CN114107530A - Salmonella detection method and kit based on droplet digital PCR technology in lettuce - Google Patents

Salmonella detection method and kit based on droplet digital PCR technology in lettuce Download PDF

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CN114107530A
CN114107530A CN202111534388.XA CN202111534388A CN114107530A CN 114107530 A CN114107530 A CN 114107530A CN 202111534388 A CN202111534388 A CN 202111534388A CN 114107530 A CN114107530 A CN 114107530A
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史贤明
方正伟
周秀娟
詹泽强
崔妍
朱瑾瑜
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Shanghai Jiaotong University
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Abstract

The invention discloses a liquid drop digital PCR detection method and a kit aiming at salmonella in lettuce, relating to the technical field of food safety detection, mainly comprising the steps of eluting salmonella in a lettuce sample, extracting DNA in an elution suspension, designing and synthesizing salmonella PCR amplification primers and probes, carrying out liquid drop PCR amplification reaction, and placing an amplified digital PCR chip in a fluorescence reader for result reading and analysis2CFU/25mL of bacterial fluid.

Description

Salmonella detection method and kit based on droplet digital PCR technology in lettuce
Technical Field
The invention relates to the technical field of food quality safety detection, in particular to a primer, a detection method and a kit for droplet digital PCR detection of salmonella in lettuce.
Technical Field
Salmonella (Salmonella) is a sporulate, capsulously, facultative anaerobic, gram-negative bacillus. It is widely distributed in nature, and can be found in warm-blooded animals or cold-blooded animals. Salmonella is one of important food-borne pathogenic bacteria, and is often transmitted by agricultural products such as meat, vegetables, eggs and the like as media, so that food poisoning is caused, and human health is harmed. After a human body is infected with salmonella, clinical symptoms such as typhoid fever, paratyphoid fever, dysentery, septicemia, gastroenteritis and the like can be caused, and the symptoms of people with low immunity (such as old people, pregnant women, infants and the like) infected with salmonella are generally serious and can die due to exhaustion if the people are not cured in time.
The outbreak of salmonella diseases is related to animal food such as polluted chicken, pork and beef, and vegetable food such as vegetables and fruits are also important carriers for the transmission of salmonella. Fruit and vegetable packages are minimally processed and ready to eat without cooking, however they are not subjected to any form of sterilization procedure and salmonella, which is not effectively inactivated, increases the frequency of food-borne disease outbreaks. Recently, in europe and other developed countries, it has been investigated that more and more salmonella infections are associated with contaminated ready-to-eat vegetables and fruits. According to statistics, the number of salmonella diseases related to fresh vegetables and fruits is increased by nearly 4.3 times in 2017 compared with 2008. Data from the american centers for disease prevention and control show that infection due to consumption of fruit and vegetables is ranked at the 3 rd position, accounting for 17.03%, in cases associated with salmonella fulminans in the united states from 2006 to 2018.
The traditional bacteriological method is adopted to detect the salmonella in the food in national standards, ISO standards and the like of all countries in the world at present, the method needs a plurality of steps, the result can be obtained in 4-7 days, the detection period is long, the workload is large, the cost is relatively high, the subjective judgment of detection personnel is relied on, the error is large, and the repeatability is poor. Therefore, in the face of increasingly complex food safety situations and continuously improved food safety control requirements, the traditional bacteriological detection method is far from meeting the requirements of current food detection, and the development of a rapid and convenient detection method becomes an urgent need for ensuring food safety.
PCR has been widely used for detecting microorganisms because of its advantages of easy and rapid operation, high sensitivity, and strong specificity. In the traditional PCR technology, target genes are subjected to PCR amplification circulation to copy thousands of progeny DNA, and then detection is performed through gel electrophoresis, so that the detection is based on an end point, and only qualitative or semi-quantitative analysis can be performed on amplification results. The real-time fluorescence quantitative PCR is to utilize fluorescence signal accumulation to monitor the whole PCR process in real time, and to quantify an unknown template through a standard curve, so that the quantitative analysis of the unknown template can be realized. Compared with fluorescent quantitative PCR, the digital PCR does not need to depend on Ct value and a standard curve, and can directly depend on the Poisson distribution principle to calculate the initial concentration of the target template in the sample. In addition, the digital PCR can effectively avoid the influence of a reaction inhibitor, and the system distribution process also greatly reduces the background sequence concentration which has a competitive effect with a target sequence, so the method is particularly suitable for detecting pathogens in complex matrixes such as food and the like. Currently, the digital PCR method has been widely used for detection of food pathogenic bacteria, Paola Cremonesi et al established a quantitative method based on droplet digital PCR, and by using 8 individual digital PCR reactions, common food-borne pathogenic bacteria in cheese, such as salmonella, listeria monocytogenes, shiga toxin-producing escherichia coli, campylobacter jejuni, etc., can be detected without selective enrichment. Yeliz et al developed a droplet digital PCR detection method using the specific target bipA of Salmonella. The Wang et al study compared the effect of fluorescent quantitative PCR and droplet digital PCR in detecting Salmonella typhimurium in milk, and compared with fluorescent quantitative PCR, droplet digital PCR has higher sensitivity, shorter pre-enrichment time and stronger inhibitor resistance. However, no relevant research on the detection of salmonella in lettuce by digital PCR has been reported.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a liquid drop digital PCR detection method for salmonella in lettuce.
The second purpose of the invention is to provide a liquid drop digital PCR detection kit aiming at salmonella in lettuce.
In order to solve the technical problems, the invention adopts the following technical scheme:
in a first aspect, the invention provides a droplet digital PCR detection method for salmonella in lettuce, which comprises the following steps:
step 1, processing a sample: eluting salmonella in the lettuce sample to obtain an elution suspension, and extracting DNA in the elution suspension to obtain a salmonella DNA sample;
step 2, designing and synthesizing salmonella PCR amplification primers and probes: the sequence of the forward primer is shown as SEQ ID NO. 1, the sequence of the reverse primer is shown as SEQ ID NO. 2, the sequence of the probe is shown as SEQ ID NO. 3, the 5 'end of the sequence of the probe is marked with a fluorescence reporter group, and the 3' end of the sequence of the probe is marked with a fluorescence quenching group;
step 3, carrying out a droplet PCR amplification reaction: adding the DNA obtained in the step (1) as a template into the primer and the probe in the step (2) to prepare a liquid drop digital PCR reaction solution, and carrying out amplification reaction;
step 4, reading fluorescence signals: and (3) placing the amplified digital PCR chip in a fluorescence reader, and reading and analyzing the result by using software.
Preferably, the lettuce samples are square small blocks of 2.5cm × 2.5cm, and the elution process comprises placing the lettuce samples into a sterile homogenizing bag, adding 25mL of sterile water, sealing the homogenizing bag, and shaking at room temperature (200r/min, 10 min).
Preferably, the extraction of DNA from the elution suspension described in step 1 is as described with reference to the TIANGEN bacterial genome extraction kit.
Preferably, the droplet digital PCR reaction system is 25. mu.L, comprising 5. mu.L of 5 XPerfect Multiplex qPCR ToughMix, 2.5. mu.L of 1. mu.M fluoroescein sodium salt, 25. mu.M forward and reverse primers, 1. mu.L each and 0.25. mu.L of 25. mu.M probe, 2. mu.L of sample DNA template and 13.25. mu.L of sterile water.
Preferably, the amplification reaction conditions in step 3 are 95 ℃ for 10 min; at 95 ℃ for 30s and at 60 ℃ for 30s, a total of 45 cycles were carried out.
On the other hand, the invention provides a liquid drop digital PCR detection kit aiming at salmonella in lettuce, which comprises liquid drop digital PCR reaction liquid and a quality control product:
the liquid drop digital PCR reaction solution contains a primer and a probe for detecting salmonella nucleic acid in lettuce, the primer and the probe comprise a forward primer shown by SEQ ID NO. 1, a reverse primer shown by SEQ ID NO. 2 and a probe shown by SEQ ID NO. 3, and two ends of the probe are marked with fluorescent groups.
Preferably, the droplet digital PCR reaction solution is a solution comprising 5 XPerfect MultiPlex qPCR Toughmix, 1. mu.M fluoroscein sodium salt, 25. mu.M forward and reverse primers, and 25. mu.M probe and sterile water.
Preferably, the quality control product comprises a negative quality control product and a positive quality control product, wherein the negative quality control product is sterile water, and the positive quality control product contains standard salmonella DNA.
As a further technical scheme, the 5 'end of the probe is labeled with a fluorescence reporter group, the 3' end of the probe is labeled with a fluorescence quenching group, the fluorescence reporter group is selected from FAM, HEX or Cy5, and the fluorescence quenching group comprises Eclipse, BHQ1 or BHQ 2.
Preferably, the probe is labeled with FAM at the 5 'end and Eclipse at the 3' end.
Compared with the prior art, the invention has the following beneficial effects:
1. the method can detect the salmonella in the lettuce without enrichment, can perform absolute quantification of the salmonella copy number, and has the detection limit as low as 5 fg/mu L and the DNA template as low as 10 fg/mu L2CFU/25mL of bacterial liquid;
2. compared with fluorescent quantitative PCR, the droplet digital PCR does not need to depend on Ct value and a standard curve, and can directly depend on the Poisson distribution principle to calculate the initial concentration of the target template in the sample. Has the advantages of high sensitivity, good specificity and high accuracy.
Drawings
FIG. 1 is a scatter diagram of a specificity experiment of a droplet digital PCR detection method for salmonella in lettuce according to the present invention;
FIG. 2 is a sensitivity experiment scatter diagram of the droplet digital PCR detection method for salmonella in lettuce according to the present invention;
FIG. 3 shows the sensitivity of the present invention for detecting pure culture of Salmonella in artificially contaminated lettuce.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Primary and primary strains
The strains used in the examples were: salmonella enteritidis ATCC 13076, Salmonella typhimurium ATCC 14028, Escherichia coli ATCC 25922, Enterobacter sakazakii ATCC 25914, Staphylococcus aureus ATCC 29213, Bacillus cereus ATCC 1220, Bacillus subtilis ATCC 6633.
Secondly, the main detecting instrument used
Naica full-automatic droplet chip-based digital PCR system (Stilla technologies, France), Sapphire chip, etc.
Third, main reagent for detection
TIANGEN DNA extraction kit, 5 XPerfectta Multiplex qPCR ToughMix, Fluoroescein sodium salt (1. mu.M) (APEXBIO), primer probe by Bao bioengineering (Dalian) Limited.
Example (b): salmonella detection method based on droplet digital PCR technology in lettuce
The method comprises the following main steps:
1. elution of Salmonella from lettuce samples
Cutting lettuce samples into square small blocks of 2.5cm × 2.5cm, placing into a sterile homogenizing bag, adding 25mL of sterile water, sealing the homogenizing bag, and shaking at room temperature for 10min at 200r/min to obtain eluted salmonella suspension.
2. Extraction of bacterial template DNA
DNA from the elution suspension was extracted using the TIANGEN bacterial genomic DNA extraction kit and as described. If the detection cannot be carried out in time, the extracted DNA can be stored at-20 ℃.
3. System preparation
Droplet digital PCR amplification system: the total volume of the reaction system of the Naica full-automatic micro-droplet chip type digital PCR system is 25 mu L, and comprises 5 XPerfectta Multiplex qPCR ToughMix 5 mu L, fluoroscein sodium salt (1 mu M)2.5 mu L, 1 mu L of each of 2 forward and reverse primers (25 mu M), 0.25 mu L of probe (25 mu M), 2 mu L of template DNA and 13.25 mu L of sterile water.
Specifically, the sequences of the designed primers and probes are shown in table 1:
TABLE 1 sequences of primers and probes
Figure BDA0003412073570000041
Figure BDA0003412073570000051
4. Droplet digital PCR reaction procedure
Reaction parameters are as follows: 10min at 95 ℃; at 95 ℃ for 30s and at 60 ℃ for 30s, a total of 45 cycles were carried out. After the system was completed, droplet dispensing and digital PCR reactions were performed. The fluorescence analysis adopts FAM single fluorescence channel, the reading and counting of the liquid drop are carried out by Naica full-automatic micro-drop chip type digital PCR system, and the liquid drop picture is analyzed to determine the fluorescence threshold value so as to calculate the number of the positive and negative liquid drops.
5. Specificity evaluation for detecting salmonella by using droplet digital PCR method
The primer probe provided by the invention is used for respectively carrying out droplet digital PCR detection on salmonella enteritidis, salmonella typhimurium, escherichia coli, enterobacter sakazakii, staphylococcus aureus, bacillus cereus, bacillus subtilis and sterile water, and the specificity of the method is verified.
As shown in fig. 1, the experimental results show that the number of droplets generated per sample is greater than 20000, consistent with the requirements of poisson distribution calculations. No positive amplification was observed with sterile water as a blank control, indicating that the system was not contaminated, amplification of FAM channel was seen in samples of salmonella enteritidis and salmonella typhimurium, while escherichia coli, enterobacter sakazakii, staphylococcus aureus, bacillus cereus, bacillus subtilis did not show positive amplification. The result shows that the primer and the probe of the liquid drop digital PCR system have good specificity and can be used for subsequent experiments. The primer probe and the liquid drop digital PCR method established by the scheme can only specifically amplify the salmonella.
6. Evaluation of DNA sensitivity of salmonella detection by droplet digital PCR method
Sequentially diluting the DNA solution of Salmonella enteritidis ATCC 13076 with concentration of 50ng/μ L according to 10 times concentration gradient, and taking the diluted DNA solution with concentration of 50 × 10-3-50×10-8The gene sequence at ng/. mu.L was detected as a template, and the amplification results are shown in FIG. 2. When the primer probe is used for carrying out sensitivity evaluation on droplet digital PCR, positive droplets can be observed to be generated in an FAM channel, fluorescence values of the positive droplets and the negative droplets are respectively gathered at one end, the limit is clear, and the generation quantity of the droplets meets the experimental requirements. The number of positive droplets decreased with decreasing template DNA concentration, when the template concentration was as low as 10-8Positive droplets can still be observed, which indicates that the sensitivity of the droplet digital PCR method on genomic DNA can reach 5 fg/. mu.L.
7. Liquid drop digital PCR method for detecting artificially polluted lettuce sample
Culturing Salmonella enteritidis ATCC 13076 in LB liquid culture medium to logarithmic phase, diluting with sterile deionized water 10 times gradient to 102CFU/mL. Get 108-1021mL of each CFU/mL salmonella bacterial liquid, and spotThe solution was dropped onto the surface of a small square of 2.5 cm. times.2.5 cm of sterile lettuce sample at about 20 spots and placed in a sterile operating table for 6 hours to dry the solution thoroughly. And putting the dried lettuce samples into a sterile homogenizing bag, adding 25mL of sterile water, sealing the homogenizing bag, then vibrating at room temperature for 10min to obtain the eluted salmonella suspension. DNA from the elution suspension was extracted using the TIANGEN bacterial genomic DNA extraction kit and as described. And (3) carrying out liquid drop digital PCR detection by using the extracted DNA as a template, wherein the detailed steps are as above, and lettuce samples without artificial pollution are used as a control group.
The amplification result is shown in figure 3, the sensitivity of the liquid drop digital PCR method for detecting the artificially polluted lettuce can reach 102CFU/25mL。
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
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Claims (10)

1. A liquid drop digital PCR detection method for salmonella in lettuce is characterized by comprising the following steps:
step 1, processing a sample: eluting salmonella in the lettuce sample to obtain an elution suspension, and extracting DNA in the elution suspension to obtain a salmonella DNA sample;
step 2, designing and synthesizing salmonella PCR amplification primers and probes: the sequence of the forward primer is shown as SEQ ID NO. 1, the sequence of the reverse primer is shown as SEQ ID NO. 2, the sequence of the probe is shown as SEQ ID NO. 3, the 5 'end of the sequence of the probe is marked with a fluorescence reporter group, and the 3' end of the sequence of the probe is marked with a fluorescence quenching group;
step 3, carrying out a droplet PCR amplification reaction: adding the DNA obtained in the step (1) as a template into the primer and the probe in the step (2) to prepare a liquid drop digital PCR reaction solution, and carrying out amplification reaction;
step 4, reading fluorescence signals: and (3) placing the amplified digital PCR chip in a fluorescence reader, and reading and analyzing the result by using software.
2. The digital PCR detection method for salmonella in lettuce according to claim 1, wherein the lettuce samples in step 1 are square small pieces of 2.5cm x 2.5cm, and the elution process comprises placing the lettuce samples in a sterile homogenizing bag, adding 25mL of sterile water, sealing the homogenizing bag, and oscillating at 200r/min for 10 min.
3. The method for detecting salmonella in lettuce by digital PCR in liquid drop as claimed in claim 1, wherein the DNA extraction process in elution suspension in step 1 is performed by referring to TIANGEN bacterial genome extraction kit.
4. The method for droplet digital PCR detection of Salmonella in lettuce according to claim 1, wherein said droplet digital PCR reaction system is 25 μ L, comprising 5 μ L5 XPerfect MultiplexqPCR ToughMix, 2.5 μ L1 μ M Fluorescein sodium salt, 25 μ M forward and reverse primers each 1 μ L and 25 μ M probe 0.25 μ L, sample DNA template 2 μ L and sterile water 13.25 μ L.
5. The method for detecting the droplet digital PCR of the salmonella in lettuce according to claim 1, wherein the amplification reaction condition in the step 3 is 95 ℃ for 10 min; 30s at 95 ℃ and 30s at 60 ℃ for 45 cycles.
6. A liquid drop digital PCR detection kit for salmonella in lettuce is characterized by comprising liquid drop digital PCR reaction liquid and a quality control product, wherein the liquid drop digital PCR reaction liquid contains a primer and a probe for detecting salmonella nucleic acid in lettuce, the primer and the probe comprise a forward primer shown by SEQ ID NO. 1, a reverse primer shown by SEQ ID NO. 2 and a probe shown by SEQ ID NO. 3, and two ends of the probe are marked with fluorescent groups.
7. The kit for droplet digital PCR detection of Salmonella in lettuce according to claim 6, wherein said droplet digital PCR reaction solution comprises 5 XPerfectta Multiplex qPCR ToughMix, 1 μ M Fluorescein sodium salt, 25 μ M forward and reverse primers, 25 μ M probe and sterile water.
8. The liquid drop digital PCR detection kit for salmonella in lettuce according to claim 6, wherein the quality control products comprise negative quality control products and positive quality control products, the negative quality control products are sterile water, and the positive quality control products contain standard salmonella DNA.
9. The kit for detecting the droplet digital PCR of the salmonella in lettuce according to claim 6, wherein the 5 'end of the probe is labeled with a fluorescence reporter group and the 3' end of the probe is labeled with a fluorescence quenching group, the fluorescence reporter group is selected from FAM, HEX or Cy5, and the fluorescence quenching group comprises Eclipse, BHQ1 or BHQ 2.
10. The kit for detecting the droplet digital PCR of the salmonella in lettuce according to claim 6, wherein FAM is labeled at the 5 'end of the probe, and Eclipse is labeled at the 3' end of the probe.
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