CN112626182A - Molecular identification method of Marneffei staphylium - Google Patents

Molecular identification method of Marneffei staphylium Download PDF

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CN112626182A
CN112626182A CN202110133526.7A CN202110133526A CN112626182A CN 112626182 A CN112626182 A CN 112626182A CN 202110133526 A CN202110133526 A CN 202110133526A CN 112626182 A CN112626182 A CN 112626182A
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高丽
叶春兵
谢祺
杨丹丹
樊红丽
代佳伟
李正伦
张米
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Yunnan Infectious Disease Hospital Yunnan Aids Care Center Yunnan Mental Health Center
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Abstract

A molecular identification method of Marneffei staphylium comprises the following steps: s1: and (3) primer determination: comprises a peripheral primer, an amplification primer and a sequencing primer; s2: and (3) PCR condition determination: determining according to the Tm value of the primer; s3: extracting sample DNA: selecting human tissue fluid as a sample; s4: amplification of nested PCR: a nested PCR amplification reaction system is a 20 mu l system; s5: and (3) agarose gel electrophoresis detection: the agarose gel electrophoresis sample is the PCR product obtained by S4; s6: purifying the target fragment product, and performing gel recovery and purification on the target fragment product with a positive PCR result in S5; s7: T-A cloning and electrophoresis detection cloning sample is DNA solution obtained in step S6; s8: sequencing: sequencing the positive sample with the correct band in the S7; s9: analysis of the homologous evolution tree: and constructing a homologous evolutionary tree by sequencing the sequence of S8, and determining the fungal species. The molecular biology technology is adopted to identify the Marneffei panoraria, the identification efficiency is high, and a reliable basis is provided for early treatment of diseases.

Description

Molecular identification method of Marneffei staphylium
Technical Field
The invention relates to the technical field of Marneffei Talaromyces identification, in particular to a molecular identification method of Marneffei Talaromyces.
Background
HIV is a retrovirus that primarily invades cells of the human immune system. It can destroy T lymphocyte in vivo, thereby blocking cellular immune process, weakening organism's immune function, leading to patient immunodeficiency, and then generating opportunistic infection, malignant tumor, etc. AIDS has spread rapidly worldwide since the first instance of AIDS reported in the United states in 1981.
With the increase of Human Immunodeficiency Virus (HIV) infected patients in recent years, clinical infection caused by Talaromyces Marneffei (TM) is second to tubercle bacillus and cryptococcus neoformans only in south east Asia, and the third place is the main pathogenic bacteria causing infection of AIDS patients. TM is the 2 nd most common conditionally pathogenic fungus after having become cryptococcus in south-east asia. The existing TM identification method mostly adopts fungus culture, and the variety of the fungus is determined by comparing the characteristics of the fungus, because the identification method is simple, the identification rate is less than 50 percent.
The invention aims at samples of opportunistic infection of HIV patients, which are inspected by special infectious disease hospitals (subsidiary infectious disease hospitals of Kunming medical university) in Yunnan province, adopts fungus general primers and designed specific fungus amplification primers to carry out PCR amplification, carry out molecular biological detection and genotyping analysis on fungi, and explore and establish an early diagnosis method of invasive fungal infection suitable for clinical popularization.
Disclosure of Invention
Aiming at the problems in the prior art, the molecular identification method of the Marneffei panoraria is provided by the invention, the identification of the Marneffei panoraria is carried out by adopting a molecular biology technology, the identification efficiency is high, the identification of the disease can be carried out in the early disease onset stage of a patient when the molecular identification method is applied to clinical experiments, the sensitivity is high, and a reliable basis is provided for the early treatment of the disease.
In order to achieve the above object, the present invention employs the following techniques:
a molecular identification method of Marneffei staphylium comprises the following steps:
s1: and (3) primer determination: the primers comprise a peripheral primer, an amplification primer and a sequencing primer, and the specific sequences are as follows:
upstream peripheral primer NSA 3F: 5'-AAACTCTGTCGTGCTGGGGATA-3', respectively;
downstream peripheral primer NLC 2R: 5'-GAGCTGCATTCCCAAACAACTC-3', respectively;
upstream amplification primer ITS 5F: 5'-GGAAGTAAAAGTCGTAACAAGG-3', respectively;
downstream amplification primer ITS 4R: 5'-TCCGTAGGTGAACCTGCGG-3', respectively;
upstream sequencing primer ITS 1F: 5'-TCCTCCGCTTATTGATATGC-3', respectively;
downstream sequencing primer ITS 4R: 5'-TCCGTAGGTGAACCTGCGG-3', respectively;
s2: and (3) PCR condition determination: determining PCR conditions according to the Tm value of the primer, wherein the PCR conditions comprise denaturation at 94 ℃/5min, denaturation at 94 ℃/30, annealing at 50 ℃/55 ℃/30S, extension at 72 ℃/1min, and total cycle is 40 cycles;
s3: extracting sample DNA:
selecting human tissue fluid as a sample;
s4: amplification of nested PCR:
the nested PCR amplification reaction system is a 20 mu l system, wherein the sample adopts eluted genomic DNA obtained in S3, the amplification conditions refer to S2, and primers used in nested PCR amplification adopt sequences in S1;
s5: and (3) agarose gel electrophoresis detection:
the sample used for agarose gel electrophoresis is a PCR product obtained from S4;
s6: purification of the product of the fragment of interest
Carrying out gel recovery and purification on the electrophoresis sample of the target fragment product with positive PCR result in S5;
s7: T-A cloning and electrophoresis detection
The cloned sample is the DNA solution obtained in the step S6, and the cloned colony is subjected to DNA extraction, nested PCR amplification and agarose gel electrophoresis detection in sequence in the steps S3, S4 and S5;
s8: sequencing: taking a positive sample with a correct strip in 50 mu l S7, and sending the positive sample to a sequencing company for sequencing;
s9: analysis of the homologous evolution tree: and (3) constructing a homologous evolutionary tree by adopting an adjacent method to the S8 sequencing sequence, and determining the fungal species.
Further, the specific operation steps of the sample DNA extraction are as follows:
firstly, sucking 120 mul of sample by using a pipette gun and adding the sample into a 1.5ml centrifuge tube;
adding 500 mul Buffer AP1 into the sample, covering the centrifuge tube cover tightly, and performing vortex oscillation for 15 s;
thirdly, adding 100 mul AP2 into the centrifuge tube, and placing for 5min after vortex oscillation for 15s to fully crack the sample DNA;
fourthly, centrifuging the lysed DNA of the sample for 10min at 12000 Xg;
fifthly, placing the preparation tube in a 2ml centrifuge tube, adding the centrifuged sample DNA in the step IV into the preparation tube, placing for 3min to ensure that the DNA is fully combined with an adsorption film in the preparation tube, and centrifuging for 1min at 12000 Xg;
sixthly, discarding the filtrate, adding 700 mul Buffer W1A into the preparation tube, standing for 2min at room temperature, and centrifuging for 30s at 12000 Xg;
seventhly, discarding the filtrate, adding 800 mul of Buffer W2 added with absolute ethyl alcohol into the preparation tube, wherein the adding ratio of the Buffer W2 to the absolute ethyl alcohol is 1:4, and centrifuging for 1min at 12000 Xg;
eighthly, abandoning the filtrate, adding 500 mul Buffer W2 into the preparation tube, and centrifuging at 12000 Xg for 1 min;
ninthly, abandoning the filtrate, putting the preparation tube back into a 2ml centrifuge tube, centrifuging for 1min at 12000 Xg, and removing redundant liquid in the preparation tube through air-throwing;
placing the preparation tube in another clean centrifugal tube with the volume of 1.5ml for 10min to completely volatilize the absolute ethyl alcohol in the preparation tube, then adding 60 mu l of Buffer TE preheated to 65 ℃ into the preparation tube, standing for 2min at room temperature, centrifuging for 1min at 12000 Xg, and discarding the supernatant to obtain the eluted genomic DNA.
Further, the nested PCR amplification reaction system comprises: rTaq enzyme: 10 μ l, RNase Free dH2O: 6 μ l, Primer F (10 μm): 1 μ l, Primer R (10 μm): 1 μ l, sample: 2 mul.
Further, the agarose gel electrophoresis procedure was as follows:
preparation of a 1xTAE buffer reagent: 800ml of pure water was placed in a beaker, and Na was weighed2EDTA•2H2Placing 37.2g of O and 242g of Tris in a beaker, heating in a 65 ℃ water bath kettle, stirring with a glass rod until the Tris is dissolved, measuring 57.1ml of glacial acetic acid, placing in the beaker, uniformly stirring, finally adding pure water to a constant volume of 1000ml to prepare 50xTAE Buffer solution, taking 20ml of 50xTAE Buffer, adding 980ml of pure water, and uniformly mixing to obtain 1xTAE Buffer solution;
② 1 percent agarose gel preparation: weighing 1g of agarose, adding 100ml of 1xTAE Buffer solution into a conical flask, placing the conical flask in a microwave oven, heating until the agarose is completely dissolved, slightly cooling, adding 5 mul of EB solution, and uniformly mixing to obtain 1% agarose;
thirdly, the cleaned electrophoresis plate and the comb are arranged in a glue tank, 1% agarose with the thickness of 0.3-0.8 cm is poured, and the electrophoresis plate and the comb are placed at room temperature for 30min to be condensed to obtain 1% agarose gel;
fourthly, slowly pulling out the comb from the 1 percent agarose gel, then putting the 1 percent agarose gel into an electrophoresis tank, and pouring 1xTAE Buffer into the electrophoresis tank to ensure that the liquid level is higher than the gel plane;
uniformly mixing 3 mul of sample with 2 mul of 6xLoading buffer, slowly adding the mixture into a glue hole, and adding 5 mul of DNA Marker 2000 into a first electrophoresis hole;
sixthly, after the sample loading is finished, the electrophoresis voltage is adjusted to be 120V, the electrophoresis is carried out at constant voltage, the current is 180mA, and the result is observed after 30 min.
Further, the specific steps of glue recovery and purification are as follows:
column balancing: putting the adsorption column into a collection pipe, adding 500 mul of balance liquid BL into the adsorption column CA2, centrifuging at 12000rpm for 1min, pouring out waste liquid in the collection pipe, and putting the adsorption column back into the collection pipe again;
cutting a target band obtained from the PCR product in the S5 from the agarose gel and putting the cut target band into a clean centrifugal tube;
thirdly, adding 400 mul of sol solution PN into the target strip, wherein the mass ratio of the target strip to the sol solution PN is 0.3-0.7: 1, carrying out 50 ℃ water bath, continuously and gently turning the centrifugal tube up and down until the gel block is completely dissolved, cooling the temperature of the gel solution to room temperature after the gel block is completely dissolved, and loading the gel solution into a column;
putting the adsorption column into another collection pipe, adding the solution obtained from the third step into the adsorption column CA2, standing at room temperature for 2min, centrifuging at 12000rpm for 1min, pouring the waste liquid in the collection pipe, and putting the adsorption column CA2 into the collection pipe again;
fifthly, adding 600 mul of rinsing liquid PW added with absolute ethyl alcohol into the adsorption column CA2, wherein the adding ratio of the rinsing liquid PW to the absolute ethyl alcohol is 1: 3-5, standing for 5 minutes at room temperature, centrifuging at 12000rpm for 1min, pouring out waste liquid in the collection pipe, and putting the adsorption column CA2 into the collection pipe;
sixthly, repeating the operation steps;
seventhly, placing the adsorption column CA2 into a collecting pipe, centrifuging at 12000rpm for 2min, removing rinsing liquid PW, placing the adsorption column CA2 into a clean centrifugal pipe, standing at room temperature for several minutes, and completely drying;
eighthly, suspending and dropwise adding 30 mul of elution buffer solution EB to the middle position of the adsorption film, keeping the adsorption film heated in a water bath at 65 ℃ when the elution buffer solution EB is dropwise added, standing at room temperature for 2min, centrifuging at 12000rpm for 2min, collecting filtrate, and obtaining DNA solution, and storing at-40 ℃.
Further, the T-A cloning method comprises the following specific steps:
preparation of LB liquid culture medium and LB solid culture medium
Weighing 1% of tryptone, 0.5% of yeast extract and 1% of NaCl in a blue-covered bottle, and additionally adding 1.5% of Agar when preparing an LB solid culture medium;
adding 480ml of deionized water into a blue-cap bottle, stirring and dissolving, and fixing the volume to 500ml after the solid is completely dissolved;
iii, adjusting the pH value of the solution to 7.0;
iv, unscrewing the blue cap bottle, and putting the prepared solution into an autoclave for autoclaving at the temperature of 121 ℃ for 20 min;
and v, screwing down a blue cover bottle cap, cooling the sterile LB culture medium at room temperature, and then putting the sterile LB culture medium into a refrigerator at 4 ℃ for later use;
activation of DH5a Strain
Placing DH5a strain preserved in a refrigerator at-80 ℃ on ice for thawing;
II, inoculating the melted DH5a strain into the prepared LB liquid culture medium in an aseptic operation platform, and culturing in a shaking table at the speed of 200rpm and the temperature of 37 ℃;
measuring the concentration of the bacterial liquid to enable the OD value to be 0.35-0.4, and then placing on ice;
③GaCl2preparation of the solution
Weighing anhydrous GaCl21.1g, placing the mixture into a conical flask, adding deionized water, stirring and dissolving, and fixing the volume to 100 ml;
II, using a 50ml syringe and a 0.22 mu m filter membrane to perform the pair of the dissolved GaCl in the sterile environment2Filtering the solution for sterilization, screwing down a bottle cap, and placing at-4 ℃ for later use;
④GaCl2solution treatment of bacterial species
Transferring the bacterial liquid in the conical flask into a 15ml centrifugal tube, centrifuging for 10min at 4000rpm and 4 ℃, pouring out the bacterial liquid, and sucking out the residual bacterial liquid by using a liquid transfer gun;
ii, 1ml of GaCl2Blowing the thallus from the solution gently, blowing and beating the thallus uniformly, and then placing the thallus on ice for 40 min;
iii 4000rpm, centrifugation at 4 ℃ for 10min, using GaCl2Blowing, beating and uniformly mixing the thalli by the solution to complete preparation of competent cells, and placing the competent cells on ice for later use;
connect
Taking 5 mul of clone sample and 1 mul of T-A carrier, and gently and uniformly blowing and beating the clone sample and the T-A carrier on ice;
II, connecting on a metal bath for 2 hours at 25 ℃;
sixthly, transformation
After the connection is finished, adding 100 mul of competent cells into the connection product through aseptic operation, gently blowing and uniformly mixing the competent cells after the addition, and placing the competent cells on ice for 30 min;
ii, finishing ice bath, performing heat shock at 42 ℃ for 90s, and immediately placing on ice;
adding 1ml of LB liquid culture medium into the transformation product, gently and slowly blowing, uniformly mixing, placing on a shaker at 180rpm, and shaking and culturing for 40min at 37 ℃;
iv, melting the solid culture medium, cooling to 50 ℃, adding 0.1 percent of Amp +, and pouring the mixture into a flat plate for later use after solidification;
centrifuging the bacterial liquid cultured by the shaking table at 5000rpm for 10min, removing supernatant, leaving 200 mu l of supernatant, and carrying out plate coating on the heavy suspension bacteria;
vi, after the plate is coated, sealing the opening and inversely placing the opening in an incubator at 37 ℃ for culturing for 12 hours;
and vii, taking 1ml of sterile centrifuge tube, sucking 600 mu l of LB liquid culture medium added with Amp +, wherein the 1ml of LB liquid culture medium contains 50 mu g of Amp +, picking the large and full single colony in the plate into the LB liquid culture medium, covering the LB liquid culture medium, placing the plate on a shaking bed at 37 ℃, and carrying out shaking culture at 160rpm for 6 hours to obtain the colony.
Further, the thickness of 1% agarose is preferably 0.5 cm.
Further, the mass ratio of the target band to the sol solution PN is preferably 0.5: 1.
Further, the ratio of the rinsing liquid PW to the absolute ethyl alcohol is preferably 1: 4.
Further, during shaking table cultivation, at first pack liquid with the box, liquid level is located the sieve top, the mode that adopts water bath heating keeps constant temperature, utilize controller control heating rod and hot plate operation simultaneously to adjust the temperature to predetermineeing the temperature, then place the blake bottle between two holding heads, receive the spring action, holding head and blake bottle outer wall elastic contact, through the clearance between the two adjustable holding heads of rotatory screw rod, be applicable to the centre gripping of different model blake bottles, the blake bottle is fixed the back, utilize the control driver to adjust the actuating lever and remove with invariable vibration frequency, carry out shaking table cultivation.
The invention has the beneficial effects that:
1. the molecular biology technology is adopted to identify the Marneffei armillaria, the identification efficiency is high, the result is accurate, and the molecular biology method can be widely applied to clinical experiments and has high application value.
2. The primer sequence specific to the Marneffei basket fungus is adopted for verification, and the method has high specificity.
3. The invention can identify the disease in the early stage of the disease onset of the patient, has high sensitivity and provides reliable basis for the early treatment of the disease.
4. The culture of the marneffei shaped fungus is carried out by adopting the constant temperature shaking table, the temperature change is reduced, the gap between the two clamping heads is adjustable, and the culture bottle clamp is suitable for clamping culture bottles of different models.
Drawings
FIG. 1 is a schematic view of the operation of the present invention.
FIG. 2 is a schematic view of the structure of the constant temperature shaking table of the present invention.
FIG. 3 is a schematic view of a clamping head according to the present invention.
FIG. 4 is a schematic view of a heating rod according to the present invention.
Fig. 5 is a schematic diagram of the structure of the sieve plate of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the following detailed description of the embodiments of the present invention is provided with reference to the accompanying drawings, but the described embodiments of the present invention are a part of the embodiments of the present invention, not all of the embodiments of the present invention.
It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, it need not be further defined and explained in subsequent figures.
In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings or orientations or positional relationships that the products of the present invention are usually placed in when used, and are only for convenience of describing the present invention and simplifying the description. The terms "first," "second," and the like are used solely to distinguish one from another and are not to be construed as indicating or implying relative importance. The terms "parallel", "perpendicular", etc. do not require that the components be absolutely parallel or perpendicular, but may be slightly inclined.
In the description of the present invention, it should also be noted that, unless otherwise explicitly specified or limited, the terms "disposed," "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; either directly or indirectly through intervening media, or through both elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
Example 1:
the invention collects 100 tissue fluids of AIDS patients as samples, each sample is divided into 3 groups, and the molecular identification of Marneffei basket fungus is carried out by adopting the steps shown in figure 1, which comprises the following steps:
s1: and (3) primer determination: the primers comprise a peripheral primer, an amplification primer and a sequencing primer, and the specific sequences are as follows:
upstream peripheral primer NSA 3F: 5'-AAACTCTGTCGTGCTGGGGATA-3', respectively;
downstream peripheral primer NLC 2R: 5'-GAGCTGCATTCCCAAACAACTC-3', respectively;
upstream amplification primer ITS 5F: 5'-GGAAGTAAAAGTCGTAACAAGG-3', respectively;
downstream amplification primer ITS 4R: 5'-TCCGTAGGTGAACCTGCGG-3', respectively;
upstream sequencing primer ITS 1F: 5'-TCCTCCGCTTATTGATATGC-3', respectively;
downstream sequencing primer ITS 4R: 5'-TCCGTAGGTGAACCTGCGG-3', respectively;
s2: and (3) PCR condition determination: determining PCR conditions according to the Tm value of the primer, wherein the PCR conditions comprise denaturation at 94 ℃/5min, denaturation at 94 ℃/30, annealing at 50 ℃/55 ℃/30S, extension at 72 ℃/1min, and total cycle is 40 cycles;
s3: extracting sample DNA:
selecting blood of AIDS patients as sample;
s4: amplification of nested PCR:
the nested PCR amplification reaction system is a 20 mu l system, wherein the sample adopts eluted genomic DNA obtained in S3, the amplification conditions refer to S2, and primers used in nested PCR amplification adopt sequences in S1;
s5: and (3) agarose gel electrophoresis detection:
the sample used for agarose gel electrophoresis is a PCR product obtained from S4;
s6: purification of the product of the fragment of interest
Carrying out gel recovery and purification on the electrophoresis sample of the target fragment product with positive PCR result in S5;
s7: T-A cloning and electrophoresis detection
The cloned sample is the DNA solution obtained in the step S6, and the cloned colony is subjected to DNA extraction, nested PCR amplification and agarose gel electrophoresis detection in sequence in the steps S3, S4 and S5;
s8: sequencing: taking a positive sample with a correct strip in 50 mu l S7, and sending the positive sample to a sequencing company for sequencing;
s9: analysis of the homologous evolution tree: and (3) constructing a homologous evolutionary tree by adopting an adjacent method to the S8 sequencing sequence, and determining the fungal species.
Further, the specific operation steps of the sample DNA extraction are as follows:
firstly, sucking 120 mul of sample by using a pipette gun and adding the sample into a 1.5ml centrifuge tube;
adding 500 mul Buffer AP1 into the sample, covering the centrifuge tube cover tightly, and performing vortex oscillation for 15 s;
thirdly, adding 100 mul AP2 into the centrifuge tube, and placing for 5min after vortex oscillation for 15s to fully crack the sample DNA;
fourthly, centrifuging the lysed DNA of the sample for 10min at 12000 Xg;
fifthly, placing the preparation tube in a 2ml centrifuge tube, adding the centrifuged sample DNA in the step IV into the preparation tube, placing for 3min to ensure that the DNA is fully combined with an adsorption film in the preparation tube, and centrifuging for 1min at 12000 Xg;
sixthly, discarding the filtrate, adding 700 mul Buffer W1A into the preparation tube, standing for 2min at room temperature, and centrifuging for 30s at 12000 Xg;
seventhly, discarding the filtrate, adding 800 mul of Buffer W2 added with absolute ethyl alcohol into the preparation tube, wherein the adding ratio of the Buffer W2 to the absolute ethyl alcohol is 1:4, and centrifuging for 1min at 12000 Xg;
eighthly, abandoning the filtrate, adding 500 mul Buffer W2 into the preparation tube, and centrifuging at 12000 Xg for 1 min;
ninthly, abandoning the filtrate, putting the preparation tube back into a 2ml centrifuge tube, centrifuging for 1min at 12000 Xg, and removing redundant liquid in the preparation tube through air-throwing;
placing the preparation tube in another clean centrifugal tube with the volume of 1.5ml for 10min to completely volatilize the absolute ethyl alcohol in the preparation tube, then adding 60 mu l of Buffer TE preheated to 65 ℃ into the preparation tube, preheating the Buffer TE to 65 ℃ before use, standing the mixture at room temperature for 2min, centrifuging the mixture at 12000 Xg for 1min, and discarding the supernatant to obtain the eluted genomic DNA.
Further, the nested PCR amplification reaction system comprises: rTaq enzyme: 10 μ l, RNase Free dH2O: 6 μ l, Primer F (10 μm): 1 μ l, Primer R (10 μm): 1 μ l, sample: 2 mul.
Further, the agarose gel electrophoresis procedure was as follows:
preparation of a 1xTAE buffer reagent: 800ml of pure water was placed in a beaker, and Na was weighed2EDTA•2H2Placing 37.2g of O and 242g of Tris in a beaker, heating in a 65 ℃ water bath kettle, stirring with a glass rod until the Tris is dissolved, measuring 57.1ml of glacial acetic acid, placing in the beaker, uniformly stirring, finally adding pure water to a constant volume of 1000ml to prepare 50xTAE Buffer solution, taking 20ml of 50xTAE Buffer, adding 980ml of pure water, and uniformly mixing to obtain 1xTAE Buffer solution;
② 1 percent agarose gel preparation: weighing 1g of agarose, adding 100ml of 1xTAE Buffer solution into a conical flask, placing the conical flask in a microwave oven, heating until the agarose is completely dissolved, slightly cooling, adding 5 mul of EB solution, and uniformly mixing to obtain 1% agarose;
thirdly, the cleaned electrophoresis plate and the comb are arranged in a glue groove, 1% agarose with the thickness of 0.5cm is poured, and the electrophoresis plate and the comb are placed at room temperature for 30min to be condensed to obtain 1% agarose gel;
fourthly, slowly pulling out the comb from the 1 percent agarose gel, then putting the 1 percent agarose gel into an electrophoresis tank, and pouring 1xTAE Buffer into the electrophoresis tank to ensure that the liquid level is higher than the gel plane;
uniformly mixing 3 mul of sample with 2 mul of 6xLoading buffer, slowly adding the mixture into a glue hole, and adding 5 mul of DNA Marker 2000 into a first electrophoresis hole;
sixthly, after the sample loading is finished, the electrophoresis voltage is adjusted to be 120V, the electrophoresis is carried out at constant voltage, the current is 180mA, and the result is observed after 30 min.
Further, the specific steps of glue recovery and purification are as follows:
column balancing: putting the adsorption column into a collection pipe, adding 500 mul of balance liquid BL into the adsorption column CA2, centrifuging at 12000rpm for 1min, pouring out waste liquid in the collection pipe, and putting the adsorption column back into the collection pipe again;
cutting a target band obtained from the PCR product in the S5 from the agarose gel and putting the cut target band into a clean centrifugal tube;
thirdly, adding 400 mul of sol solution PN into the target strip, wherein the mass ratio of the target strip to the sol solution PN is 0.5:1, carrying out water bath at 50 ℃, continuously and gently turning the centrifuge tube up and down until the gel blocks are completely dissolved, and lowering the temperature of the gel solution to room temperature and then loading the gel solution to a column after the gel blocks are completely dissolved;
putting the adsorption column into another collection pipe, adding the solution obtained from the third step into the adsorption column CA2, standing at room temperature for 2min, centrifuging at 12000rpm for 1min, pouring the waste liquid in the collection pipe, and putting the adsorption column CA2 into the collection pipe again;
fifthly, adding 600 mul of rinsing liquid PW added with absolute ethyl alcohol into the adsorption column CA2, wherein the adding ratio of the rinsing liquid PW to the absolute ethyl alcohol is 1:4, standing for 5 minutes at room temperature, centrifuging at 12000rpm for 1min, pouring waste liquid in the collection pipe, and putting the adsorption column CA2 into the collection pipe;
sixthly, repeating the operation steps;
seventhly, placing the adsorption column CA2 into a collecting pipe, centrifuging at 12000rpm for 2min, removing rinsing liquid PW, placing the adsorption column CA2 into a clean centrifugal pipe, standing at room temperature for several minutes, and completely drying;
eighthly, suspending and dropwise adding 30 mul of elution buffer solution EB to the middle position of the adsorption film, keeping the adsorption film heated in a water bath at 65 ℃ when the elution buffer solution EB is dropwise added, standing at room temperature for 2min, centrifuging at 12000rpm for 2min, collecting filtrate, and obtaining DNA solution, and storing at-40 ℃.
Further, the T-A cloning method comprises the following specific steps:
preparation of LB liquid culture medium and LB solid culture medium
Weighing 1% of tryptone, 0.5% of yeast extract and 1% of NaCl in a blue-covered bottle, and additionally adding 1.5% of Agar when preparing an LB solid culture medium;
adding 480ml of deionized water into a blue-cap bottle, stirring and dissolving, and fixing the volume to 500ml after the solid is completely dissolved;
iii, adjusting the pH value of the solution to 7.0;
iv, unscrewing the blue cap bottle, and putting the prepared solution into an autoclave for autoclaving at the temperature of 121 ℃ for 20 min;
and v, screwing down a blue cover bottle cap, cooling the sterile LB culture medium at room temperature, and then putting the sterile LB culture medium into a refrigerator at 4 ℃ for later use;
activation of DH5a Strain
Placing DH5a strain preserved in a refrigerator at-80 ℃ on ice for thawing;
II, inoculating the melted DH5a strain into the prepared LB liquid culture medium in an aseptic operation platform, and culturing in a shaking table at the speed of 200rpm and the temperature of 37 ℃;
measuring the concentration of the bacterial liquid to enable the OD value to be 0.35-0.4, and then placing on ice;
③GaCl2preparation of the solution
Weighing anhydrous GaCl21.1g, placing the mixture into a conical flask, adding deionized water, stirring and dissolving, and fixing the volume to 100 ml;
II, using a 50ml syringe and a 0.22 mu m filter membrane to perform the pair of the dissolved GaCl in the sterile environment2Filtering the solution for sterilization, screwing down a bottle cap, and placing at-4 ℃ for later use;
④GaCl2solution treatment of bacterial species
Transferring the bacterial liquid in the conical flask into a 15ml centrifugal tube, centrifuging for 10min at 4000rpm and 4 ℃, pouring out the bacterial liquid, and sucking out the residual bacterial liquid by using a liquid transfer gun;
ii, 1ml of GaCl2Blowing the thallus from the solution gently, blowing and beating the thallus uniformly, and then placing the thallus on ice for 40 min;
iii 4000rpm, centrifugation at 4 ℃ for 10min, using GaCl2Blowing, beating and uniformly mixing the thalli by the solution to complete preparation of competent cells, and placing the competent cells on ice for later use;
connect
Taking 5 mul of clone sample and 1 mul of T-A carrier, and gently and uniformly blowing and beating the clone sample and the T-A carrier on ice;
II, connecting on a metal bath for 2 hours at 25 ℃;
sixthly, transformation
After the connection is finished, adding 100 mul of competent cells into the connection product through aseptic operation, gently blowing and uniformly mixing the competent cells after the addition, and placing the competent cells on ice for 30 min;
ii, finishing ice bath, performing heat shock at 42 ℃ for 90s, and immediately placing on ice;
adding 1ml of LB liquid culture medium into the transformation product, gently and slowly blowing, uniformly mixing, placing on a shaker at 180rpm, and shaking and culturing for 40min at 37 ℃;
iv, melting the solid culture medium, cooling to 50 ℃, adding 0.1 percent of Amp +, and pouring the mixture into a flat plate for later use after solidification;
centrifuging the bacterial liquid cultured by the shaking table at 5000rpm for 10min, removing supernatant, leaving 200 mu l of supernatant, and carrying out plate coating on the heavy suspension bacteria;
vi, after the plate is coated, sealing the opening and inversely placing the opening in an incubator at 37 ℃ for culturing for 12 hours;
and vii, taking 1ml of sterile centrifuge tube, sucking 600 mu l of LB liquid culture medium added with Amp +, wherein the 1ml of LB liquid culture medium contains 50 mu g of Amp +, picking the large and full single colony in the plate into the LB liquid culture medium, covering the LB liquid culture medium, placing the plate on a shaking bed at 37 ℃, and carrying out shaking culture at 160rpm for 6 hours to obtain the colony.
Further, during shaking table cultivation, adopt the shaking table structure as shown in fig. 2~5, including box 1, the inside diapire of box 1 is equipped with heating rod 101 that is S-shaped, the inside lateral wall of box 1 is equipped with hot plate 102, heating rod 101 top is equipped with sieve 2, 2 top surface cards of sieve are equipped with many pairs of fixture block 201, fixture block 201 top surface is connected with a pair of spring 202, the spring 202 top is connected with grip block 203, grip block 203 threaded connection has the screw rod 204 that the level set up, screw rod 204 end connection has curved holding head 205, two screw rods 204 that are located same pair of fixture block 201 top are located same vertical plane, 2 bottom symmetries of sieve are equipped with a plurality of mount pads 3, mount pad 3 is inserted and is equipped with actuating lever 301, the actuating lever 301 other end is connected with driver 302, driver 302 is located box 1 bottom, heating rod 101, hot plate.
Shaking table during operation, at first pack liquid with box 1, the liquid level is located sieve 2 top, the mode that adopts water bath heating keeps constant temperature, utilize controller 4 control heating rod 101 and hot plate 102 operation simultaneously to adjust the temperature to predetermineeing the temperature, then place the blake bottle between two holding head 205, receive the effect of spring 202, holding head 205 and blake bottle outer wall elastic contact, through the adjustable two holding head 205 clearances of rotatory screw rod 204, be applicable to the centre gripping of different model blake bottles, the blake bottle is fixed the back, utilize control driver 302 to adjust actuating lever 301 and remove with invariable vibration frequency, carry out shaking table cultivation.
Example 2:
the same sample and the same operation steps as those in example 1 were adopted in this example, except that the thickness of 1% agarose in this example was 0.3cm, the mass ratio of the target band to the sol solution PN was 0.3:1, and the addition ratio of the rinsing solution PW to the absolute ethyl alcohol was 1: 3.
Example 3:
the same sample and the same operation steps as those in example 1 were adopted in this example, except that the thickness of 1% agarose in this example was 0.8cm, the mass ratio of the target band to the sol solution PN was 0.7:1, and the addition ratio of the rinsing solution PW to the absolute ethyl alcohol was 1: 5.
Sequencing a product which meets the requirement of a target fragment and is obtained by agarose gel electrophoresis in the step 7 of the embodiment, constructing a homologous evolution tree for a sequencing sequence through homologous evolution tree analysis, and determining the kind of fungi, wherein the identification result of the Marneffei basket fungus is shown as the following figure:
Figure DEST_PATH_IMAGE001
the result shows that the identification rate of the Marneffei basket fungus is more than 90% by adopting the method of the invention, the clinical application value is higher, and the identification rate is the highest when the thickness of 1% agarose is 0.5cm, the mass ratio of a target band to a sol solution PN is 0.5:1, and the adding ratio of a rinsing liquid PW to absolute ethyl alcohol is 1: 4.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and it is apparent that those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (10)

1. A molecular identification method of Marneffei staphylium is characterized by comprising the following steps:
s1: and (3) primer determination: the primers comprise a peripheral primer, an amplification primer and a sequencing primer, and the specific sequences are as follows:
upstream peripheral primer NSA 3F: 5'-AAACTCTGTCGTGCTGGGGATA-3', respectively;
downstream peripheral primer NLC 2R: 5'-GAGCTGCATTCCCAAACAACTC-3', respectively;
upstream amplification primer ITS 5F: 5'-GGAAGTAAAAGTCGTAACAAGG-3', respectively;
downstream amplification primer ITS 4R: 5'-TCCGTAGGTGAACCTGCGG-3', respectively;
upstream sequencing primer ITS 1F: 5'-TCCTCCGCTTATTGATATGC-3', respectively;
downstream sequencing primer ITS 4R: 5'-TCCGTAGGTGAACCTGCGG-3', respectively;
s2: and (3) PCR condition determination: determining PCR conditions according to the Tm value of the primer, wherein the PCR conditions comprise denaturation at 94 ℃/5min, denaturation at 94 ℃/30, annealing at 50 ℃/55 ℃/30S, extension at 72 ℃/1min, and total cycle is 40 cycles;
s3: extracting sample DNA:
selecting human tissue fluid as a sample;
s4: amplification of nested PCR:
the nested PCR amplification reaction system is a 20 mu l system, wherein the sample adopts eluted genomic DNA obtained in S3, the amplification conditions refer to S2, and primers used in nested PCR amplification adopt sequences in S1;
s5: and (3) agarose gel electrophoresis detection:
the sample used for agarose gel electrophoresis is a PCR product obtained from S4;
s6: purification of the product of the fragment of interest
Carrying out gel recovery and purification on the electrophoresis sample of the target fragment product with positive PCR result in S5;
s7: T-A cloning and electrophoresis detection
The cloned sample is the DNA solution obtained in the step S6, and the cloned colony is subjected to DNA extraction, nested PCR amplification and agarose gel electrophoresis detection in sequence in the steps S3, S4 and S5;
s8: sequencing: taking a positive sample with a correct strip in 50 mu l S7, and sending the positive sample to a sequencing company for sequencing;
s9: analysis of the homologous evolution tree: and (3) constructing a homologous evolutionary tree by adopting an adjacent method to the S8 sequencing sequence, and determining the fungal species.
2. The molecular identification method of Marneffei staphylium according to claim 1, characterized in that: the concrete operation steps of the sample DNA extraction are as follows:
firstly, sucking 120 mul of sample by using a pipette gun and adding the sample into a 1.5ml centrifuge tube;
adding 500 mul Buffer AP1 into the sample, covering the centrifuge tube cover tightly, and performing vortex oscillation for 15 s;
thirdly, adding 100 mul AP2 into the centrifuge tube, and placing for 5min after vortex oscillation for 15s to fully crack the sample DNA;
fourthly, centrifuging the lysed DNA of the sample for 10min at 12000 Xg;
fifthly, placing the preparation tube in a 2ml centrifuge tube, adding the centrifuged sample DNA in the step IV into the preparation tube, placing for 3min to ensure that the DNA is fully combined with an adsorption film in the preparation tube, and centrifuging for 1min at 12000 Xg;
sixthly, discarding the filtrate, adding 700 mul Buffer W1A into the preparation tube, standing for 2min at room temperature, and centrifuging for 30s at 12000 Xg;
seventhly, discarding the filtrate, adding 800 mul of Buffer W2 added with absolute ethyl alcohol into the preparation tube, wherein the adding ratio of the Buffer W2 to the absolute ethyl alcohol is 1:4, and centrifuging for 1min at 12000 Xg;
eighthly, abandoning the filtrate, adding 500 mul Buffer W2 into the preparation tube, and centrifuging at 12000 Xg for 1 min;
ninthly, abandoning the filtrate, putting the preparation tube back into a 2ml centrifuge tube, centrifuging for 1min at 12000 Xg, and removing redundant liquid in the preparation tube through air-throwing;
placing the preparation tube in another clean centrifugal tube with the volume of 1.5ml for 10min to completely volatilize the absolute ethyl alcohol in the preparation tube, then adding 60 mu l of Buffer TE preheated to 65 ℃ into the preparation tube, standing for 2min at room temperature, centrifuging for 1min at 12000 Xg, and discarding the supernatant to obtain the eluted genomic DNA.
3. The molecular identification method of Marneffei staphylium according to claim 1, characterized in that: nested PCR amplification reaction system comprises: rTaq enzyme: 10 μ l, RNase Free dH2O: 6 μ l, Primer F (10 μm): 1 μ l, Primer R (10 μm): 1 μ l, sample: 2 mul.
4. The molecular identification method of Marneffei staphylium according to claim 1, characterized in that: the agarose gel electrophoresis procedure was as follows:
preparation of a 1xTAE buffer reagent: 800ml of pure water was placed in a beaker, and Na was weighed2EDTA•2H2Placing 37.2g of O and 242g of Tris in a beaker, heating in a 65 ℃ water bath kettle, stirring with a glass rod until the Tris is dissolved, measuring 57.1ml of glacial acetic acid, placing in the beaker, uniformly stirring, finally adding pure water to a constant volume of 1000ml to prepare 50xTAE Buffer solution, taking 20ml of 50xTAE Buffer, adding 980ml of pure water, and uniformly mixing to obtain 1xTAE Buffer solution;
② 1 percent agarose gel preparation: weighing 1g of agarose, adding 100ml of 1xTAE Buffer solution into a conical flask, placing the conical flask in a microwave oven, heating until the agarose is completely dissolved, slightly cooling, adding 5 mul of EB solution, and uniformly mixing to obtain 1% agarose;
thirdly, the cleaned electrophoresis plate and the comb are arranged in a glue tank, 1% agarose with the thickness of 0.3-0.8 cm is poured, and the electrophoresis plate and the comb are placed at room temperature for 30min to be condensed to obtain 1% agarose gel;
fourthly, slowly pulling out the comb from the 1 percent agarose gel, then putting the 1 percent agarose gel into an electrophoresis tank, and pouring 1xTAE Buffer into the electrophoresis tank to ensure that the liquid level is higher than the gel plane;
uniformly mixing 3 mul of sample with 2 mul of 6xLoading buffer, slowly adding the mixture into a glue hole, and adding 5 mul of DNA Marker 2000 into a first electrophoresis hole;
sixthly, after the sample loading is finished, the electrophoresis voltage is adjusted to be 120V, the electrophoresis is carried out at constant voltage, the current is 180mA, and the result is observed after 30 min.
5. The molecular identification method of Marneffei staphylium according to claim 1, characterized in that: the specific steps of glue recovery and purification are as follows:
column balancing: putting the adsorption column into a collection pipe, adding 500 mul of balance liquid BL into the adsorption column CA2, centrifuging at 12000rpm for 1min, pouring out waste liquid in the collection pipe, and putting the adsorption column back into the collection pipe again;
cutting a target band obtained from the PCR product in the S5 from the agarose gel and putting the cut target band into a clean centrifugal tube;
thirdly, adding 400 mul of sol solution PN into the target strip, wherein the mass ratio of the target strip to the sol solution PN is 0.3-0.7: 1, carrying out 50 ℃ water bath, continuously and gently turning the centrifugal tube up and down until the gel block is completely dissolved, cooling the temperature of the gel solution to room temperature after the gel block is completely dissolved, and loading the gel solution into a column;
putting the adsorption column into another collection pipe, adding the solution obtained from the third step into the adsorption column CA2, standing at room temperature for 2min, centrifuging at 12000rpm for 1min, pouring the waste liquid in the collection pipe, and putting the adsorption column CA2 into the collection pipe again;
fifthly, adding 600 mul of rinsing liquid PW added with absolute ethyl alcohol into the adsorption column CA2, wherein the adding ratio of the rinsing liquid PW to the absolute ethyl alcohol is 1: 3-5, standing for 5 minutes at room temperature, centrifuging at 12000rpm for 1min, pouring out waste liquid in the collection pipe, and putting the adsorption column CA2 into the collection pipe;
sixthly, repeating the operation steps;
seventhly, placing the adsorption column CA2 into a collecting pipe, centrifuging at 12000rpm for 2min, removing rinsing liquid PW, placing the adsorption column CA2 into a clean centrifugal pipe, standing at room temperature for several minutes, and completely drying;
eighthly, suspending and dropwise adding 30 mul of elution buffer solution EB to the middle position of the adsorption film, keeping the adsorption film heated in a water bath at 65 ℃ when the elution buffer solution EB is dropwise added, standing at room temperature for 2min, centrifuging at 12000rpm for 2min, collecting filtrate, and obtaining DNA solution, and storing at-40 ℃.
6. The molecular identification method of Marneffei staphylium according to claim 1, characterized in that: the T-A cloning method comprises the following specific steps:
preparation of LB liquid culture medium and LB solid culture medium
Weighing 1% of tryptone, 0.5% of yeast extract and 1% of NaCl in a blue-covered bottle, and additionally adding 1.5% of Agar when preparing an LB solid culture medium;
adding 480ml of deionized water into a blue-cap bottle, stirring and dissolving, and fixing the volume to 500ml after the solid is completely dissolved;
iii, adjusting the pH value of the solution to 7.0;
iv, unscrewing the blue cap bottle, and putting the prepared solution into an autoclave for autoclaving at the temperature of 121 ℃ for 20 min;
and v, screwing down a blue cover bottle cap, cooling the sterile LB culture medium at room temperature, and then putting the sterile LB culture medium into a refrigerator at 4 ℃ for later use;
activation of DH5a Strain
Placing DH5a strain preserved in a refrigerator at-80 ℃ on ice for thawing;
II, inoculating the melted DH5a strain into the prepared LB liquid culture medium in an aseptic operation platform, and culturing in a shaking table at the speed of 200rpm and the temperature of 37 ℃;
measuring the concentration of the bacterial liquid to enable the OD value to be 0.35-0.4, and then placing on ice;
③GaCl2preparation of the solution
Weighing anhydrous GaCl21.1g, placing the mixture into a conical flask, adding deionized water, stirring and dissolving, and fixing the volume to 100 ml;
ii, using 50ml under sterile environmentSyringe and 0.22 μm filter for dissolved GaCl2Filtering the solution for sterilization, screwing down a bottle cap, and placing at-4 ℃ for later use;
④GaCl2solution treatment of bacterial species
Transferring the bacterial liquid in the conical flask into a 15ml centrifugal tube, centrifuging for 10min at 4000rpm and 4 ℃, pouring out the bacterial liquid, and sucking out the residual bacterial liquid by using a liquid transfer gun;
ii, 1ml of GaCl2Blowing the thallus from the solution gently, blowing and beating the thallus uniformly, and then placing the thallus on ice for 40 min;
iii 4000rpm, centrifugation at 4 ℃ for 10min, using GaCl2Blowing, beating and uniformly mixing the thalli by the solution to complete preparation of competent cells, and placing the competent cells on ice for later use;
connect
Taking 5 mul of clone sample and 1 mul of T-A carrier, and gently and uniformly blowing and beating the clone sample and the T-A carrier on ice;
II, connecting on a metal bath for 2 hours at 25 ℃;
sixthly, transformation
After the connection is finished, adding 100 mul of competent cells into the connection product through aseptic operation, gently blowing and uniformly mixing the competent cells after the addition, and placing the competent cells on ice for 30 min;
ii, finishing ice bath, performing heat shock at 42 ℃ for 90s, and immediately placing on ice;
adding 1ml of LB liquid culture medium into the transformation product, gently and slowly blowing, uniformly mixing, placing on a shaker at 180rpm, and shaking and culturing for 40min at 37 ℃;
iv, melting the solid culture medium, cooling to 50 ℃, adding 0.1 percent of Amp +, and pouring the mixture into a flat plate for later use after solidification;
centrifuging the bacterial liquid cultured by the shaking table at 5000rpm for 10min, removing supernatant, leaving 200 mu l of supernatant, and carrying out plate coating on the heavy suspension bacteria;
vi, after the plate is coated, sealing the opening and inversely placing the opening in an incubator at 37 ℃ for culturing for 12 hours;
and vii, taking 1ml of sterile centrifuge tube, sucking 600 mu l of LB liquid culture medium added with Amp +, wherein the 1ml of LB liquid culture medium contains 50 mu g of Amp +, picking the large and full single colony in the plate into the LB liquid culture medium, covering the LB liquid culture medium, placing the plate on a shaking bed at 37 ℃, and carrying out shaking culture at 160rpm for 6 hours to obtain the colony.
7. The molecular identification method of Marneffei staphylium according to claim 4, wherein: the thickness of 1% agarose is preferably 0.5 cm.
8. The molecular identification method of Marneffei staphylium according to claim 5, wherein: the mass ratio of the target band to the sol solution PN is preferably 0.5: 1.
9. The molecular identification method of Marneffei staphylium according to claim 5, wherein: the ratio of the rinsing liquid PW to the absolute ethyl alcohol is preferably 1: 4.
10. The molecular identification method of Marneffei staphylium according to claim 6, wherein: during shaking table cultivation, at first pack liquid with box (1), the liquid level is located sieve (2) top, the mode that adopts the water bath heating keeps constant temperature, utilize controller (4) control heating rod (101) and hot plate (102) to move simultaneously and adjust the temperature to predetermineeing the temperature, then place the blake bottle between two holding head (205), receive spring (202) effect, holding head (205) and blake bottle outer wall elastic contact, through clearance between two adjustable holding head (205) of rotatory screw rod (204), be applicable to the centre gripping of different model blake bottles, the blake bottle is fixed the back, utilize control driver (302) to adjust actuating lever (301) and remove with invariable vibration frequency, carry out shaking table cultivation.
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Cited By (2)

* Cited by examiner, † Cited by third party
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CN114657242A (en) * 2022-03-16 2022-06-24 广州医科大学附属第一医院 Application of GPR33 gene in assessment of marneffei Talaromyces susceptible population
CN114657242B (en) * 2022-03-16 2022-11-11 广州医科大学附属第一医院 Application of GPR33 gene in assessment of marneffei Talaromyces susceptible population

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