CN110093430A - It is a kind of for detecting the high-throughput quantification detection kit of bathing beach pathogenic bacteria - Google Patents
It is a kind of for detecting the high-throughput quantification detection kit of bathing beach pathogenic bacteria Download PDFInfo
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Abstract
The invention discloses a kind of for detecting the high-throughput quantification detection kit of bathing beach pathogenic bacteria, feature is to include 12 kinds for detecting the LAMP detection primer group of bathing beach pathogenic bacteria, feature is gene order as shown in SEQ ID NO.1~NO.48, further includes sample template, 10 × reaction buffer, MgSO4、dNTP、BstArchaeal dna polymerase and distilled water, advantage are that high specificity, high sensitivity, flux are high, highly reliable, at low cost and without false negative result.
Description
Technical field
The present invention relates to a kind of for detecting the quick detection kit of bathing beach pathogenic bacteria, more particularly, to a kind of use
In the high-throughput quantification detection kit of detection bathing beach pathogenic bacteria.
Background technique
It entertains disease caused by the microorganism infection human body in water body and is referred to as amusement (leisure) waterborne disease
(recreational waterborne illness,RWIs).They may result in the portions such as diarrhea and lung, eye, ear, stomach
The infection of position.Existing research shows that the most common disease in bathing beach is intestines problem.People swallows or otherwise touches
Contaminated water may be infected.Every midsummer more and more people choose bathing beach and carry out aquatic recreational amenities, and
And sanitary sewage, industrial wastewater etc. increase year by year to the discharge amount of ocean, more threaten the health of bathing beach amusement crowd
Risk.Developed country 60, the human healthy risk assessment's work for just carrying out coastal seawater outdoor bathing place the seventies, and this current side of China
The research report in face is still rare, in addition China's seashore wire length, and coastal cities are numerous, therefore are highly desirable to common disease in outdoor bathing place
Pathogenic microorganism is monitored, and rationally assesses corresponding human health risk.Currently, both at home and abroad not yet disclose it is any about with
In the correlative study report of the high-throughput quantification detection kit of detection bathing beach pathogenic bacteria.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of high specificity, high sensitivity, flux it is high, it is highly reliable,
The high-throughput quantification detection kit for being used to detect bathing beach pathogenic bacteria at low cost, without false negative result.
The technical scheme of the invention to solve the technical problem is: a kind of for detecting bathing beach pathogenic bacteria
High-throughput quantification detection kit, including 12 kinds for detecting the LAMP detection primer group of bathing beach pathogenic bacteria, gene sequence
Column are as shown in table 1 below:
Table 1
The LAMP reaction system of the detection kit and the final concentration of each ingredient are as follows: 1 μ L of sample template DNA,
2.5 μ 10 × reaction buffers of L, 1.5 μ L concentration are the MgSO of 100mM4, 3.5 μ L concentration are the dNTP of 10mM, 1 μ L concentration
For 8 U/ μ l Bst DNA polymerase, 1 μ L FIP/BIP primer sets solution, 1 μ L F3/B3 primer sets solution and 4 μ L glycine betaines,
Then 25 μ L are supplied with distilled water；Wherein the FIP/BIP primer sets solution be respectively Aeromonas caviae shown in table 1,
Hydrophila, Escherichia coli O 157: H7, listeria monocytogenes, salmonella, vibrio parahaemolytious, Vibrio vulnificus, molten algae
The FIP/BIP primer sets solution of vibrios, streptococcus fecalis, Shigella, staphylococcus aureus and vibrio cholerae 01, each primer are whole
Concentration is 40 μM, and the F3/B3 primer sets solution is respectively Aeromonas caviae shown in table 1, hydrophila, big
Enterobacteria O157:H7, listeria monocytogenes, salmonella, vibrio parahaemolytious, Vibrio vulnificus, vibrio alginolyticus, excrement hammer
The F3/B3 primer sets solution of bacterium, Shigella, staphylococcus aureus and vibrio cholerae 01, each primer final concentration are 5 μM.
The detection kit further includes color developing agent, and the color developing agent is calcein/manganese chloride solution or hydroxyl
Base naphthol blue solution or SYBR-Green I, wherein calcein concentration is 75 μM in calcein/manganese chloride solution, chlorination
Manganese concentration is 500 μM；The hydroxynaphthol blue solution concentration be 150 μM, the calcein/manganese chloride solution or
The additive amount of the hydroxynaphthol blue solution is 1 hole μ L/, and the additive amount of the SYBR-Green I color developing agent is 5 μ L/
Hole.
Compared with the prior art, the advantages of the present invention are as follows: the present invention is a kind of for detecting the height of bathing beach pathogenic bacteria
The primer specific of throughput quantification detection kit design is strong, rapid amplifying can be carried out, by fluorescent dye according to the more of double-stranded DNA
Few to determine the amount of cause of disease template, to reach quantitative detection, the time just completes entire detection in 2-3h, have specificity
By force, the advantages of high sensitivity, flux are high, highly reliable, at low cost, without false negative result.
Detailed description of the invention
Fig. 1 is 12 kinds for detecting bathing beach pathogenic bacteria LAMP specific detection as a result, wherein left side is electrophoretogram, right
Side is Visual retrieval figure, A: Aeromonas caviae testing result；B: Aeromonas hydrophila testing result；C: Escherichia coli
O157:H7 testing result；D: listeria monocytogenes testing result；E: Salmeterol fluticasone propionate result；F: vibrio parahaemolytious inspection
Survey result；G: Vibrio vulnificus testing result；H: vibrio alginolyticus testing result；I: streptococcus fecalis testing result；J: shiga's bacterial examination
Survey result；K: staphylococcus aureus testing result；L: comma bacillus (01) testing result；0:DNA Marker；1: cavy gas
Monad；2: Aeromonas hydrophila；3: Escherichia coli O 157: H7；4: listeria monocytogenes；5: salmonella；6: secondary molten
Blood vibrios；7: Vibrio vulnificus；8: vibrio alginolyticus；9: streptococcus fecalis；10: Shigella；11: staphylococcus aureus；12: cholera
Vibrios (01)；13: negative control；
Fig. 2 is 12 kinds for detecting bathing beach pathogenic bacteria LAMP sensitivity technique result figure；Wherein left side is electrophoretogram, right
Side is Visual retrieval figure, A: Aeromonas caviae testing result；B: Aeromonas hydrophila testing result；C: Escherichia coli
O157:H7 testing result；D: listeria monocytogenes testing result；E: Salmeterol fluticasone propionate result；F: vibrio parahaemolytious inspection
Survey result；G: Vibrio vulnificus testing result；H: vibrio alginolyticus testing result；I: streptococcus fecalis testing result；J: shiga's bacterial examination
Survey result；K: staphylococcus aureus testing result；L: comma bacillus (01) testing result；0:DNA Marker；1: negative right
According to；8-2: being respectively 10 times of diluted plasmid templates of concentration gradient.
Fig. 3 is sample detection instance graph, and wherein A is microflow controlled biochip detection knot of the hydroxynaphthol blue as indicator
Fruit, B are microflow controlled biochip testing result of the hydroxynaphthol blue as indicator；1: Aeromonas caviae；2: thermophilic aqueous vapor list
Born of the same parents bacterium；3: Escherichia coli O 157: H7；4: listeria monocytogenes；5: salmonella；6: vibrio parahaemolytious；7: Vibrio vulnificus；
8: vibrio alginolyticus；9: streptococcus fecalis；10: Shigella；11: staphylococcus aureus；12: comma bacillus (01)；13: negative right
According to.
Specific embodiment
The present invention will be described in further detail below with reference to the embodiments of the drawings.
Specific embodiment one
It is a kind of for detecting the high-throughput quantification detection kit of bathing beach pathogenic bacteria, including 12 kinds for detecting bathing beach
The LAMP detection primer group of pathogenic bacteria, gene order are as shown in table 1 below:
Table 1
Said gene target position point design: gene design primer special between seed selection, conservative in kind is because of external primers F3
Equally have an opportunity in conjunction with the F3c in template and extend, displaces the complementary single strand of complete FIP connection.At this point, on FIP
F1c can be achieved with it is complementary with this single-stranded upper Fl, pass through self base pairing formed cyclic structure.Likewise, downstream primer BIP
There is the synthesis similar to primers F IP and F3 with B3, this just provides possibility to form the single-stranded structure of dumbbell shaped.At this point, 3 '
The Fl section at end will carry out DNA synthesis using itself as template, be formed under the archaeal dna polymerase effect with strand displacement capability
Stem loop structure.
It is target according to the above-mentioned specific gene segment for selecting 12 kinds of bathing beach pathogenic bacteria, application
The synthesis of PRIMEREXPLORER V5 software Design primers, primer is completed by Shanghai Sheng Gong bioengineering Co., Ltd.By primer
It is compared with target, carries out a large amount of screening tests and analysis, obtain special, sensitive, stable above-mentioned F3, B3, FIP, BIP
LAMP detection primer group；Will design synthesis 4 primers carry out the reaction time and temperature optimization experiment after, determine amplification efficiency and
The best reaction condition of specificity.
Specific embodiment two
It is a kind of for detecting the high-throughput quantification detection kit of bathing beach pathogenic bacteria, LAMP reaction system and each ingredient
Final concentration is as follows: 1 μ L of sample template DNA, 2.5 μ 10 × reaction buffers of L, and 1.5 μ L concentration are the MgSO of 100mM4, 3.5
μ L concentration is the dNTP of 10mM, and 1 μ L concentration is 8 U/ μ l Bst DNA polymerase, 1 μ L FIP/BIP primer sets solution, 1 μ L
F3/B3 primer sets solution and 4 μ L glycine betaines, then supply 25 μ L with distilled water；Wherein FIP/BIP primer sets solution is respectively
Aeromonas caviae shown in table 1, hydrophila, Escherichia coli O 157: H7, listeria monocytogenes, salmonella, pair
Hemolysis vibrion, Vibrio vulnificus, vibrio alginolyticus, streptococcus fecalis, Shigella, staphylococcus aureus and vibrio cholerae 01
FIP/BIP primer sets solution, each primer final concentration are 40 μM, and F3/B3 primer sets solution is respectively cavy gas shown in table 1
Monad, hydrophila, Escherichia coli O 157: H7, listeria monocytogenes, salmonella, vibrio parahaemolytious, wound arc
The F3/B3 primer sets solution of bacterium, vibrio alginolyticus, streptococcus fecalis, Shigella, staphylococcus aureus and vibrio cholerae 01, respectively
Primer final concentration is 5 μM.
Above-mentioned detection kit further includes color developing agent, and color developing agent is calcein/manganese chloride solution or hydroxynaphthol blue
Solution or SYBR-Green I, wherein calcein concentration is 75 μM in calcein/manganese chloride solution, and manganese chloride concentration is
500μM；The hydroxynaphthol blue solution concentration is 150 μM, calcein/manganese chloride solution or hydroxynaphthol blue solution
Additive amount is 1 hole μ L/, and the additive amount of SYBR-Green I color developing agent is 5 holes μ L/.Being additionally provided with negative control is water, positive right
According to the DNA for double-strand.
Specific embodiment three
A kind of high-throughput quantification carried out using above-mentioned two detection kit of specific embodiment for detecting bathing beach pathogenic bacteria
The method of detection, comprising the following steps:
Measuring samples are taken, sample template DNA is extracted according to commercially available DNA of bacteria extracts kit, takes 1 μ L of sample template DNA, press
It is formed according to LAMP reaction system and 2.5 μ 10 × reaction buffers of L, 1.5 μ L MgSO is added4, 3.5 μ L dNTP, 1 μ L Bst DNA
Polymerase, 1 μ L FIP/BIP primer sets solution, 1 μ L F3/B3 primer sets solution and 4 μ L glycine betaines, are then supplied with distilled water
It is mixed after 25 μ L, taking 20 μ L of mixed liquor to add to each well of sample detection chip, (wherein primer sets solution is respectively cavy
Aeromonas, hydrophila, Escherichia coli O 157: H7, listeria monocytogenes, salmonella, vibrio parahaemolytious, wound
Vibrios, vibrio alginolyticus, streptococcus fecalis, Shigella, staphylococcus aureus and vibrio cholerae 01 12 kinds of bathing beaches cause a disease
The primer sets solution of bacterium, the primer sets solution of each bathing beach pathogenic bacteria are accordingly added in a well, in addition adding yin
Property control well, it is 13 that the well for participating in reaction, which has altogether), then 1 μ L color developing agent is added in each hole, opens instrument
Power switch is completed to screen self-test, opens reaction chamber cap, chip is put into instrument reaction chamber, reaction chamber cap, screen are covered
Upper click START button, instrument carry out automatically, and reaction is waited to terminate, and detection menu interface, selection inspection are entered after instrument prompt
It surveys, instrument can read the color data of chip reaction chamber automatically, and (positive is blue when use hydroxynaphthol blue as color developing agent, negative
Property is pansy；Using calcein be color developing agent when, green for the positive, yellow be feminine gender) and according to rgb value carry out concentration
Conversion, finally provides the copy number of contained bacterial nucleic acid in each hole sample.
If when using SYBR-Green I developing solution, 20 μ L of mixed liquor is taken to add to each sample-adding of sample detection chip
Hole.Power switch device is opened, is completed to screen self-test, reaction chamber cap is opened, chip is put into instrument reaction chamber, is covered anti-
Chamber cap is answered, START button is clicked on screen, instrument carries out automatically, and reaction is waited to terminate, and takes out chip, and each well adds 5
The working solution of the SYBR-Green I of μ L.Menu interface, selection detection are detected into instrument, instrument can read chip reaction automatically
The color data (green is the positive, orange for feminine gender) of room simultaneously carries out concentration conversion according to rgb value, finally provides in each sample
The copy number of contained bacterial nucleic acid.
Fig. 1 is that left side is 12 kinds of electrophorograms for being used to detect after the LAMP reaction of bathing beach pathogenic bacteria, and right side is to adopt
It is green in aforementioned manners using 12 kinds after SYBR-Green I dyestuff for detecting the LAMP reaction result of bathing beach pathogenic bacteria
Color is the positive, orange for feminine gender.As shown in Figure 1, there was only corresponding bathing beach pathogenic bacteria LAMP in sample detection chip
Detection primer is the positive, and blank control and remaining non-purpose bacterium testing result are feminine gender.
Specific embodiment four
Sensitivity test: by 10 times of gradient dilutions of plasmid respectively containing 12 kinds of pathogenic bacteria specific fragments, each dilution is taken to carry out
LAMP amplified reaction, the detection of 12 kinds of pathogenic bacteria LAMP methods are limited to 10-70 copies/μ L.As shown in Fig. 2, 8 being in figure
Original concentration, 8-2 are respectively 10 times of diluted templates, after carrying out LAMP reaction, use electrophoresis respectively and use SYBR-Green I
Dyeing observes result.Initial concentration A: 6.23 × 107Copies/μ L, B:2.14 × 107Copies/μ L, C:5.22
×107Copies/μ L, D:3.79 × 107Copies/μ L, E:2.06 × 107Copies/μ L, F:4.52 × 107
Copies/μ L, G:1.95 × 107Copies/μ L, H:1.86 × 107Copies/μ L, I:3.53 × 107 copies /μ
L, J:3.90 × 107Copies/μ L, K:2.27 × 107Copies/μ L, L:4.19 × 107 copies /μL。
Specific embodiment five
Sample pre-treatments are carried out according to related national standard method by doubtful foodstuff samples 2 to be measured, prepare DNA profiling, sample warp
After LAMP reacts (centrifugal type microfludic biochip) detection, A sample is shown that B passes through SYBR- using hydroxynaphthol blue
Green I dyeing.As shown in figure 3, containing listeria monocytogenes and vibrio parahaemolytious, B sample in sample A as the result is shown
In product, contain salmonella and vibrio alginolyticus.It is verified through physiological and biochemical test, the pathogenic bacteria containing detection in two above sample
(Fig. 3).
Above description is not limitation of the present invention, and the present invention is also not limited to the example above.The art it is common
Within the essential scope of the present invention, the variations, modifications, additions or substitutions made also should belong to protection of the invention to technical staff
Range.
Claims (3)
1. a kind of for detecting the high-throughput quantification detection kit of bathing beach pathogenic bacteria, it is characterised in that: including 12 kinds with
In the LAMP detection primer group of detection bathing beach pathogenic bacteria, gene order is as shown in the table
。
2. it is according to claim 1 a kind of for detecting the high-throughput quantification detection kit of bathing beach pathogenic bacteria,
Be characterized in that: the LAMP reaction system of the detection kit and the final concentration of each ingredient are as follows: 1 μ L of sample template DNA,
2.5 μ 10 × reaction buffers of L, 1.5 μ L concentration are the MgSO of 100mM4, 3.5 μ L concentration are the dNTP of 10mM, 1 μ L concentration
For 8 U/ μ l Bst DNA polymerase, 1 μ L FIP/BIP primer sets solution, 1 μ L F3/B3 primer sets solution and 4 μ L glycine betaines,
Then 25 μ L are supplied with distilled water；Wherein the FIP/BIP primer sets solution is respectively cavy gas described in claim 1
Monad, hydrophila, Escherichia coli O 157: H7, listeria monocytogenes, salmonella, vibrio parahaemolytious, wound arc
The FIP/BIP primer sets solution of bacterium, vibrio alginolyticus, streptococcus fecalis, Shigella, staphylococcus aureus and vibrio cholerae 01,
Each primer final concentration is 40 μM, the F3/B3 primer sets solution be respectively Aeromonas caviae shown in claim 1,
Hydrophila, Escherichia coli O 157: H7, listeria monocytogenes, salmonella, vibrio parahaemolytious, Vibrio vulnificus, molten algae
The F3/B3 primer sets solution of vibrios, streptococcus fecalis, Shigella, staphylococcus aureus and vibrio cholerae 01, each primer are dense eventually
Degree is 5 μM.
3. it is according to claim 2 a kind of for detecting the high-throughput quantification detection kit of bathing beach pathogenic bacteria,
Be characterized in that: the detection kit further includes color developing agent, and the color developing agent is calcein/manganese chloride solution or hydroxyl
Base naphthol blue solution or SYBR-Green I, wherein calcein concentration is 75 μM in calcein/manganese chloride solution, chlorination
Manganese concentration is 500 μM；The hydroxynaphthol blue solution concentration be 150 μM, the calcein/manganese chloride solution or
The additive amount of the hydroxynaphthol blue solution is 1 hole μ L/, and the additive amount of the SYBR-Green I color developing agent is 5 μ L/
Hole.
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