Perera et al. Rapid Identification of Helicoverpa armigera and Helicoverpa zea (Lepidoptera:
Noctuidae) Using Ribosomal RNA Internal Transcribed Spacer 1.
Detailed protocol for detecting Helicoverpa armigera in pools of up to 25 samples.
1. Reagents and supplies.
a. NaCl
b. EDTA
c. Trizma Base
d. HCl
e. PCR plates to match requirements of the real time PCR instrument
f. Optical film for sealing real-time PCR plates.
g. Deep-well 0.8 or 1 ml 96-well plates or skirted PCR plates for homogenizing
samples.
h. Silicone gaskets for sealing deep-well plates or foil seals for PCR plates.
i. Stainless steel ball bearings (bleached, washed, and sterilized)
i. 2 mm for small sample pools homogenized in PCR plates
ii. 4 mm for 0.8 or 1 ml deep-well plates for large sample pools
j. Mini-BeadBeater or equivalent instrument for homogenizing samples.
k. Centrifuge equipped with a swing-bucket rotor for 96-well plates.
l. SYBR Fast or TaqMan 2X master mix or any suitable PCR reagent (optimized for
the instrument and specific requirements of individual laboratories).
m. Primer cocktail (20X) containing 8 µM each of primers 3373 and 3377, 4 µM of
primer 3374, and 2 µM each of primers 3695 and 3696.
2. Prepare “Squish” buffer (modified from Gloor et al, 1993) for 100 ml
a. 1 ml of 1 M Tris-HCl, pH 8.2 (final concentration 10 mM Tris-HCl)
b. 100 µl of 0.5M EDTA (final concentration 0.5 mM EDTA)
c. 1.25 ml of 1M NaCl (final concentration 12.5 mM NaCl)
Add 97.65 ml of distilled deionized water and autoclave for 15 min.
Note: We used 25µl of the modified squish buffer per adult leg or neonate. DNA
concentration in lysates in pools containing multiple samples could be increased by
using less squish buffer per sample.
3. If using adults moths from pheromone traps, up to 25 legs could be homogenized in a
deep-well plate
a. Place a stainless steel ball bearing on the bottom of each well
b. Pull one leg from each of the sample to be pooled and place in a well of the 96well plate
Note: Make sure to keep all the samples pooled together (e.g. place them
in a plastic bag and label with plate number and the well number)
c. Place a stainless steel ball bearing on top of the samples and add up to 25 µl of
squish buffer per sample (use less if higher DNA concentrations are needed).
d. Seal the deep-well plate or PCR plate containing samples using a silicone mat or a
foil seal, respectively.
e. Secure the sealed sample plate to the holder of the mini-Bead Beater.
Homogenize the samples for 2-3 min and centrifuge the plate briefly to bring the
liquid to the bottom of plate
f. Incubate the plate in an incubator set to 80 ºC or a thermal cycler (PCR plates) for
15 min. (note: the PCR master mix (step 4.a) could be prepared during this
incubation period)
g. Centrifuge the sample plate at maximum possible speed (depending on the
centrifuge and 96-well plate type) for 10 min to pellet debris. Transfer one µl the
tissue lysate to a PCR plate containing reagents using a multi-channel pipette.
Tissue homogenate can be stored in a -20 ºC freezer for up to 2 weeks if needed to
re-use. Up to two µl of the tissue lysate per 20nµl reaction could be used if
desired. However, increased salt concentrations will increase the Tm of the
amplicons (i.e. shift peaks to the right) and may alter the derivative dissociation
curves profiles.
4. Prepare the PCR master mix following manufacturer’s instructions. Instructions for 100
reactions (20 µl/reaction) using a 2X master mix and 1 µl of tissue lysate per reaction are
given below. If more tissue lysate was to be used, reduce the volume of water
accordingly to obtain a total of 2,000.0 µl.
a. 1,000.0 µl 2X master mix
100.0 µl 20X primer cocktail
X µl ROX (optional)
Y µl SYTO-9 or SYBR Green (if not included in the master mix)
Z µl Water to final volume of 1,900.0 µl.
b. Dispense 19 µl of the reaction mix (from 4.a ) to each well of a 96-well PCR
plate appropriate for the real-time PCR instrument being used.
c. Dispense 1 µl DNA (tissue lysate) to each well using a multi-channel pipette (step
3.g).
5. Program the real-time PCR instrument for amplification step. Alternatively, amplification
can be performed using a standard thermal cycler programmed with the thermal cycling
profile given in step 5.c.
a. Start a new document and provide a name for the file.
b. Select SRBR or SYTO-9 as the detector (make sure that the instrument is
calibrated for the dye being used)
c. Set the amplification cycling to 30 S initial denaturing step at 95 ºC, 35-40 cycles
of denaturing step of 5s at 95 ºC and annealing/extension step of 30 S at 60 ºC.
6. Program the real-time PCR instrument for dissociation curve analysis.
a. Open a new document and delete automatically inserted amplification thermal
profile.
b. Add a dissociation analysis to the document. Dissociation curve parameters are
automatically set as follows:
i. Initial denaturing step of 15 S at 95 ºC followed by annealing step of 1
min at 65 ºC
ii. Ramp temperature to 95 ºC at a rate of 0.1 ºC/S.
c. Set up samples and add the detector of your choice (e.g SYBR) to the samples.
d. For ABI instruments, make sure to select “none” for passive reference dye (which
is set to ROX by default).
e. Save the file and start the instrument.
f. At the end of run, save the file and analyze using HRM software.