Molecular detection of wolbachia in the tricolor book

Experimental reagent

Chloroform [Chongqing Chuandong Chemical Group Co., Ltd.]

Isopropanol [Chongqing Chuandong Chemical Group Co., Ltd.]

Anhydrous ethanol [Chongqing Chuandong Chemical Group Co., Ltd.]

Isoamyl alcohol [Shanghai Chemical Reagent Co., Ltd.]

Bromophenol blue [Shanghai San Aisi Reagent Co., Ltd.]

Xylene cyanide FF [Amersco Co.]

Ethidium bromide [Amersco Co.]

Saturated Phenol (pH 7.0) [ Tianjin, H&Y Bio. Co. Ltd]

Taq DNA Polymerase [ Tianjin, H&Y Bio. Co. Ltd]

Sodium lauryl sarcosinate [ Tianjin, H&Y Bio. Co. Ltd]

Sodium lauryl sulfate [ Tianjin, H&Y Bio. Co. Ltd]

Tri-light methylaminomethane [ Tianjin, H&Y Bio. Co. Ltd]

Ethylenediaminetetraacetic acid [ Tianjin, H&Y Bio. Co. Ltd]

Saturated phenol [ Tianjin, H&Y Bio. Co. Ltd]

100 bp Ladder DNA Marker [ Tianjin, H&Y Bio. Co. Ltd]

Agarose [ Tianjin, H&Y Bio. Co. Ltd]

Laboratory equipment
SW-CJ-CO purification workbench [Suzhou Purification Equipment Co., Ltd.]

FA1004A electronic balance [Shanghai Jingtian Electronic Instrument Factory]

70 type ion water purifier [Shanghai Yarong Biochemical Instrument Factory]

SZ-93 automatic double pure water distiller [Wuxi Keda Instrument Factory]

Mikro22R high speed freezing centrifuge [Whatman Biometra]

5415D Bench Top Centrifuge [Eppendorf]

SS-325 Autoclave [Tomy Kogyo Co. Ltd]

YLE-2000 digital display electric heating constant temperature water bath [Shanghai Yuejin Medical Instrument Factory]

pHS-4C type acidity meter [Chengdu Ark Technology Development Co., Ltd.]

2002 type oscillator [Changzhou Guohua Electric Co., Ltd.]

Vortex Mixer [Northern Tongzheng Biotechnology Development Company]

Gradient thermal cycler [Whatman biometra]

Gel Imaging System [Shanghai Four Star Biotechnology Industrial Co., Ltd.]

MDF-382E cryopreservation box [Sanyo Electric Biomedical Co., Ltd]

DYY 12 type computer three constant multi-purpose electrophoresis instrument [Beijing Liuyi Instrument Factory]

SmartSpecTM 3000 Nucleic Acid Concentration Analyzer [BIO-RAD]

Experimental Materials

The tricolor book was collected from the wild and established in the laboratory.

Experimental procedure

1. Preparation of total DNA of tricolor book

(1) Place 50 stacks in a 1.5 mL centrifuge tube, and thoroughly grind the test insects in a liquid nitrogen atmosphere with a pestle and add 200 μL of DNA extraction lysate.

(2) Add 2.5 μL of proteinase K solution (20 mg/mL), and bath at 50 ° C for 2 h (or overnight).

(3) Shake well (about 20 min) with an equal volume of balanced phenol (Tris-HC1 saturated phenol, pH 7.8) and store at 20 ° C for 5-10 min.

(4) Centrifuge at 4 ° C for 10 min, and transfer the viscous aqueous phase to a clean centrifuge tube.

(5) Extract once with an equal volume of phenol: chloroform (1:1), phenol: chloroform: isoamyl alcohol (25:24:1) and chloroform, and centrifuge (9000 r/min 10 min) to take the supernatant. .

(6) A 0.2 volume volume of sodium acetate solution (10 mol/L) and a double volume of absolute ethanol were added, and the DNA was sufficiently precipitated by standing at -20 ° C for 2 hours.

(7) Centrifugation (12000 r/min 10 min), the supernatant was discarded, and the pellet was washed twice with 70% ethanol (4 ° C).

(8) After the pellet was air-dried (as much as possible to remove ethanol), the DNA was resuspended in TE buffer (pH 8.0), and gently shaken at 37 ° C to facilitate DNA resuspension to fully dissolve the DNA.

(9) Add Rnase to a final concentration of 1 μg/mL, and incubate at 37 ° C for 1 hr.

(10) Extract according to (4)~(9), precipitate, resuspend the DNA, and store in 4 μL of double-digested water or TE at 4 °C.

(11) 4 μL of DNA was mixed with 2 μl of 6x loading buffer, and spotted onto a 1.4% agarose gel and electrophoresed at 2.5-5 v/cm.

(12) EB staining at 0.5 μg/mL for 15-30 min, photographed under UV light.

The prepared DNA concentration was measured by a SmartSpecTM 3000 nucleic acid concentration analyzer. It was required to repeatedly wash the cuvette with 100 μL/time of sterile water and measure the absorbance of water, and then dilute the DNA solution by 50 times for detection. Requires OD = 1.7 or so.

2. Long PCR detection of symbiotic bacteria Wolbachia in three-color book

The 20uL reaction system of Long PCR includes:

10 x buffer
5 μL

25 mM MgCl2
2 μL

10 mM dNTP
0.7 μL

Pwo enzyme 1 U

Taq DNA
5 U

Template 10 ng

10μM primer 1.6 μL

wsp-F, 5'-TGG TCC AAT AAG TGA TGA AAG AAA CTA GCT A

wsp-R, 5'-AAAAAT TAAACG CTA CTC CAG CTT CTG CAC

Replenish the reaction system to 20 μL with sterile water

The amplification conditions were: 94 ° C for 2 min pre-denaturation, 94 ° C l0 sec, 65 ° C 30 sec, 68 ° C lmin, 10 cycles; 94 ° C l0 sec, 65 ° C 30 sec, 68 ° C lmin. 25 cycles, each cycle extending at 68 ° C for 20 sec.

After the completion of the amplification, 5 μL of the PCR product was detected by 1.5% agarose gel electrophoresis (voltage 5 v/cm, electrophoresis buffer was 1 X TAE), and the results were observed under a Bio-Rad gel imaging system.

3. Determination and analysis of sequences

The Wolbachia wsp gene sequence was obtained by direct sequencing of the Long PCR product from Shanghai. DNA sequence searches and homology comparisons were made using the BLAST tool (NCBI website). The phylogenetic tree is reconstructed by the Maximum Likelihood Method (ML) in the program, and the bootstrap test confidence of the nodes in the phylogenetic tree is estimated by 1000 repeated calculations. The phylogenetic analysis of the fragment of the Wsp gene of Wolbachia in the obtained trichrome scorpion was performed, and the fragments of the Wsp gene of Wolbachia in other hosts were obtained by GenBank.

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