Jun 28, 2017
Version 1
Identification of Diatraea spp (Lepidoptera: Crambidae) based on Cytochrome oxidase II and SSCP V.1
- Gloria Patricia Barrera1,
- Deisy Liseth Toloza1,
- Laura Fernanda Villamizar1,
- Carlos Espinel.1
- 1Centro de investigación Tibaitatá, Corporación Colombiana de Investigación Agropecuaria CORPOICA, Km 14 Vía Mosquera 250047, Cundinamarca, Colombia. AgResearch Ltd. Lincoln Research Centre, Christchurch 8140, New Zealand.
Protocol Citation: Gloria Patricia Barrera, Deisy Liseth Toloza, Laura Fernanda Villamizar, Carlos Espinel. 2017. Identification of Diatraea spp (Lepidoptera: Crambidae) based on Cytochrome oxidase II and SSCP. protocols.io https://dx.doi.org/10.17504/protocols.io.ipqcdmw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: June 28, 2017
Last Modified: February 26, 2018
Protocol Integer ID: 6608
Keywords: Diatraea, SSCP, PCR
Abstract
Diatraea spp. (Lepidoptera: Crambidae) are a group of insects considered as an agriculture pest in many economically relevant crops such as sugarcane, sorghum, corn and rice. Currently, identification is based on the male genitalia. However, the availability of specimens collected from field and subjectivity on the character recognition can seriously hamper species identification, and therefore result in inadequate pest management. We described a protocol based on a fragment of the cytochrome oxidase subunit II (CO II) mitochondrial gene and Single-Strand Conformation Polymorphism (SSCP) methodology to properly recognizing each species and in consequence is proposed as an effective tool in Diatraea species identification.
Guidelines
- Simon C, Frati F, Beckenback A, Crespi B, Liu H, Flook P. Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved PCR primers. Ann Entomol Soc Am. 1994; 87: 651-701.
- Lange CL, Scott KD, Graham GC, Sallam MN, Allsopp PG. Sugarcane moth borers (Lepidoptera: Noctuidae and Pyraloidea): phylogenetics constructed using COII and 16S mitochondrial partial gene sequences. Bull Entomol Res. 2004; 94(5): 457-464
- Palacio-Cortés AM., Zarbin PH, Takiya DM, Bento JM, Guidolin AS, Consoli FL. Geographic variation of sex pheromone and mitochondrial DNA in Diatraea saccharalis (Fab., 1794) (Lepidoptera: Crambidae). J Insect Physiol. 2010; 56: 1624-1630.
Materials
MATERIALS
AcrylamideP212121
bind silaneMerck MilliporeSigma (Sigma-Aldrich)Catalog #17-1330-01
Safety warnings
Acrylamide solution may cause cancer, heritable genetic damage, possible risk of impaired fertility.
It is toxic if swallowed, and may causeserious damage to health by prolonged exposure through inhalation, in contact with skin and if swallowed Harmful by inhalation and in contact with skin. Irritating to eyes and skin and may cause sensitisation by skin contact
Dissection, DNA extraction and COII fragment amplification:
Abdomens from insects must be dissected and maintained in ethanol at 70 % v/v in sterile distilled water to DNA extractions. DNA of insects must be obtained from complete abdomen by using commercial kit (DNeasy Blood & Tissue Kit, QIAGEN) and quantified by spectrophotometry (Nanodrop 1000, Thermo-Fisher).
COII amplification: In vitro amplifications of CO II fragments must be carried out by the Polymerase Chain Reaction method (PCR) in standard conditions (final volume 25 μL) using Taq polymerase (PROMEGA), 50 ng of template and the primers previously described and used in Diatraea: A-298 (5’- ATTGGACATCAATGATATTGA-3’) and B-tLYS (5’ GTTTAAGAGACCAGTACTTG-3’) (Simon et al. 1994; Lange et al. 2004, Palacio et al. 2010). Thermal cycling was performed using the following conditions: 1 cycle at 95 °C for 3 min; 34 cycles at 95 °C for 10 s; 53 °C for 45 s, and 72 °C for 30 s; and one cycle at 72 °C for 5 min. Aliquots of amplification products were resolved in 1% w/v agarose gel electrophoresis and later stained with SYBR-Safe (Invitrogen). Amplicons will be used for SSCP methodology.
Single-Strand Conformation Polymorphism (SSCP)
Single-Strand Conformation Polymorphism (SSCP)
Preparing electrophoresis equipment
For this protocol the Biorad Sequi-Gen® GT (38 x 50 cm) was used. The detailed instructions for assembly are in http://www.cmu.edu.cn/cmu/upl_files/327/2007111485016635.pdf
To prepare Sequi-gen glass plate (IPC) and outer plates follow the Biorad manual instructions. Additionally, after cleaning, add silicone and shine the IPC (rub until silicone residue is completely removed). The outer plate must be prepared with bind silane (See: Reagent Preparation) and after cleaned two times with ethanol.
Note: Tips and paper towels used for the treatment of outer plate should be discarded immediately after use avoiding contamination of IPC.
Assembly the glass plate sandwich and slowly inject the polyacrylamide solution (6%, 49:1) with a 100 mL syringe avoiding bubbles formation. To prepare polyacrylamide solution and electrophoresis buffers see Reagent Preparation.
Preparing sample and running electrophoresis
After PCR of COII fragment, prepare the sample by mixing 9 μL of loading buffer (95% v/v Formamide, 20mM EDTA, 0.05% w/v bromophenol blue and 0.05% w/v Xylene-cyanol, in distilled water),) with 1 μL of PCR product. Denature for 5 minutes at 95 °C and immediately after cool on ice for 3 minutes. Add approximately 1.3 μL of PCR product mix into the wells. Place the electrodes caps and connect to the power supply. Apply 600 volts and run for 8 hours.
Staining the gel (See Reagent Preparation).
Stain gel using and adapted silver nitrate method. Briefly, gels fix for 3 min (10% v/v ethanol, 1% v/v acetic acid in distilled water), oxide for 3 min (1.5 % v/v nitric acid in distilled water), stain for 20 min (0.1% w/v silver nitrate, 0.045 % v/v formaldehyde in distilled water), reveal for approximately 5 min (3% w/v NaCO3, 0.02 % v/v formaldehyde in distilled water), and stop reaction by adding acetic acid solution (5 % v/v in distilled water).
REAGENT PREPARATION
Bind silane Solution:
Absolute ethanol 95%
Acetic acid glacial 5 %
Bind-Silane 0.13%
Apply 1mL to outer glass
Acrylamide bisacrylamide Stock (49:1; 40%)
Acrylamide 39.2 g
Bisacrylamide 0.8 g
Distilled water to 100 mL
6% polyacrylamide solution (49:1)
Acrylamide bisacrylamide stock 15 mL
10X TBE Solution 10 mL
Distilled water 75 m
Distilled water to 100 mL
Immediately before use add Ammonium Persulfate (10%) 850 ul and TEMED108 ul
TBE buffer (10X)
Tris base 53.85 g
Boric acid 27.51 g
0.5M EDTA (pH: 8.0) 20 mL
Add water to 500 mL
STAINING POLYACRYLAMIDE GELS
FIXATION SOLUTION
Absolute ethanol 150 mL
Acetic acid glacial 15 mL
Distilled water 1335 mL
Add water to 1500 mL
Leave for three minutes. Wash with distilled water for three minutes.
OXIDATION SOLUTION
Nitric acid 22.5 ml
Distilled water 1477.5 ml
Add water to 1500 ml
Leave for three minutes. Wash with distilled water for three minutes.
NITRATE SOLUTION
Silver nitrate 1.5 g
Formaldehyde 1.8 mL
Add distilled water to 1500 mL
Leave for twenty minutes. Wash with distilled water for ten seconds.
REVEALED SOLUTION
Sodium carbonate 30 g
Formaldehyde 540 ul
Add distilled water to 1000 mL
Leave until the gel reveals.
STOP SOLUTION
Acetic acid glacial 75 mL
Distilled water 1425 mL
Add distilled water to 1500 mL
Leave for four minutes. Wash with distilled water for four minutes.