CN111154633A - Kit for quickly extracting and amplifying bacterial nucleic acid carried by high-frequency cross-border vector insects - Google Patents
Kit for quickly extracting and amplifying bacterial nucleic acid carried by high-frequency cross-border vector insects Download PDFInfo
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Abstract
The kit for quickly extracting and amplifying the bacterial nucleic acid carried by the high-frequency cross-border vector insects belongs to the technical field of biology, and is characterized in that the overall structure of the kit for quickly extracting and amplifying the bacterial nucleic acid carried by the high-frequency cross-border vector insects comprises a liquid containing bottle (1), a freezing bottle (2), a normal temperature box (3), a freezing box (4), a reagent box cover (5), a reagent box body (6), a kit hinge (7), a kit lock catch (8) and a liquid containing bottle fixing plate (9); the used reagents comprise PCR reaction solution, high-success-rate PCR enzyme, lysis buffer solution and label primers, and are characterized in that: the nucleotide sequence of the tag primer used for amplifying the target fragment is shown as Seq ID No: 1 and Seq ID No: 2, respectively.
Description
Technical Field
The invention relates to a kit for quickly extracting and amplifying bacterial nucleic acid carried by high-frequency cross-border vector insects, belonging to the technical field of biology.
Background
The high-frequency cross-border vector organisms carry a large amount of bacteria and pathogens, and the current detection of the bacterial pathogens carried by the vector organisms mainly adopts a microorganism automatic identification system and 16SrRNA, but the methods have defects. The second-generation sequencing technology is widely used for high-throughput detection of environmental microorganisms, but no sample preparation method and standard specially used for second-generation sequencing detection of vector-carrying bacteria exist at present, the conventional general method cannot extract enough genome DNA from the vector-carrying bacteria to complete subsequent experiments, and a proper standard gene is not screened out to be used for detecting the vector-carrying bacteria. How to extract enough genomic DNA from the vector-carrying bacteria to complete subsequent experiments and to screen out a suitable standard gene for detecting the vector-carrying bacteria becomes a major problem to be solved urgently? Therefore, the invention discloses a kit for quickly extracting and amplifying bacterial nucleic acid carried by high-frequency cross-border vector insects.
Disclosure of Invention
In order to overcome the difficulty that how to extract enough genome DNA from the vector-carrying bacteria to complete subsequent experiments and screen out a proper standard gene for detecting the vector-carrying bacteria, the invention provides a high-frequency cross-border vector insect-carried bacterial nucleic acid rapid extraction amplification kit. The kit aims to provide a method and a kit for extracting and amplifying bacterial nucleic acid carried by high-frequency cross-border vector insects, which have the advantages of rapidness, high yield, high repeatability and good effect.
The technical scheme adopted by the invention for solving the technical problems is as follows:
the invention relates to a kit for quickly extracting and amplifying bacterial nucleic acid carried by high-frequency cross-border vector insects, which has an integral structure consisting of a liquid containing bottle 1, a freezing bottle 2, a normal temperature box 3, a freezing box 4, a reagent box cover 5, a reagent box body 6, a reagent box hinge 7, a reagent box lock catch 8 and a liquid containing bottle fixing plate 9, wherein the liquid containing bottle 1 is used for containing buffer solution and ddH2The bottle for the liquid reagents such as O and the like which can be stored at normal temperature is made of glass or plastic and comprises a bottle body and a bottle cap, the total volume of the liquid is 100 plus 150 ml, the lower part of the bottle body is a bottle body, the bottle body is cylindrical, the inner surface of the bottle body is smooth, the diameter of the cylindrical bottle body is 5-10 cm, and the height of the cylindrical bottle body is 5-10 cm; the upper part of the bottle body is a bottleneck, the lower part of the bottleneck is in a circular truncated cone shape, the lower edge of the circular truncated cone is connected with the upper edge of the bottle body, the upper edge of the circular truncated cone is connected with the lower edge of the upper part of the bottleneck, the upper part of the bottleneck is in a cylindrical shape, the diameter of the cylindrical bottleneck is 3-5 cm, the height of the cylindrical bottleneck is 2-3 cm, and the outer surface of the upper part of the bottleneck; all the liquid bottles 1 are placedIn the room temperature chamber 3. The freezing bottle 2 is a bottle for containing liquid reagents such as dNTP, high-success-rate PCR enzyme, forward primers, reverse primers, template DNA and the like which are stored at normal temperature, is made of glass or plastic, and consists of a bottle body and a bottle cap, wherein the total volume of the contained liquid is 0.5-10 ml, the lower part of the bottle body is a bottle body, the bottle body is cylindrical, the inner surface of the bottle body is smooth, the diameter of the cylindrical bottle body is 1-3 cm, and the height of the cylindrical bottle body is 3-5 cm; the upper part of the bottle body is a bottleneck, the lower part of the bottleneck is in a circular truncated cone shape, the lower edge of the circular truncated cone is connected with the upper edge of the bottle body, the upper edge of the circular truncated cone is connected with the lower edge of the upper part of the bottleneck, the upper part of the bottleneck is in a cylindrical shape, the diameter of the cylindrical bottleneck is 0.5-1 cm, the height of the cylindrical bottleneck is 0.5-1 cm, and the outer surface of the upper part of the bottleneck; all the vials 2 are placed in the freezer compartment 4. The freezing box 4 is a foam box with an ice bag inside; the upper part of the freezing box 4 is a freezing box cover 11, the lower part is a freezing box body 10, and the freezing bottle 2 and the ice bag maintaining low temperature are placed in the freezing box body 10; the thickness of the four peripheral walls of the refrigerator cover 11 is 1-2 cm, and the thickness of the top wall of the refrigerator cover 11 is 1.5-3 cm; the thickness of the four peripheral walls of the refrigerator body 10 is 1.5-3 cm, the thickness of the bottom wall is 2-3 cm, the inner side of the upper edge of the four peripheral walls of the refrigerator body 10 is upwards provided with a bulge with the thickness of 0.5 cm, and the height of the bulge is 1-2 cm. The constant temperature box 3 is a space left after the freezing box 4 is placed in the reagent box body 6; the normal temperature box 3 is internally provided with a liquid bottle fixing plate 9, the liquid bottle fixing plate 9 is arranged at the middle part of the normal temperature box 3 and is made of paper, plastics, aluminum alloy or stainless steel, round holes are arranged alternately on the liquid bottle fixing plate, and the diameter of the round holes is 5-10 cm. The kit for quickly extracting and amplifying the bacterial nucleic acid carried by the high-frequency cross-border vector insect is made of plastic, aluminum alloy or stainless steel and is cuboid, the length of the cuboid is 15-30 cm, the width of the cuboid is 10-20 cm, the upper part of the kit for quickly extracting and amplifying the bacterial nucleic acid carried by the high-frequency cross-border vector insect is a reagent box cover 5, the lower part of the kit is a reagent box body 6, and the reagent box cover 5 and the reagent box body 6 are movably connected together through a reagent box hinge 7. The thickness of the peripheral wall of the reagent box cover 5 is 1-10 mm, and the thickness of the top wall is 3-15 mm. The thickness of the four peripheral walls of the reagent box body 6 is 3-15 mm, the thickness of the bottom wall is 5-15 mm, and the inner side of the upper edge of the peripheral walls of the reagent box body 6 is upwards provided with a bulge and a protrusion with the thickness of 0.5-1 mmThe height of the screw is 0.5-1 cm. The kit hinge 7 is a known hinge and comprises 2 hinge pieces, wherein the 2 hinge pieces are connected through a shaft, each hinge piece is made of stainless steel and is in a cuboid shape, the length of the cuboid is 2-3 cm, the width of the cuboid is 1-2 cm, and the thickness of the cuboid is 0.5-1 mm. The structure of the reagent box lock 8 is the same as the well-known lock structure, a movable lock nose is fixed on the outer surface of the side wall of the reagent box cover 5, a rectangular hole is arranged on the lock nose, a fixed lock is fixed on the outer surface of the side wall of the reagent box body 6, and an elastic strip capable of retracting inwards is arranged on the lock.
The reagent used by the kit for quickly extracting and amplifying the bacterial nucleic acid carried by the high-frequency cross-border vector insect comprises PCR reaction liquid, high-success-rate PCR enzyme, lysis buffer and a label primer, and is characterized by comprising the following steps:
(1) collection of bacteria carried on body surface of high-frequency cross-border medium organism
Placing non-visceral tissues such as hair, feet and ears of 1 vector insect or mouse in a proper centrifuge tube, adding 0.9% NaCl solution capable of immersing the high-frequency cross-border vector organism, vortex oscillating at 800r/min for 30s, and centrifuging for a short time. The supernatant was collected and transferred to a new centrifuge tube for 13 min and centrifuged at 800 Xg for 2 min. The supernatant was discarded and the precipitate was retained. Repeat the above steps 3 times. The collected bacterial precipitates were added to 500. mu.L of 0.9% NaCl solution, suspended, and then combined and stored at 4 ℃ for further use.
The waste liquid and the waste are collected and discarded after autoclaving.
(2) Collection of bacteria carried in high-frequency cross-border medium organism
If the bacteria in the rat body are extracted, the internal organ tissues are taken for experiment, and the step is directly carried out without the treatment of the step (1).
Transferring the high-frequency cross-border medium organism treated in the step (1) into a new sterilized centrifugal tube, adding 75% ethanol which can immerse the high-frequency cross-border medium organism, soaking for 5min, and repeating for 3 times; transferring the high-frequency cross-border vector organisms into another new sterilized centrifuge tube, adding 0.9% NaCl solution capable of immersing the high-frequency cross-border vector organisms, performing vortex oscillation at 800r/min for 30s, discarding supernatant, and repeating the steps for 3 times.
The samples were placed in a biosafety cabinet and air dried for 15 min. The sample was transferred to a new centrifuge tube with sterile forceps and the specimen was ground to a paste.
Adding 3mL of physiological saline, performing vortex oscillation at 800r/min for 30s, centrifuging for a short time, transferring the supernatant into a new 10mL centrifuge tube, centrifuging for 2min at 13,800 Xg, discarding the supernatant, and leaving the precipitate. Repeating the above collection steps for 3 times, adding 500 μ L of 0.9% NaCl solution into the collected bacterial precipitate, suspending, mixing, and storing at 4 deg.C.
The waste liquid and the waste are collected and discarded after autoclaving.
(3) Collection of whole high-frequency cross-border medium biological carried bacteria
1 high-frequency cross-border medium organism or tissue is taken and placed in a proper centrifuge tube, and the specimen is ground into paste.
Adding a proper amount of 0.9% NaCl solution according to the size of the sample, carrying out vortex oscillation at 800r/min for 30s, carrying out short-time centrifugation, transferring the supernatant into a new centrifuge tube, carrying out centrifugation at 13,800 Xg for 2min, discarding the supernatant, and leaving a precipitate. Repeating the above collection steps for 3 times, adding 500 μ L of 0.9% NaCl solution into the collected bacterial precipitate, suspending, mixing, and storing at 4 deg.C.
The waste liquid and the waste are collected and discarded after autoclaving.
(4) Extraction of bacterial genomic DNA
The tube 13800 Xg filled with the bacterial solution was centrifuged for 2min, and the supernatant was transferred and autoclaved. The collected bacteria were extracted using a bacterial genomic DNA extraction kit (containing lysozyme digestion) or using an automatic nucleic acid extractor, and the extracted genomic DNA was stored in a refrigerator at 4 ℃ as template DNA for future use.
(5) Amplification of fragments of interest
Taking the genomic DNA obtained in the step (4) as a template, and adopting a tag primer: the forward primer 341F5 '-TCAGAGCTACTCCTACGGGAGGCAGCAG-3' and the reverse primer 806R5 '-GGACTACHVGGGTWTCTAAT-3' are used for PCR amplification to obtain the V3-V4 hypervariable region gene sequence of 16S of the high-frequency cross-border vector organism carrying bacteria.
PCR amplification system 50. mu.L: 25. mu.L of buffer (2X), 2.5. mu.L of dNTP (2mmol/L), 1. mu.L of high success rate PCR enzyme (1U/. mu.L), 1. mu.L of forward primer (20. mu. mol/L)L, 1. mu.L of reverse primer (20. mu. mol/L), 3. mu.L of template DNA, ddH2O 16.5μL。
PCR amplification conditions: pre-denaturation at 94 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 50 ℃ for 30s, and extension at 68 ℃ for 30s for 30 cycles; extending for 10min at 68 ℃; keeping the temperature at 10 ℃.
(6) Determination of PCR product concentration
Detecting the concentration and the quality of the PGR product by using a DNA concentration determinator, respectively determining the absorption values of the sample solution at 260nm, 280nm, 230nm and 270nm by taking a DNA dissolving solution as a reference, and calculating the ratios of A260/A280, A260/A230 and A260/A270, wherein the DNA sample meeting the following conditions can be regarded as high-quality genomic DNA; the A260/A280 is 1.8-1.9, the A260/A230 is more than 2.0, and the A260/A270 is 1.1-1.3.
The total amount of DNA of the PCR product was required to calculate the DNA concentration using a double-stranded DNA in which A260-1 corresponds to 50. mu.g/mL≥2 mug, the concentration is more than or equal to 50 ng/muL.
The invention has the beneficial effects that: according to the kit for rapidly extracting and amplifying the bacterial nucleic acid carried by the high-frequency cross-border vector insects, bacteria carried by the high-frequency cross-border vector organisms, especially unknown bacterial pathogens are detected as far as possible by researching a sample preparation method for carrying the bacteria by the high-frequency cross-border vector organisms by using a second-generation sequencing high-throughput sequencing technology, so that the basic condition that the high-frequency cross-border vector organisms carry the pathogens is mastered in time, the detection effect and efficiency are improved, and the detection cost is reduced; the method for collecting bacteria carried inside and outside a high-frequency cross-border vector organism is optimized, the label primers which can be used for second-generation sequencing are designed, the reaction system and the reaction conditions are optimized, the bacteria species carried by the high-frequency cross-border vector insects can be detected at one time, and particularly the species identification of unknown bacteria is realized. The method has the advantages of good stability, high amplification efficiency, and particularly great reliability and adaptability.
Drawings
The invention is further described below with reference to the accompanying drawings.
FIG. 1 is a schematic diagram of the overall structure of a kit for rapid extraction and amplification of bacterial nucleic acid carried by high-frequency cross-border vector insects.
In the figure, the position of the upper end of the main shaft,1. the kit comprises a liquid containing bottle, 2. a freezing bottle, 3. a normal temperature box, 4. a freezing box, 5. a reagent box cover, 6. a reagent box body, 7. a reagent box hinge, 8. a reagent box lock catch, 9. a liquid containing bottle fixing plate, 10. a freezing box body and 11. a freezing box cover. Wherein the reagent not requiring cryopreservation is stored in a liquid bottle, and comprises buffer solution and ddH2O; the reagent to be preserved at low temperature is placed in a freezing bottle and contains dNTP, PGR enzyme with high success rate, forward primer, reverse primer and template DNA.
FIG. 2 is a diagram of a labeled primer of a kit for rapid extraction and amplification of bacterial nucleic acid carried by high-frequency cross-border vector insects.
Detailed Description
In the context of this specification, unless otherwise indicated, any terms used herein have the meanings commonly understood in the art by those skilled in the art, and experimental procedures without specification to details are carried out according to conventional experimental procedures or according to the instructions recommended by the supplier.
Example one
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
The overall structure of the bacterial nucleic acid rapid extraction amplification kit carried by the high-frequency cross-border vector insects comprises a liquid containing bottle 1, a freezing bottle 2, a normal temperature box 3, a freezing box 4, a reagent box cover 5, a reagent box body 6, a reagent box hinge 7, a kit lock catch 8, a liquid containing bottle fixing plate 9, a freezing box body 10 and a freezing box cover 11, wherein the liquid containing bottle 1 contains buffer solution and ddH2The bottle for the liquid reagents such as O and the like which can be stored at normal temperature is made of glass or plastic and comprises a bottle body and a bottle cap, the total volume of the liquid is 100 plus 150 ml, the lower part of the bottle body is a bottle body, the bottle body is cylindrical, the inner surface of the bottle body is smooth, the diameter of the cylindrical bottle body is 5-10 cm, and the height of the cylindrical bottle body is 5-10 cm; the upper part of the bottle body is a bottle neck, the lower part of the bottle neck is in a circular truncated cone shape, the lower edge of the circular truncated cone is connected with the upper edge of the bottle body, the upper edge of the circular truncated cone is connected with the lower edge of the upper part of the bottle neck, the upper part of the bottle neck is in a cylindrical shape, the diameter of the cylindrical bottle neck is 3-5 cm, the height of the cylindrical bottle neck is 2-3 cm, the outer surface of the upper part of the bottle neck is provided withThe lines are meshed, so that the bottle cap can seal the bottle body after the bottle cap is screwed on the bottle neck, and liquid contained in the liquid containing bottle 1 is prevented from overflowing; all the liquid bottles 1 are placed in the normal temperature box 3. The freezing bottle 2 is a bottle for containing liquid reagents such as dNTP, high-success-rate PCR enzyme, forward primers, reverse primers, template DNA and the like which are stored at normal temperature, is made of glass or plastic, and consists of a bottle body and a bottle cap, wherein the total volume of the contained liquid is 0.5-10 ml, the lower part of the bottle body is a bottle body, the bottle body is cylindrical, the inner surface of the bottle body is smooth, the diameter of the cylindrical bottle body is 1-3 cm, and the height of the cylindrical bottle body is 3-5 cm; the upper part of the bottle body is a bottle neck, the lower part of the bottle neck is in a circular truncated cone shape, the lower edge of the circular truncated cone is connected with the upper edge of the bottle body, the upper edge of the circular truncated cone is connected with the lower edge of the upper part of the bottle neck, the upper part of the bottle neck is in a cylindrical shape, the diameter of the cylindrical bottle neck is 0.5-1 cm, the height of the cylindrical bottle neck is 0.5-1 cm, the outer surface of the upper part of the bottle neck is provided with a protruding thread so as to be meshed with the protruding thread on the inner surface of the side wall of the bottle cap, and therefore after the bottle cap; all the vials 2 are placed in the freezer compartment 4. The freezing box 4 is a foam box with an ice bag inside, the low temperature in the freezing box 4 is maintained by the ice bag, once the bacterial nucleic acid rapid extraction amplification kit carried by the high-frequency cross-border vector insects is bought to a hand, the freezing box 4 is taken out of the kit as soon as possible and placed in a refrigerating layer of a refrigerator for long-term storage for later use; the freezing box 4 is made of foam, the upper part is a freezing box cover 11, the lower part is a freezing box body 10, and the freezing bottle 2 and the ice bag maintaining low temperature are placed in the freezing box body 10; the thickness of the four peripheral walls of the refrigerator cover 11 is 1-2 cm, the thickness of the top wall of the refrigerator cover 11 is 1.5-3 cm, the refrigerator cover 11 can be buckled with the refrigerator 10 after covering the upper part of the refrigerator 10, and after the refrigerator cover 11 is buckled with the refrigerator 10, the gap on the outer surface is sealed by using a transparent adhesive tape, so that the cavity in the refrigerator 10 can be separated from the outside, and the cavity in the refrigerator 10 can be kept in a low-temperature state for a long time; the thickness of the four peripheral walls of the refrigerator body 10 is 1.5-3 cm, the thickness of the bottom wall is 2-3 cm, the inner side of the upper edge of the four peripheral walls of the refrigerator body 10 is upwards provided with a bulge with the thickness of 0.5 cm, and the height of the bulge is 1-2 cm, so that the refrigerator is convenientThe lid 11 and the freezer compartment 10 snap together well. The constant temperature box 3 is the space left after the freezing box 4 is placed in the reagent box body 6 and is used for containing the liquid containing bottle 1; the normal temperature box 3 is internally provided with a liquid bottle fixing plate 9, the liquid bottle fixing plate 9 is arranged at the middle part of the normal temperature box 3 and is made of paper, plastic, aluminum alloy or stainless steel, round holes are alternately arranged on the normal temperature box, the diameter of each round hole is 5-10 cm, and after the liquid bottle 1 is placed in the round hole on the liquid bottle fixing plate 9, the liquid bottle fixing plate 9 can play the purpose of stabilizing and isolating the liquid bottle 1. The kit for quickly extracting and amplifying the bacterial nucleic acid carried by the high-frequency cross-border vector insect is made of plastic, aluminum alloy or stainless steel and is cuboid, the length of the cuboid is 15-30 cm, the width of the cuboid is 10-20 cm, the upper part of the kit for quickly extracting and amplifying the bacterial nucleic acid carried by the high-frequency cross-border vector insect is a reagent box cover 5, the lower part of the kit is a reagent box body 6, and the reagent box cover 5 and the reagent box body 6 are movably connected together through a reagent box hinge 7 so that the reagent box cover 5 can be lifted or fall down when articles in the kit are taken out and placed. The thickness of the peripheral wall of the reagent box cover 5 is 1-10 mm, the thickness of the top wall is 3-15 mm, the reagent box cover 5 can be buckled with the reagent box body 6 after being covered on the upper part of the reagent box body 6, after the reagent box cover 5 and the reagent box body 6 are buckled together, the reagent box lock catch 8 on the outer side of the other side wall opposite to the reagent box hinge 7 can buckle the reagent box cover 5 and the reagent box body 6 together, and articles placed in the reagent box body 6 cannot fall off. The thickness of the peripheral wall of the reagent box body 6 is 3-15 mm, the thickness of the bottom wall is 5-15 mm, the inner side of the upper edge of the peripheral wall of the reagent box body 6 is upwards provided with a bulge with the thickness of 0.5-1 mm, and the height of the bulge is 0.5-1 cm, so that the reagent box cover 5 and the reagent box body 6 can be well buckled together. The kit hinge 7 is a known hinge and comprises 2 hinge pieces, wherein the 2 hinge pieces are connected through a shaft, each hinge piece is made of stainless steel and is in a cuboid shape, the length of the cuboid is 2-3 cm, the width of the cuboid is 1-2 cm, and the thickness of the cuboid is 0.5-1 mm. The structure of the kit lock 8 is the same as the well-known lock structure, the movable lock nose is fixed on the outer surface of the side wall of the reagent box cover 5, the lock nose is provided with a rectangular hole, the immovable lock is fixed on the outer surface of the side wall of the reagent box body 6, the lock is provided with an elastic strip which can retract inwards, after the lock nose is pressed on the lock, the elastic strip extends out, and a test is carried outThe reagent box cover 5 and the reagent box body 6 are buckled together; when the reagent box cover is required to be opened, the elastic strip is pressed down and retracted into the lock catch, and then the lock nose can be separated from the lock catch, so that the reagent box cover 5 and the reagent box body 6 are separated.
KOD FX Neo polymerase was purchased from Shanghai Biotech, Inc., Toyobo. Primer synthesis is completed by Dalian Co., Ltd, and DNA sequencing is completed by Huada gene.
Example 1 effect verification: rapid detection and identification of bacterial species in and out of fly carrier
In 2018, 12 fly samples are collected in four seasons in the whole year for experiments, and the sample specification is shown in table 1. Flies stored in the refrigerator were removed and bacteria carried in and out of the fly body were collected. The method comprises the following specific operation steps:
TABLE 1 sample information
1 collection of bacteria carried on the surface of the fly body:
1 fly is taken and placed in a moderate centrifuge tube, 2mL of 0.9% NaCl solution is added, vortex oscillation is carried out at 800r/min for 30s, and the mixture is centrifuged for a short time. The supernatant was collected and transferred to a new centrifuge tube for 13 min and centrifuged at 800 Xg for 2 min. The supernatant was discarded and the precipitate was retained. Repeat the above steps 3 times. The collected bacterial precipitates were added to 500. mu.L of 0.9% NaCl solution, suspended, and then combined and stored at 4 ℃ for further use.
The waste liquid and the waste are collected and discarded after autoclaving.
2 Collection of bacteria carried in fly
Transferring the sample treated by the step 1 into a new sterilized centrifugal tube, adding 75% ethanol of a 3ML substance, soaking for 5min, and repeating for 3 times; the sample was transferred to another fresh sterilized centrifuge tube, then 2mL of 0.9% NaCl solution was added, vortexed at 800r/min for 30s, the supernatant was discarded, and the above procedure was repeated 3 times.
The samples were placed in a biosafety cabinet and air dried for 15 min. The sample was transferred to a new centrifuge tube with sterile forceps and the specimen was ground to a paste.
Adding 3mL of physiological saline, performing vortex oscillation at 800r/min for 30s, centrifuging for a short time, transferring the supernatant into a new 10mL centrifuge tube, centrifuging for 2min at 13,800 Xg, discarding the supernatant, and leaving the precipitate. Repeating the above collection steps for 3 times, adding 500 μ L of 0.9% NaCl solution into the collected bacterial precipitate, suspending, mixing, and storing at 4 deg.C.
The waste liquid and the waste are collected and discarded after autoclaving.
(4) Extraction of bacterial genomic DNA
The bacterial genome DNA extraction kit is used for containing lysozyme for digestion or extracting the bacterial genome DNA by using an automatic nucleic acid extractor, and the extracted genomic DNA is stored in a refrigerator at 4 ℃ and used as template DNA for later use.
(5) Amplification of
Taking the genomic DNA obtained in the step 4 as a template, and adopting a label primer: the forward primer (341F5 '-TCAGAGCTACTCCTACGGGAGGCAGCAG-3') and the reverse primer (806R5 '-GGACTACHVGGGTWTCTAAT-3') are used for PCR amplification to obtain the gene sequence of the 16S V3-V4 hypervariable region of the high-frequency cross-border vector organism carrying bacteria.
PCR amplification system 50. mu.L: 2X 25. mu.L of buffer, 2.5. mu.L of dNTP 2mmol/L, 1. mu.L of high success rate PCR enzyme 1U/. mu.L, 1. mu.L of forward primer, 1. mu.L of reverse primer, 3. mu.L of template DNA, and 16.5. mu.L of ddH2O 16.5.
PCR amplification conditions: pre-denaturation at 94 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 50 ℃ for 30s, and extension at 68 ℃ for 30s for 30 cycles; extending for 10min at 68 ℃; keeping the temperature at 10 ℃.
(6) Determination of PCR product concentration
And (3) carrying out quantitative and purity analysis on bacterial DNA (deoxyribonucleic acid) extracted from a fly sample and a PCR (polymerase chain reaction) product by using a full-wavelength microplate reader, wherein the target fragment quantity of the PCR product is higher than 1.5 mu g, and the purity is 1.7-1.9, so that the library building requirement of second-generation sequencing is met. The results are shown in Table 2.
TABLE 2 bacterial dsDNA concentration and 260/280 nucleic acid purity
(7) High throughput sequencing results
405162 high-quality sequences were obtained from all the Drosophila samples, and from the number of sequences obtained from each sample, the high-quality sequences obtained from the samples were 10328 at minimum and 89572 at maximum, and these sequences were subjected to OTU clustering analysis at 97% similarity to obtain 331 OTUs, the number of which varies from 52 to 199, as shown in Table 3.
TABLE 3 sample sequence number and OTU statistics
The number of species identified by the bacteria carried by the 12 drosophila samples was 79, including 5 pathogenic bacteria and 13 conditional pathogenic bacteria, and the distribution is shown in table 4, the number of Escherichia coli (Escherichia coli) is the highest among all pathogenic bacteria, followed by aroma-like aroma bacteria (aroid odorimiums), both of which are significantly more numerous in the fourth quarter than in the other three quarters, and are the highest among drosophila rubripes; lactococcus garvieae (Lactococcus garvieae) was most abundant in the third quarter and most detected in Sarcophaga peregrina.
TABLE 4 distribution of pathogenic bacteria and conditioned pathogens in each sample
Note: is marked with "＊"is conditioned byPathogenic bacteria, marked by "＊＊"is a pathogen.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined by the appended claims and their equivalents.
Claims (3)
1. The bacterial nucleic acid rapid extraction and amplification kit carried by the high-frequency cross-border vector insects is characterized in that the overall structure of the bacterial nucleic acid rapid extraction and amplification kit carried by the high-frequency cross-border vector insects is composed of a liquid containing bottle (1), a freezing bottle (2), a normal temperature box (3), a freezing box (4), a reagent box cover (5), a reagent box body (6), a kit hinge (7), a kit lock catch (8) and a liquid containing bottle fixing plate (9); the used reagents comprise PCR reaction solution, high-success-rate PCR enzyme, lysis buffer solution and tag primers, and are characterized in that the target fragment is amplified by using the tag primers, and the nucleotide sequences of the tag primers are shown as Seq ID No: 1 and Seq ID No: 2, respectively.
2. The kit for rapidly extracting and amplifying the bacterial nucleic acid carried by the high-frequency cross-border vector insect according to claim 1, which is characterized in that: the overall structure of the high-frequency cross-border vector insect carried bacteria nucleic acid rapid extraction amplification kit is composed of a liquid containing bottle (1), a freezing bottle (2), a normal temperature box (3), a freezing box (4), a reagent box cover (5), a reagent box body (6), a kit hinge (7), a kit lock catch (8) and a liquid containing bottle fixing plate (9), wherein the liquid containing bottle (1) is used for containing buffer liquid and a liquid containing bottle fixing plate (9)ddH2The bottle for the liquid reagents such as O and the like which can be stored at normal temperature is made of glass or plastic and comprises a bottle body and a bottle cap, the total volume of the liquid is 100 plus 150 ml, the lower part of the bottle body is a bottle body, the bottle body is cylindrical, the inner surface of the bottle body is smooth, the diameter of the cylindrical bottle body is 5-10 cm, and the height of the cylindrical bottle body is 5-10 cm; the upper part of the bottle body is a bottleneck, the lower part of the bottleneck is in a circular truncated cone shape, the lower edge of the circular truncated cone is connected with the upper edge of the bottle body, the upper edge of the circular truncated cone is connected with the lower edge of the upper part of the bottleneck, the upper part of the bottleneck is in a cylindrical shape, the diameter of the cylindrical bottleneck is 3-5 cm, the height of the cylindrical bottleneck is 2-3 cm, and the outer surface of the upper part of the bottleneck; all the liquid bottles (1) are placed in the normal temperature box (3); the freezing bottle (2) is a bottle for containing liquid reagents such as dNTP, high-success-rate PCR enzyme, forward primers, reverse primers, template DNA and the like which are stored at normal temperature, is made of glass or plastic, and consists of a bottle body and a bottle cap, the total volume of the contained liquid is 0.5-10 ml, the lower part of the bottle body is a bottle body, the bottle body is cylindrical, the inner surface of the bottle body is smooth, the diameter of the cylindrical bottle body is 1-3 cm, and the height of the cylindrical bottle body is 3-5 cm; the upper part of the bottle body is a bottleneck, the lower part of the bottleneck is in a circular truncated cone shape, the lower edge of the circular truncated cone is connected with the upper edge of the bottle body, the upper edge of the circular truncated cone is connected with the lower edge of the upper part of the bottleneck, the upper part of the bottleneck is in a cylindrical shape, the diameter of the cylindrical bottleneck is 0.5-1 cm, the height of the cylindrical bottleneck is 0.5-1 cm, and the outer surface of the upper part of the bottleneck; all the freezing bottles (2) are placed in a freezing box (4); the freezing box (4) is a foam box with an ice bag inside; the upper part of the freezing box (4) is a freezing box cover (11), the lower part is a freezing box body (10), and the freezing bottle (2) and the ice bag maintaining low temperature are placed in the freezing box body (10); the thickness of the four peripheral walls of the refrigerator cover (11) is 1-2 cm, and the thickness of the top wall of the refrigerator cover (11) is 1.5-3 cm; the thickness of the four peripheral walls of the refrigerator body (10) is 1.5-3 cm, the thickness of the bottom wall is 2-3 cm, the inner side of the upper edge of the peripheral walls of the refrigerator body (10) is upwards provided with a bulge with the thickness of 0.5 cm, and the height of the bulge is 1-2 cm; the constant temperature box (3) is the space left after the freezing box (4) is placed in the reagent box body (6); a liquid bottle fixing plate (9) is arranged in the normal temperature box (3), the liquid bottle fixing plate (9) is arranged in the middle of the normal temperature box (3) and is made of paper, plastic, aluminum alloy or stainless steel, and the liquid bottle fixing plate are alternately arranged on the paper, plastic, aluminum alloy or stainless steelThe diameter of the round hole is 5-10 cm; the kit for quickly extracting and amplifying the bacterial nucleic acid carried by the high-frequency cross-border vector insect is made of plastic, aluminum alloy or stainless steel and is cuboid, the length of the cuboid is 15-30 cm, the width of the cuboid is 10-20 cm, the upper part of the kit for quickly extracting and amplifying the bacterial nucleic acid carried by the high-frequency cross-border vector insect is a reagent box cover (5), the lower part of the kit is a reagent box body (6), and the reagent box cover (5) and the reagent box body (6) are movably connected together by means of a kit hinge (7); the thickness of the peripheral wall of the reagent box cover (5) is 1-10 mm, and the thickness of the top wall is 3-15 mm; the thickness of the peripheral wall of the reagent box body (6) is 3-15 mm, the thickness of the bottom wall is 5-15 mm, the inner side of the upper edge of the peripheral wall of the reagent box body (6) is upwards provided with a bulge with the thickness of 0.5-1 mm, and the height of the bulge is 0.5-1 cm; the kit hinge (7) is a known hinge and comprises 2 hinge pieces, wherein the 2 hinge pieces are connected by a shaft, each hinge piece is made of stainless steel and is in a cuboid shape, the length of the cuboid is 2-3 cm, the width of the cuboid is 1-2 cm, and the thickness of the cuboid is 0.5-1 mm; the structure of the kit lock catch (8) is the same as the well-known lock catch structure, a movable lock nose is fixed on the outer surface of the side wall of the kit cover (5), a rectangular hole is arranged on the lock nose, a fixed lock catch is fixed on the outer surface of the side wall of the kit body (6), and an elastic strip capable of retracting inwards is arranged on the lock catch.
3. The kit for rapidly extracting and amplifying the bacterial nucleic acid carried by the high-frequency cross-border vector insect according to claim 1, which is characterized in that: the kit for quickly extracting and amplifying the bacterial nucleic acid carried by the high-frequency cross-border vector insects comprises the following steps:
(1) collecting bacteria carried on the surface of the high-frequency cross-border medium organism: placing 1 tissue (non-visceral tissue such as hair, feet and ears) of a vector insect or mouse in a proper centrifuge tube, adding 0.9% NaCl solution capable of immersing the high-frequency cross-border vector organism, performing vortex oscillation at 800r/min for 30s, and centrifuging for a short time; collecting supernatant, transferring to a new centrifuge tube, and centrifuging for 2min at 13,800 Xg; discarding the supernatant and leaving the precipitate; repeating the steps for 3 times; adding 500 μ L of 0.9% NaCl solution into the collected bacterial precipitate, suspending, mixing, and storing at 4 deg.C; collecting waste liquid and wastes, and discarding the wastes after autoclaving;
(2) collecting bacteria carried in high-frequency cross-border medium organisms: if bacteria in the rat body are extracted, the internal organ tissue is taken for experiment, and the step is directly carried out without the treatment of the step (1); transferring the high-frequency cross-border medium organism treated in the step (1) into a new sterilized centrifugal tube, adding 75% ethanol which can immerse the high-frequency cross-border medium organism, soaking for 5min, and repeating for 3 times; transferring the high-frequency cross-border vector organisms into another new sterilized centrifugal tube, adding 0.9% NaCl solution capable of immersing the high-frequency cross-border vector organisms, performing vortex oscillation at 800r/min for 30s, discarding supernatant, and repeating the steps for 3 times; placing the sample in a biological safety cabinet and airing for 15 min; transferring the sample into a new centrifugal tube by using a sterilization forceps, and grinding the sample into paste; adding 3mL of normal saline, carrying out vortex oscillation at 800r/min for 30s, centrifuging for a short time, transferring the supernatant into a new 10mL centrifuge tube, centrifuging for 2min at 13,800 Xg, discarding the supernatant, and leaving a precipitate; repeating the above collection steps for 3 times, adding 500 μ L of 0.9% NaCl solution into the collected bacterial precipitate, suspending, mixing, and storing at 4 deg.C; collecting waste liquid and wastes, and discarding the wastes after autoclaving;
(3) collecting bacteria carried by the whole high-frequency cross-border medium organism: putting 1 high-frequency cross-border medium organism or tissue into a proper centrifugal tube, and grinding the sample into paste; adding a proper amount of 0.9% NaCl solution according to the size of a sample, carrying out vortex oscillation at 800r/min for 30s, centrifuging for a short time, transferring the supernatant into a new centrifugal tube, centrifuging for 2min at 13,800 Xg, removing the supernatant, and leaving a precipitate; repeating the above collection steps for 3 times, adding 500 μ L of 0.9% NaCl solution into the collected bacterial precipitate, suspending, mixing, and storing at 4 deg.C; collecting waste liquid and wastes, and discarding the wastes after autoclaving;
(4) extraction of bacterial genomic DNA: centrifuging 13800 Xg of the centrifugal tube filled with the bacterial liquid for 2min, transferring supernatant and carrying out autoclaving treatment; extracting the bacterial genome DNA of the collected bacteria by using a bacterial genome DNA extraction kit (containing lysozyme digestion) or using an automatic nucleic acid extractor, and storing the extracted genomic DNA in a refrigerator at 4 ℃ as template DNA for later use;
(5) amplification of the fragment of interest: taking the genomic DNA obtained in the step (4) as a template, and adopting a tag primer: forward primer 341F (5' -TCAG)AGCTACTCCTACGGGAGGCAGCAG-3 ') and a reverse primer 806R (5 ' -GGACTACHVGGGTWTCTAAT-3 ') to obtain a V3-V4 hypervariable region gene sequence of 16S of the high-frequency cross-border vector organism carrying bacteria; PCR amplification System (50. mu.L): 25. mu.L of buffer (2X), 2.5. mu.L of dNTP (2mmol/L), 1. mu.L of high success rate PCR enzyme (1U/. mu.L), 1. mu.L of forward primer (20. mu. mol/L), 1. mu.L of reverse primer (20. mu. mol/L), 3. mu.L of template DNA, ddH2O16.5 mu L; PCR amplification conditions: pre-denaturation at 94 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 50 ℃ for 30s, and extension at 68 ℃ for 30s for 30 cycles; extending for 10min at 68 ℃; keeping the temperature at 10 ℃;
(6) and (3) determining the concentration of the PCR product: detecting the concentration and the quality of the PCR product by using a DNA concentration determinator, respectively determining the absorption values of the sample solution at 260nm, 280nm, 230nm and 270nm by taking a DNA dissolving solution as a reference, and calculating the ratios of A260/A280, A260/A230 and A260/A270, wherein the DNA sample meeting the following conditions can be regarded as high-quality genomic DNA: the A260/A280 is 1.8-1.9, the A260/A230 is more than 2.0, and the A260/A270 is 1.1-1.3; the DNA concentration was calculated using a double-stranded DNA in which A260-1 corresponds to 50. mu.g/mL, and the total amount of DNA in the PCR product was required to be 2. mu.g or more and the concentration was 50 ng/. mu.L or more.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102634453A (en) * | 2012-05-08 | 2012-08-15 | 厦门大学附属中山医院 | FQ-PCR (fluorescent quantitative-polymerase chain reaction) detection kit for bmp gene of treponema pallidum and preparation method thereof |
| CN206359521U (en) * | 2016-12-26 | 2017-07-28 | 上海新培晶医学检验所有限公司 | A kind of kit for being used to detect Dubin Johnson syndrome gene mutations |
| CN108410971A (en) * | 2018-06-04 | 2018-08-17 | 上海市食品药品检验所 | A kind of bacterial nucleic acid sequencing identification method and bacterial identification kits based on DNA characteristics sequence |
| CN209412237U (en) * | 2018-06-04 | 2019-09-20 | 上海市食品药品检验所 | A kind of bacterial identification kits based on DNA characteristics sequence |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102634453A (en) * | 2012-05-08 | 2012-08-15 | 厦门大学附属中山医院 | FQ-PCR (fluorescent quantitative-polymerase chain reaction) detection kit for bmp gene of treponema pallidum and preparation method thereof |
| CN206359521U (en) * | 2016-12-26 | 2017-07-28 | 上海新培晶医学检验所有限公司 | A kind of kit for being used to detect Dubin Johnson syndrome gene mutations |
| CN108410971A (en) * | 2018-06-04 | 2018-08-17 | 上海市食品药品检验所 | A kind of bacterial nucleic acid sequencing identification method and bacterial identification kits based on DNA characteristics sequence |
| CN209412237U (en) * | 2018-06-04 | 2019-09-20 | 上海市食品药品检验所 | A kind of bacterial identification kits based on DNA characteristics sequence |
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