CN106222274B - quick detection method for hollisgilettia - Google Patents
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Abstract
The invention discloses specific primers for Holisgerettia, which are external primers, internal primers and pair loop primers designed according to a conserved region of a parahemolytic arc strain specific gene fur, and also discloses a rapid detection method for Holisgerettia, which comprises the following steps of extracting DNA of the Holisgerettia, verifying peripheral primers, establishing a loop-mediated isothermal amplification reaction system, establishing a loop-mediated isothermal amplification program, and detecting fluorescence signals of amplification products2CFU/mL; the specificity is strong, only the hollisgilliet bacteria are detected, and the detection results of other pathogenic bacteria are negative; the detection is rapid, the detection speed is higher than that of real-time fluorescence quantitative PCR, and the detection efficiency is obviously improved; and detecting the amplification product by a real-time fluorescent quantitative PCR instrument for 20-40 min.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a detection method of vibrio parahaemolyticus.
Background
The Holisgeridiella is also named Vibrio hollisae (Vh), is formally renamed as Holisgeridiella in 2003, is of the Geriteria, is a gram-negative, facultative anaerobic and halophilic pathogenic bacterium, is a smaller bacillus, is widely distributed in marine products of sea, sea mouths, near coast and water areas, is a marine dominant bacterium, has pathogenicity, is 1 of 12 pathogenic vibrios, is a third pathogenic microorganism and has strong toxicity, the Holisgeridiella causes main pathogenic toxins including heat-resistant hemolytic toxins and heat-resistant related hemolytic toxins, wherein the toxins have enterotoxicity, hepatotoxicity and cytotoxicity effects, can also produce heat-resistant enterotoxins, can elongate Chinese hamster ovary cells and can cause water accumulation of mice, also has the capacity of adhering to eukaryotic cells, can enter the blood circulation to cause bacteremia or precooking of patients, and can cause serious edema due to enterotoxemia, and is caused by the food poisoning, the food poisoning caused by the Holisgeridiella, the food poisoning, the enterotoxemia is caused by the accumulation of the enterotoxemia, the food water loss, the food edema is increased by the enterotoxemia, the accumulation of the enterotoxemia, the food edema is caused by the accumulation of the enterotoxemia, the small intestine, the.
The established detection method of the Monte's hollisae includes a traditional method of a bacteria-increasing culture method, a maximum possible count method and a PCR method, a loop-mediated isothermal amplification technology is which is researched by researchers at home and abroad in recent years and is also a method developed faster than , the detection of pathogenic organisms such as bacteria, viruses and parasites is popularized by , and is popularized in field inspection and basic laboratories, at present, the method is used for detecting vibrio parahaemolyticus, Listeria monocytogenes, vibrio vulnificus, Vibrio fluvialis, Vibrio alginolyticus, Escherichia coli, O-shigella, Salmonella , tubercle bacillus and staphylococcus aureus, and no report for detecting the Monte's hollisae exists.
In view of the above, the present inventors have studied and designed a rapid detection method for species of Montmorillosis hollisi, and have resulted therefrom.
Disclosure of Invention
An th object of the present invention is to provide specific primers for use in L.hollisae.
The second purpose of the invention is to provide a detection method for kinds of Horisingnerettia.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problem is as follows:
specific primers for the hollisneritina are outer primers, inner primers and ring primers which are designed according to the conserved region of the specific gene fur of the parahemolytic arc strain;
the external primer is:
the forward outer primer GHF3 shown in the sequence table SEQ ID NO. 1: GGCAACTGTCTACCGTGTAC, respectively;
the reverse outer primer GHB3 shown in the sequence table SEQ ID NO. 2: GCTGTGATTCGTCAGTCTGA, respectively;
the inner primer is:
a forward inner primer GHFIP shown in a sequence table SEQ ID NO. 3: GTCGCCAGCTCAAACACAGATTTTTTTAACCAGTTTGATGATGCCGG, respectively;
the reverse inner primer GHBIP shown in the sequence table SEQ ID NO. 4:
CACCACCATGACCACTTGGTTTGTTTTTATCTCTCTGGCGCTGCTC；
the pair of loop primers is as follows:
a forward loop primer GHLF shown in a sequence table SEQ ID NO. 5: CCACCTTCAAAATGGTGACGGGT, respectively;
a reverse loop primer GHLB shown in the sequence table SEQ ID NO. 6: GTCGAGTCATCGAGTTTACGGACG are provided.
quick detection method of hollisnery monterella, comprising the following steps:
step , extraction of template DNA of Montmorillonella hollisi
Carrying out thermal cracking or bacterial genome DNA extraction by using a 100 ℃ water bath for 10min, wherein the kit is purchased from TIANGEN Xiamen Taijing biotechnology limited company, and total DNA of the hollisberg Monte bacteria is extracted as a template;
step two, verification of outer primers
Using to amplify the peripheral primer GHF3/GHB3 to the template, sequencing the amplified product, wherein if the amplified fragment of the peripheral primer is the nucleic acid sequence of the conserved region of the Monte bacterium hollisae fur, the peripheral primer is suitable and can be used for the subsequent loop-mediated isothermal amplification method experiment;
step three, establishment of loop-mediated isothermal amplification reaction system
A25-microliter isothermal reaction system comprises 12.5 microliter reaction liquid RM (2X), 0.8 microliter inner primer, 0.1 microliter outer primer, 0.4 microliter loop primer, 1.0 microliter Bst polymerase, 0.5 microliter fluorescent dye I, 1.0 microliter DNA template and ultrapure water added to 25 microliter, wherein the reaction liquid RM, the Bst polymerase and the fluorescent dye I are commercial DNA amplification kits and are purchased from DiAustralian Biotech Limited;
step four, loop-mediated isothermal amplification program
Placing a PCR tube to be reacted in a real-time fluorescence quantitative PCR instrument, and selecting a FAM channel for detection, wherein the reaction procedure comprises 1 cycle of reaction at 63 ℃ for 30 s; 40 cycles: reacting at 63 ℃ for 1min, and collecting a fluorescence signal at 16 s;
step five, judging the detection result of the amplification product
If the "S" type amplification curve is present, the amplification is positive, that is, nucleic acid amplification is performed, and if the "S" type amplification curve is absent, the amplification is negative, that is, nucleic acid amplification is not performed.
Compared with the prior art, the invention has the following beneficial effects:
1. high sensitivity, minimum detection is 102CFU/mL；
2. The specificity is strong, only the hollisgilliet bacteria are detected, and the detection results of other pathogenic bacteria are negative;
3. the detection is rapid, the detection speed is higher than that of real-time fluorescence quantitative PCR, and the detection efficiency is obviously improved;
4. the amplification product is detected by a real-time fluorescent quantitative PCR instrument and can be completed within 20-40 min, and the method is particularly suitable for basic experiments.
Drawings
FIG. 1 is an electrophoretogram of loop-mediated isothermal amplification products of Montelella hollisi; wherein, M: d2000 DNAmarker; 1: (ii) amplification products; 2: negative control;
FIG. 2 is a diagram showing the detection result of the real-time fluorescence quantitative PCR instrument using loop-mediated isothermal amplification; wherein, 1: loop-mediated isothermal amplification products of the hollisnery monterella; 2: negative control;
FIG. 3 is a specific electrophoresis diagram of loop-mediated isothermal amplification detection of Salmonella choleraesuis, wherein 1 is Salmonella choleraesuis, and non-Gh comprises Vibrio parahaemolyticus, Halomonas halophilus, Vibrio alginolyticus, Vibrio fluvialis, Vibrio vulnificus, Vibrio mimicus, Vibrio freudenreichii, Vibrio harveyi, Vibrio anguillarum, Enterobacter sakazakii, Listeria monocytogenes, Salmonella paratyphi A , and Escherichia coli.
Detailed Description
Example 1 design and Synthesis of primers
Three pairs of primers are designed according to the conserved region of the hollynereis species specific gene fur, and the sequences are as follows:
the external primers are:
forward outer primer GHF 3: GGCAACTGTCTACCGTGTAC, respectively;
reverse outer primer GHB 3: GCTGTGATTCGTCAGTCTGA, respectively;
the inner primer is:
forward inner primer GHFIP: GTCGCCAGCTCAAACACAGATTTTTTTAACCAGTTTGATGATGCCGG, respectively;
reverse inner primer GHBIP: CACCACCATGACCACTTGGTTTGTTTTTATCTCTCTGGCGCTGCTC, respectively;
the pair of loop primers is as follows:
forward loop primer GHLF: CCACCTTCAAAATGGTGACGGGT, respectively;
reverse loop primer GHLB: GTCGAGTCATCGAGTTTACGGACG, respectively;
the hollisnery monte bacteria species specificity gene fur is an important gene for coding and regulating the iron absorption protein of hollisnery monte bacteria, is ubiquitous in the hollisnery monte bacteria and has species specificity.
Example 2 establishment of LAMP method
Extracting template DNA of the hollisnery monterella:
a thermal cracking or bacterial genome DNA extraction kit is prepared by using 100 ℃ water bath for 10min, and the kit is purchased from TIANGEN Xiamen Taijing biotechnology limited company, and total DNA of the hollisberg Monte bacteria is extracted to be used as a template.
And (3) verifying the outer primer:
and (3) amplifying the template by using as a peripheral primer GHF3/GHB3, and sequencing an amplified product, wherein if the amplified fragment of the peripheral primer is a nucleic acid sequence of a conserved region of the Monte bacterium hollisae fur, the peripheral primer is proper and can be used for a subsequent loop-mediated isothermal amplification method experiment.
Establishing a loop-mediated isothermal amplification reaction system:
a25. mu.L isothermal reaction system included: reaction RM (2X) 12.5. mu.L; inner primer 0.8. mu.M; 0.1 mu M of outer primer; loop primer 0.4. mu.M; bst polymerase 1.0. mu.L; 0.5 mu L of fluorescent dye I; 1.0 μ L of DNA template; adding ultrapure water to 25 μ L; the reaction solution, Bst polymerase and fluorescent dye I are commercialized DNA amplification kits.
Loop-mediated isothermal amplification procedure:
LAMP reaction program: placing a PCR tube to be reacted in a real-time fluorescence quantitative PCR instrument, and selecting a FAM channel for detection, wherein the reaction procedure comprises 1 cycle of reaction at 63 ℃ for 30 s; and (4) 40 circulation: the reaction was carried out at 63 ℃ for 1min, and the fluorescence signal was collected starting at 16 s. After the reaction, the amplification product was subjected to electrophoresis, and the results are shown in FIG. 1.
And (3) judging the detection result of the amplification product: if the "S" type amplification curve is present, it is determined to be positive (nucleic acid amplification), and if the "S" type amplification curve is absent, it is determined to be negative (nucleic acid amplification is absent). The detection result of the real-time fluorescence quantitative PCR instrument of the loop-mediated isothermal amplification is shown in figure 2.
Example 3 detection of specificity by LAMP method
In order to verify the specificity in step , vibrio parahaemolyticus, halophilic monad, vibrio alginolyticus, vibrio fluvialis, vibrio vulnificus, vibrio mimicus, vibrio freundii, vibrio harveyi, vibrio anguillarum, enterobacter sakazakii, listeria monocytogenes, salmonella paratyphi bacteria and escherichia coli are respectively selected as control bacteria to be tested, and as shown in figure 3, the control bacteria result is negative.
LAMP for detecting the sensitivity of vibrio parahaemolyticus. The concentration of the pure culture is calculated by counting the plate colonies, the pure culture is diluted by 10 times of gradient, the diluted culture is thermally cracked by water bath at 100 ℃ for 10min to extract genome, and 1 mu L of the extracted genome is used for detecting the sensitivity of the LAMP reaction. The minimum detection limit of the invention is 10 after detection2CFU/mL。
All modifications which can be derived or suggested by a person skilled in the art from the present disclosure are to be considered within the scope of the invention.
Claims (1)
- The rapid detection method of hollisneritina is characterized in that the detection method is not a disease diagnosis method and comprises the following steps:outer primer, inner primer and ring primer designed according to the conserved region of the parahemolytic arc strain specific gene fur;the external primer is:the forward outer primer GHF3 shown in the sequence table SEQ ID NO. 1: GGCAACTGTCTACCGTGTAC, respectively;the reverse outer primer GHB3 shown in the sequence table SEQ ID NO. 2: GCTGTGATTCGTCAGTCTGA, respectively;the inner primer is:a forward inner primer GHFIP shown in a sequence table SEQ ID NO. 3:GTCGCCAGCTCAAACACAGATTTTTTTAACCAGTTTGATGATGCCGG；the reverse inner primer GHBIP shown in the sequence table SEQ ID NO. 4:CACCACCATGACCACTTGGTTTGTTTTTATCTCTCTGGCGCTGCTC；the pair of loop primers is as follows:a forward loop primer GHLF shown in a sequence table SEQ ID NO. 5: CCACCTTCAAAATGGTGACGGGT, respectively;a reverse loop primer GHLB shown in the sequence table SEQ ID NO. 6: GTCGAGTCATCGAGTTTACGGACG, respectively;step , extracting template DNA of the Monte bacterium hollisae:carrying out thermal cracking or bacterial genome DNA extraction by using a 100 ℃ water bath for 10min, wherein the kit is purchased from TIANGEN Xiamen Taijing biotechnology limited company, and total DNA of the hollisberg Monte bacteria is extracted as a template;step two, verifying the outer primer:using to amplify the peripheral primer GHF3/GHB3 to the template, sequencing the amplified product, wherein if the amplified fragment of the peripheral primer is the nucleic acid sequence of the conserved region of the Monte bacterium hollisae fur, the peripheral primer is suitable and can be used for the subsequent loop-mediated isothermal amplification method experiment;step three, establishing a loop-mediated isothermal amplification reaction system:A25-mu-L constant-temperature reaction system comprises 2 XRM 12.5 mu L of reaction liquid, 0.8 mu M of inner primer, 0.1 mu M of outer primer, 0.4 mu M of loop primer, 1.0 mu L of Bst polymerase, 0.5 mu L of fluorescent dye I, 1.0 mu L of DNA template and ultrapure water added to 25 mu L, wherein the reaction liquid RM, the Bst polymerase and the fluorescent dye I are commercial DNA amplification kits purchased from Australian Biotech, Inc. ;step four, a loop-mediated isothermal amplification program:placing a PCR tube to be reacted in a real-time fluorescence quantitative PCR instrument, and selecting a FAM channel for detection, wherein the reaction procedure comprises 1 cycle of reaction at 63 ℃ for 30 s; 40 cycles: reacting at 63 ℃ for 1min, and collecting a fluorescence signal at 16 s;step five, judging the detection result of the amplification product:if the "S" type amplification curve is present, the amplification is positive, that is, nucleic acid amplification is performed, and if the "S" type amplification curve is absent, the amplification is negative, that is, nucleic acid amplification is not performed.
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