopting a TaqMan probe method, and a double-quenching-group method is adopted for designing.
The primer sequence is as follows:
an upstream primer InlA-R: 5'-GAGACCGTGTCTGTTACATTCGTT-3', respectively;
the downstream primer InlA-F: 5'-ATGCTCAGGCAGCTACAATTACAC-3' are provided. Further, in the step a, the sequence of the probe InlA-probe is as follows: FAM-CCTATTAAT × CAGATTTTTACAGAYRCAGCTCTAGCGG-BHQ1, wherein, represent ZEN quenching group.
Step a, extracting whole genome nucleic acid from food by using a genome extraction kit, dissolving the whole genome nucleic acid by using 50ul of deionized water, performing primary quantification of the whole genome by using NanoDrop, calculating a possible copy number according to the molecular weight of Listeria monocytogenes, performing gradient dilution on a dissolved whole genome nucleic acid solution, and mixing the dissolved whole genome nucleic acid solution with a PCR reaction mixed solution and a primer probe sequence to form a reaction premixed solution;
b, injecting the reaction premixed solution into a digital PCR reaction chip, scraping the reaction premixed solution into micropores of the reaction chip by using a special scraper, and injecting mineral oil to physically isolate the reaction micropores from the outside;
step c, capping the digital PCR reaction chip, and then placing the chip injected with the reaction premix into an integrated digital PCR instrument for PCR reaction, wherein the reaction conditions are as follows:
the reaction temperature was 95 ℃ for 5 minutes;
and (3) cyclic reaction: the reaction temperature is 95 ℃ for 30 seconds; the reaction temperature is 55 ℃ and the duration is 30 seconds; the reaction temperature was 72 ℃ for 1 minute and cycled 45 times.
Specifically, a CCD camera is used for photographing a digital PCR chip before reaction to calculate the effective hole entering rate of the reaction premix; after the reaction was completed, a photograph was taken with a CCD camera, and the photograph was processed to convert into a digital signal, and then the absolute quantification of the number of positive DNA copies was calculated.
Specifically, in the embodiment of the invention, before the reaction, the reaction premixed liquid is photographed by the CCD camera to obtain the volume V of the reaction premixed liquid entering the reaction bin, so that the effective hole entering rate of the reaction premixed liquid can be calculated conveniently. The CCD camera acquires a standard image T1 of the reaction premixed liquid in the reaction chip before injecting the reaction premixed liquid into the micropores, and acquires a first image area S1 through the first image T1; after the injection is started, acquiring a second image T2 of the reaction premixed liquid in the reaction chip at a first preset time T1, and acquiring a second image area S2 through the second image T2, wherein the second preset time is T2; acquiring a third image T3 of the reaction premixed liquid in the reaction chip at a second preset time, and acquiring a third image area S3 through the third image T3, wherein the third preset time is T3; and acquiring an nth image of the reaction premixed liquid in the reaction chip at nth preset time, and acquiring the area Sn of the nth image through the nth image, wherein the nth preset time is tn.
Specifically, by acquiring the first flow rate v1 of the reaction premix from the image,
v1＝(S1-S2)x h/(t2-t1) (1)
wherein, the first image area S1, the second image area S2, the first predetermined time t1, and the second predetermined time t2, h represents the height of the reaction premixed solution, which is a predetermined standard value, determined by the structure of the chip itself, and is a fixed value.
The second flow rate v2 of the reaction premix is obtained from the above image,
V2＝(S2-S3)x h/(t3-t2) (2)
in the formula, the second image area S2, the third image area S3, the second predetermined time t2, the third predetermined time t3, and h represents the height of the reaction premixed solution, which is a predetermined standard value, determined by the structure of the chip itself, and is a fixed value.
Obtaining the n-1 th flow velocity v (n-1) of the reaction premixed liquid through the image,
V(n-1)＝(Sn-S(n-1))x h/[tn-t(n-1)](3)
in the formula, the nth image area S (n-1) and the nth image area Sn, the nth-1 preset time is t (n-1), the nth preset time is tn, h represents the height of the reaction premixed liquid, which is a preset standard value and is determined by the structure of the chip, and is a fixed value.
By obtaining the average flow rate Vm of the reaction premix [ V1+ V2+ … … + V (n-1) ]/(n-1) as described above,
in the formula, the first flow rate V1 of the reaction premix, the second flow rate V2 of the reaction premix, and the n-1 th flow rate V (n-1) of the reaction premix are set.
Specifically, the CCD camera continuously takes pictures, and the cutoff time t0 when the area of the reaction premixed liquid of the taken image is 0 is obtained, then the initial volume Q1 of the reaction premixed liquid is calculated and obtained,
Q1＝Vm x t0 (4)
in the formula, the reaction premix has an average flow rate Vm and a cut-off time t 0.
In order to accurately determine the volume Q of the reaction premix, the embodiment of the present invention further considers the error value of the reaction premix generated by the micro-pore leakage of the chip, wherein the reaction chip of the embodiment is provided with a first micro-pore 11, a second micro-pore 12, and a third micro-pore 13, the cross-sectional area of each micro-pore is S0, when the cut-off time t0 is reached, the height h1 between the liquid level of the reaction premix in the first micro-pore 11 and the surface of the first micro-pore, the height h2 between the liquid level of the reaction premix in the second micro-pore 12 and the surface of the second micro-pore, and the height h3 between the liquid level of the reaction premix in the third micro-pore 13 and the surface of the third micro-
Obtaining the error amount Q0 of the reaction premix liquid as h1 x S0+ h2 x S0+ h3 x S0 (5)
Wherein the height h1 between the liquid level of the reaction premixed liquid in the first micropore and the surface of the first micropore, the height h2 between the liquid level of the reaction premixed liquid in the second micropore and the surface of the second micropore, and the height h3 between the liquid level of the reaction premixed liquid in the third micropore and the surface of the third micropore, and the cross-sectional area of each micropore is S0.
The volume of the finally obtained reaction premix, Q ═ Q1+ Q0 (6)
The initial volume of the reaction premix Q1 and the error amount of the reaction premix Q0.
In the embodiment of the invention, the flow error of the reaction premixed liquid caused by the micropores is used as a part of the finally obtained reaction premixed liquid, so that accurate calculation is realized.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A method for detecting the amount of listeria monocytogenes in a food product, comprising:
step a, extracting whole genome nucleic acid from food by using a genome extraction kit, dissolving by using deionized water, carrying out preliminary quantification of the whole genome, calculating a possible copy number according to the molecular weight of the Listeria monocytogenes, carrying out gradient dilution on a dissolved whole genome nucleic acid solution, and then selecting a proper concentration to mix with a PCR reaction mixed solution and a primer probe sequence to form a reaction premixed solution;
b, injecting the reaction premixed solution into a digital PCR reaction chip, scraping the reaction premixed solution into micropores of the reaction chip by using a special scraper, and injecting mineral oil to physically isolate the reaction micropores from the outside;
step c, sealing the digital PCR reaction chip, and then placing the chip injected with the reaction premix into an integrated digital PCR instrument for PCR reaction;
wherein, the primer probe fragment in the step a is positioned on the inl A gene in the genome of the Listeria monocytogenes, and the size of the amplified fragment is 118 bp;
c, shooting the digital PCR chip by using a CCD camera before carrying out PCR reaction in the step c to calculate the effective hole entering rate of the reaction premix; after the PCR reaction was completed, a photograph was taken with a CCD camera, and then the photograph was processed to convert into a digital signal and the absolute quantification of the number of positive DNA copies was calculated.
2. The method of claim 1, wherein in step a,
the primer sequence is as follows:
an upstream primer InlA-R: 5'-GAGACCGTGTCTGTTACATTCGTT-3', respectively;
the downstream primer InlA-F: 5'-ATGCTCAGGCAGCTACAATTACAC-3' are provided.
3. The method for detecting the number of Listeria monocytogenes in food according to claim 2, wherein in step a, the sequence of the probe InlA-probe is: FAM-CCTATTAAT × CAGATTTTTACAGAYRCAGCTCTAGCGG-BHQ1, wherein, represent ZEN quenching group.
4. The method of claim 1, wherein in step c, the PCR reaction conditions are as follows:
pre-denaturation: the reaction temperature was 95 ℃ for 5 minutes;
and (3) cyclic reaction: the reaction temperature is 95 ℃ for 30 seconds; the reaction temperature is 55 ℃ and the duration is 30 seconds; the reaction temperature was 72 ℃ for 1 minute and cycled 45 times.
5. The method according to claim 4, wherein the PCR reaction is performed in a PCR reaction apparatus, the PCR reaction apparatus comprises a reaction chip, a dichroic mirror holder, a light source and the CCD camera, wherein the reaction chip is disposed under the dichroic mirror holder, the dichroic mirror is disposed on the dichroic mirror holder, the dichroic mirror is disposed at an angle of 45 degrees, the light source is disposed at one side of the dichroic mirror, and the CCD camera is disposed above the dichroic mirror for taking a picture of the reaction chip.
6. The method of claim 5, wherein before PCR in step c, the CCD camera obtains a first image T1 of the reaction premix in the reaction chip before injecting the reaction premix into the microwell, and the first image area S1 is obtained from the first image T1; after the injection is started, acquiring a second image T2 of the reaction premixed liquid in the reaction chip at a first preset time T1, and acquiring a second image area S2 through the second image T2, wherein the second preset time is T2; acquiring a third image T3 of the reaction premixed liquid in the reaction chip at a second preset time, and acquiring a third image area S3 through the third image T3, wherein the third preset time is T3; and acquiring an nth image of the reaction premixed liquid in the reaction chip at nth preset time, and acquiring the area Sn of the nth image through the nth image, wherein the nth preset time is tn.
7. The method of claim 6, wherein in step c, the pre-mix is subjected to image acquisition at a first flow rate v1,
v1＝(S1-S2)x h/(t2-t1) (1)
wherein, the first image area S1, the second image area S2, the first preset time t1, the second preset time t2, h represents the height of the reaction premixed liquid;
a second flow rate v2 of the reaction premix was obtained,
V2＝(S2-S3)x h/(t3-t2) (2)
wherein, the second image area S2, the third image area S3, the second preset time t2, the third preset time t3, and h represents the height of the reaction premixed solution;
obtaining the n-1 flow velocity v (n-1) of the reaction premixed liquid,
V(n-1)＝(Sn-S(n-1))x h/[tn-t(n-1)](3)
in the formula, the n-1 image area S (n-1), the n-1 image area Sn, the n-1 preset time is t (n-1), the n-1 preset time is tn, and h represents the height of the reaction premix.
8. The method of claim 7, wherein the amount of Listeria monocytogenes in the food product is determined by,
obtaining the average flow speed Vm of the reaction premix liquid as [ V1+ V2+ … … + V (n-1) ]/(n-1),
in the formula, the first flow rate V1 of the reaction premix, the second flow rate V2 of the reaction premix, and the n-1 th flow rate V (n-1) of the reaction premix are set.
9. The method of claim 8, wherein the CCD camera obtains the cut-off time t0 when the area of the reaction premix in the captured image is 0, and then obtains the initial volume Q1 of the reaction premix by calculation,
Q1＝Vm x t0 (4)
in the formula, the reaction premix has an average flow rate Vm and a cut-off time t 0.
10. The method of claim 9, wherein the amount of Listeria monocytogenes in the food product is determined by,
the CCD camera respectively obtains the height h1 of the liquid level of the reaction premixed liquid in the first micropore and the surface of the first micropore on the reaction chip, the height h2 of the liquid level of the reaction premixed liquid in the second micropore and the surface of the second micropore, and the height h3 of the liquid level of the reaction premixed liquid in the third micropore and the surface of the third micropore, and then
Obtaining the error amount Q0 of the reaction premix liquid as h1 x S0+ h2 x S0+ h3 x S0 (5)
Wherein the height h1 between the liquid level of the reaction premixed liquid in the first micropore and the surface of the first micropore, the height h2 between the liquid level of the reaction premixed liquid in the second micropore and the surface of the second micropore, the height h3 between the liquid level of the reaction premixed liquid in the third micropore and the surface of the third micropore, and the cross-sectional area of each micropore is S0;
the volume of the finally obtained reaction premix, Q ═ Q1+ Q0 (6)
The initial volume of the reaction premix Q1 and the error amount of the reaction premix Q0.
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