WS/T 594-2018Test methods of fly resistance to insecticides-A molecular method for detecting insensitive ace allele in the Musca domestica (English PDF)

Foreword

This standard was drafted in accordance with the rules given in GB/T 1.1-2009.

This standard is mainly drafted by. Institute of Zoology, Chinese Academy of Sciences, Beijing Municipal Center for Disease Control and Prevention, Yangzhou University, China Disease Pre-Prevention and Control Center of Infectious Disease Prevention and Control Center, China Agricultural University, Military Medical Research Institute of the Chinese People's Liberation Army Academy of Military Sciences Institute of Diseases, Shanghai Municipal Center for Disease Control and Prevention.

1 Scope

This standard specifies the detection method of the housefly insensitive acetylcholinesterase allele.

This standard applies to the detection of the 342 amino acid locus genotype of Musca domestica acetylcholinesterase.

2 Terms and definitions

The following terms and definitions apply to this document.

Acetylcholinesterase acetyl cholinesterase A hydrolase present in an organism, encoded by the acetylcholinesterase gene ace.

Allele

Different forms of the same gene present in the same species are referred to as alleles.

342G, 342V and 342A

342G, 342V and 342A respectively indicate that the amino acid at the 342th amino acid residue position of Musca domestica acetylcholinesterase is glycine (Gly), Proline (Val) and alanine (Ala).

3 Sample collection

The housefly adults were collected from the scene by trapping or net trapping, and immersed in 100% ethanol solution and brought back to the laboratory. Soaking the sample can be Store at 4 °C for a short period of time (within 6 months) and store at -20 °C for a long time.

4 Primers

The sequence of primer F is CGGTGCATTTGGGTTTCTAC, and the sequence of primer R is CGTAACCGCAGAGTCGCGCTG. Primer design reference Record A. Prepare at a concentration of 10 µmol/L and store in a freezer at -20 °C.

5 Instruments

5.1 PCR (polymerase chain reaction) instrument.

5.2 Agarose gel electrophoresis instrument.

5.3 Gel Imager.

5.4 Constant temperature water bath.

6 Main reagents

6.1 DNA polymerase (Taq enzyme).

6.2 Restriction enzyme Bsp1286I.

6.3 Restriction enzyme Eae I.

7 Operating steps

7.1 Add 10× PCR buffer (containing 100 mmol/L Tris-HCl, 500 mmol/L KCl, 15 mmol/L MgCl2) to the PCR tube.

2.5 µL, dNTP (2.5 mmol/L) 2.0 µL, primer F 0.5 µL, primer R 0.5 µL, genomic DNA of Musca domestica 1 µL (100 ng ~ 300 ng, extraction method can be found in Appendix B, can also be extracted with commercially available DNA extraction kits or other similar methods) and Taq Enzyme (2 to 5 units), add up to 25 µL with sterile water, and mix.

7.2 Put the PCR tube into the PCR machine and set the PCR reaction procedure to. 94 °C for 3 min; 94 °C for 30 s, 50 °C for 30 s, 72 °C.

30 s, 35 cycles; 72 °C for 10 min. Run the program.

7.3 After the end of the PCR run, 5 µL of the PCR product was electrophoresed on a 1.5% agarose gel, and the voltage was set at 160 V.

After 20 min, it was detected under ultraviolet light, and a PCR product with a clear single 609 bp band was used for the following digestion reaction.

7.4 Two PCR reactions were performed on the PCR product of the qualified sample (that is, the electrophoresis product was 609 bp), and the reaction A was treated with Bsp1286I.

Reaction B is digested with the Eae I enzyme, and the enzyme digestion reaction system is set according to the instructions for use of the enzyme product (see Appendix C).

7.5 The digested product was loaded onto a 1.5% agarose gel at a voltage of 160 V, electrophoresed for 20 min and examined under UV light.

Measurement.

8 Genotype determination

The genotype of acetylcholinesterase in Musca domestica was determined according to the electrophoresis results of the digested products (see Appendix D), as shown in Table 1.

Table 1 Genotypic determination of acetylcholinesterase in Musca domestica

Reaction I (with Bsp1286I digestion) Reaction II (with Eae I digestion) Acetylcholinesterase 342 Amino acid site genotype 361 bp, 248 bp 609 bp sensitive homozygote (G/G) 609bp 609bp insensitive type I homozygous (V/V) 609bp 361bp, 248bp insensitive type II homozygote (A/A) 609bp, 361bp, 248bp 609bp insensitive type I/sensitive heterozygote (G/V) 609bp, 361bp, 248bp 609bp, 361bp, 248bp insensitive type II/sensitive heterozygote (G/A) 609bp 609bp, 361bp, 248bp insensitive type I/insensitive type II heterozygote (V/A) Note. G indicates that the amino acid residue is Gly (glycine); V indicates that the amino acid residue is Val (valine); A indicates that the amino acid residue is Aal (C Amino acid).

Appendix A

(informative appendix) Primer design reference sequence 342V CGGTGCATTTGGGTTTCTACATCTTTCACCGGTTATGCCAGGTTTTGAAGAAGAAGCTCC 342A CGGTGCATTTGGGTTTCTACATCTTTCACCGGTTATGCCAGGTTTTGAAGAAGAAGCTCC 342G CGGTGCATTTGGGTTTCTACATCTTTCACCGGTTATGCCAGGTTTTGAAGAAGAAGCTCC 342V CGGCAACGTGGGTCTTTGGGATCAGGCCTTGGCCTTGCGTTGGCTGAAGGAGAATGCCCG 342A CGGCAACGTGGGCCTCTGGGATCAGGCCTTGGCCTTGCGTTGGCTTAAGGAGAATGCCCG 342G CGGCAACGTGGGCCTTTGGGATCAGGCCTTGGCCTTGCGTTGGCTGAAGGAGAATGCCCG 342V TGCATTTGGCGGCAATCCGGAATGGATGACGCTGTTTGGTGAATCGGCTGGTTCGAGTTC 342A TGCATTTGGCGGCAATCCGGAATGGATGACGCTGTTTGGTGAATCGGCTGGTTCGAGTTC 342G TGCATTTGGCGGCGATCCCGAATGGATGACGCTGTTTGGTGAATCGGCTGGTTCGAGTTC 342V CGTGAATGCTCAACTGATGTCGCCGGTAACGCGTGGCCTGGTCAAACGTGGTATGATGCA 342A CGTGAATGCTCAACTGATGTCGCCGGTAACGCGTGGCCTGGTCAAACGTGGCATGATGCA 342G CGTGAATGCTCAACTGATGTCGCCGGTAACGCGTGGCCTTGTCAAACGTGGCATGATGCA 342V GTCGGTCACAATGAATGCTCCCTGGAGCCACATGACATCAGAGAAGGCGGTTGAAATTGG 342A GTCGGCCACAATGAATGCTCCCTGGAGCCACATGACATCAGAGAAGGCGGTTGAAATTGG 342G GTCGGGCACAATGAATGCTCCCTGGAGCCACATGACATCAGAGAAGGCGGTTGAAATTGG 342V TAAAGCTTTGGTAAATGACTGTAACTGTAATGCCTCATTGTTACCGGTAAGCAGTGGAGT 342A TAAAGCTTTGGTAAATGACTGTAACTGTAATGCCTCATTGTTACCGGTAAGCAGTGGAGT 342G TAAAGCTTTGGTAAATGACTGTAACTGTAATGCCTCATTGTTACCGGTAAGCAGTGGAGT 342V ATTTATGCGACTATACGAATTTTTCTTTGCTGATGAATTTTATTTTCCTCTTTTTTGTGT 342A ATTTATGCGACTATACGAATTTTTCTTTGCTGATGAATTTTATTTTCCTCTTTTTTGTGT 342G ATTTATGCGATTACACGAATTATTCTTTGCTGATGAATTTTATTTTCGGCTTCTTTGTGT 342V TTTCAACTTAGGAAAATCCACAAGCTGTCATGGCTTGCATGCGACAGGTTGATGCGAAAA 342A TTTCAACTTAGGAAAATCCACAAGCTGTCATGGCTTGCATGCGACAGGTTGATGCGAAAA 342G TTTCAACTTAGGAAAATCCCCAAGCTGTCATGGCTTGCATGCGACAGGTTGATGCGAAAA 342V CAATTTCTGTCCAACAATGGAACTCGTATTCTGGAATTTTAAGTTATCCCTCGGCCCCAA 342A CAATTTCTGTCCAACAATGGAACTCGTATTCTGGAATTTTAAGTTATCCCTCGGCCCCAA 342G CAATTTCTGTCCAACAATGGAACTCGTATTCTGGAATTTTAAGTTTTCCCTCGGCCCCAA 342V CTATAGATGGAGCATTTTTGCCCGCAGATCCAATGACACTGTTGAAAACAGCAGATCTTA 342A CTATAGATGGAGCATTTTTGCCCGCAGATCCAATGACACTGTTGAAAACAGCAGATCTTA 342G CTATAGATGGTGCATTTTTGCCCGCAGATCCAATGACACTGTTGAAAACAGCAGATCTTA 342V GCGGTTACG 342A GCGGTTACG 342G GCGGTTACG Note. The underline corresponds to the upstream primer and the downstream primer sequence (reverse complementation); the box contains the codon for the 342 amino acid.

Appendix B

(informative appendix) Housefly genomic DNA extraction method B.1 Take a single-headed housefly, remove the abdomen, place it in a 1.5 mL volume centrifuge tube (eppendorf tube), and add 0.5 mL of freshly prepared Lysate (containing 100 mmol/L Tris-HCl pH 8.0, 10 mmol/L EDTA, 50 mmol/L NaCl, 1% SDS, 0.15 mmol/L spermine, 0.5 mmol/L spermidine and 2 U/mL proteinase K), milled the worms and placed in a warm bath at 60 °C.

Take it out after 20 minutes.

B.2 Add 75 µL of 8 M potassium acetate, mix and place on ice for 10 min.

B.3 Centrifuge at 14000 g for 5 min, transfer 400 µL of the supernatant to a new centrifuge tube.

B.4 Add 800 µL of ice-cold 100% ethanol and let stand for 10 min at room temperature.

B.5 Centrifuge for 10 min, discard the supernatant, and centrifuge to wash with 500 µL of 70% ethanol.

B.6 Centrifuge for 5 min, after sedimentation and drying (can be placed in a clean bench, open ventilation), add 50 µL of sterilized water or TE buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH=8.0) dissolved. The extracted DNA sample can be used directly for PCR. sample Store at 4 °C for long-term storage at -20 °C.

Appendix C

(informative appendix) Enzyme digestion system setting example the setting of C.1 digestion reaction system is shown in Table C.1.

Table C.1 Digestion reaction system

Ingredient 10×NEBuffer 4 100×BSA Bsp1286I Eae I PCR product sterilized water Reaction A 2 µL 0.5 µL 0.5 µL 0 1 µg to 20 µL Reaction B 2 µL 0 0 0.5 µL 1 µg to 20 µL Note. Restriction enzyme Bsp1286I, EaeI was used. 10×NEBuffer 4 and 100×BSA are provided with the enzyme, of which 1×NEBuffer 4 containing 50 mmol/L KAc, 20 mmol/L Tris-Ac, 10 mmol/L Mg (Ac) 2, 1 mmol/L DDT, pH 7.9 (25 ° C as determined), 100 × BSA was 10 mg/mL of BSA (bovine serum albumin).

C.2 Warm bath at 37 ° C, time 2h ~ 4h, the reaction is over, directly used for electrophoresis detection.

Appendix D

(informative appendix) Enzyme digestion results of different genotype PCR products of 347 acetylcholinesterase in Musca domestica the PCR product digestion results of different genotypes of 347 acetylcholinesterase in Musca domestica are shown in Figure D.1.

G--amino acid residue is Gly (glycine);

V--amino acid residue is Val (valine);

A--amino acid residue is Aal (alanine);

G/G--sensitive homozygous;

V/V--insensitive type I homozygous;

A/A--insensitive type II homozygous;

G/V--insensitive type I/sensitive heterozygotes;

G/A--insensitive type II/sensitive heterozygote;

V/A--insensitive type I/insensitive type II heterozygote;

1--Bsp1286I digestion product map;

2-EaeI digestion product map.

Note. The rightmost lane is the DNA molecular weight marker.

Figure D.1 Digestion results of PCR products of different genotypes of 342 sites in Musca domestica acetylcholinesterase