2ND PART TRAINING-COURSE LAMP AND RT-PCR AT CNRA
INSTRUCTORS: DR NDEDE YANKEY AND DR. YAIMA AROCHA ROSETE
(September 21st to 25th 2015)
SAMPLING EQUIPMENT REQUIRED
1. Hand-held GPS unit
2. Digital camera
3. Cordless drill driver with rechargeable batteries, e.g., DeWalt DC720, 18 V, and battery charger.
4. 8 mm diameter, 6.5 in. (16.5 cm) long multipurpose drill bits.
5. Portable propane torch.
6. Small hammer.
7. Clean re-sealable plastic bags.
8. Polypropylene wash bottle(s) filled with deionized water.
9. Polypropylene wash bottle(s) filled with 70% Ethanol.
10. Portable coolers with crushed ice.
11. Disposable gloves
12. Permanent markers pens
13. Laboratory wrapping paper
14. Aluminium foil
15. Insect collection set
16. Sweep nets
17. Spectrophotometer.
LAB EQUIPMENT AND MISCELLANEOUS REQUIRED AT CNRA.
1. Parafilm roll
2. RT-PCR machine with laptop
3. SYBR Green JumpStart Taq Ready mix (Sigma, USA)
4. LAMP machine (Genie II) with laptop and software licence key
5. Micro-centrifuge
6. Mini centrifuge (for strips of 8)
7. Centrifuge with rotor for multi-well plates
8. 96-well RT-PCR micro-plates and micro-sealing films (taken from Canada)
9. LFD buffer C containing steel ball bearings (Forsite Diagnostics, UK) (taken from Canada)
10. Isothermal master mix (GenSys, UK)
11. Genie® strips (strips of 8)
12. PCR cabinet
13. 2uL, 10uL, 20uL, 100uL, 1000uL tips
14. At least three sets of 2uL, 10uL, 20uL, 100uL, 1000uL pipettes
15. 0.2mL, 0.5mL, 1.5mL, 2 mL filter microtubes and racks
16. Permanent marker pens for labelling
17. Sterile deionized water
18. PCR grade RNAse-free water
19. 70% Ethanol
20. 0.5% Sodium hypochlorite
21. Eppendorf PCR coolers
LAB CONSUMABLES REQUIRED
1. LAMP Primers
CSPWD 23S assay
CSPWDF3: TAGAGGAAGGGCCTATAGCTCAGT
CSPWDB3: GTATCGCCGTTAATTGCGTC
CSPWDFIP: TGAATAAGAGGAATATGGTATGGGTGTGGTAGAGCACACGCTTGATAAG
CSPWDBIP: TCTCTAATGACACACCAATGAAGGACATCGGCTCTTAGTGCCAAG
CSPWDFL: GGACTTGAAACCATTGACCG
CSPWDBL: AAGGGCGTACAGTGGATGC
COX assay (for coconut DNA)
COX F3: TAT GGG AGC CGT TTT TGC
COX B3: AAC TGC TAA GRG CAT TCC
COX FIB: ATG GAT TTG RCC TAA AGT TTC AGG GCA GGA TTT CAC TAT TGG GT
COX BIP: TGC ATT TCT TAG GGC TTT CGG ATC CRG CGT AAG CAT CTG
COX FL: ATG TCC GAC CAA AGA TTT TAC C
COX BL: GTA TGC CAC GTC GCA TTC C
2. RT-PCR Primers
Cn18sF3: CTGTGATGCCCTTAGATGTTCTG
Cn18sR3: ACCAACAATTGCAATGATCTATCC
RTribF1: CGAATGGGGCAACCTACTAC
RTribR1: CGGATTCGCTCGTCACTAC
CnA: AACGAGACCTCAGCCTGCTA
CnB: GCGATCCGAACACTTCACC
RTA: GGCACTAAGAGCCGATGAAG
RTB: TCCTCCCATCTTCCAACAAG
FIELD AND LABORATORY METHODS
SAMPLING LEAFLETS AND PETIOLE
1. Collect one sample per palm
2. Cut down one frond.
3. Pull four leaves from different points along the rachis.
4. Cut a section of the petiole.
5. Put samples into sealable plastic bags.
6. Transport collected material to the lab.
7. Once in the lab, use a sterilized scalpel blade to strip off the laminas from the leaflets leaving
only the midrib.
8. Chop the midrib into small pieces with a clean sterilized scalpel blade.
9. Label samples accordingly.
SAMPLING INFLORESCENCES
1. Climb a palm and cut down the inflorescence.
2. Transport right after sampling the whole inflorescence to the lab
3. Once in the lab, immediately cut a clean section of the inflorescence with a sterilized kitchen
knife.
4. Chop it into small pieces with a sterilized scalpel blade.
5. Label samples accordingly.
SAMPLING TRUNK BORINGS
1. Attach a multipurpose drill bit to a cordless electric drill.
Note: An electric drill and a portable generator are fine if cordless electric drill is not available.
2. Wash sterilize the bit with 0.5% sodium hypochlorite.
3. Flame sterilize the bit using a propane torch and then cool it with a stream of deionized water.
Begin sampling by drilling a shallow pilot hole in the lower stem (around 1m above the ground
level) to remove the outermost layer of pseudobark.
4. Discard these tissues.
Note: The dead outermost layer of pseudobark varies in thickness according to the age,
condition, and species of palms. These dead tissues may contribute DNA sources from non-target
Gram-positive bacteria that may interfere with the specificity of PCR assays that incorporate
phytoplasma universal rRNA gene primer pairs.
5. Resume drilling incrementally through the pilot hole into the interior stem to a final depth of
~15 cm while using a back-and-forth motion to dislodge shavings.
6. Passively collect one sample of shavings (5g) per palm into a clean sealable plastic freezer bag
positioned at a safe distance below the rim of the drill hole.
Note: For coconut or other host palms with minimal pseudobark, a hole drilled to a depth of 15
cm provides at least 3 g of stem shavings. Samples yield sufficient DNA for diagnostic.
7. Once sampling is completed, the stem can be sealed, if necessary, by tapping a piece of wooden
dowel into the hole. This will prevent copious sap bleeding and provide a barrier against
invasion by pests.
Note: The vasculature of monocots, combined with enhanced sensitivity of nested PCR assays,
makes single-point sampling of palms possible. Reliable pathogen detection is unaffected by the
8.
9.
10.
11.
12.
13.
14.
location of stem sampling sites. Excision of tissues from the basal stem is cosmetically desirable
for palms in ornamental or amenity plantings with questionable cases of disease.
Use water to thoroughly wash residual tissues from the drill bit.
Wash sterilize with 0.5% Sodium hypochlorite solution and flame sterilize and cool the bit again
before sampling the next palm.
Collect sawdust from 3 different levels with 3 different boring points along the stem for palms
exhibiting apparent symptoms.
Document the location (GPS coordinates) and image of each sampled palm species.
Store packaged samples on ice during field collection and at 4°C for no longer than 24–48 h prior
to laboratory processing. For longer periods, keep samples at -20°C.
Ship stem samples harvested within 24h after by overnight courier to laboratories for diagnostic
testing show no apparent deterioration in quality due to growth of secondary microbial
contaminants.
Preserve samples from remote locations requiring extended travel times by immersion in CTAB
extraction buffer to reach diagnostic laboratories.
SAMPLING INSECT VECTORS
1. Insects will be collected by “tubing” single insects directly from the plants.
2. Insects will be also collected by using nets to sweep specimens from the fronds or branches of
larger trees.
3. Place individual insects into 95% ethanol after collection.
4. Representative specimens of insects collected will be sorted by family Cicadellidae, Fulgoroidea,
Derbidae, Delphacidae, Flatidae and Lophopidae.
5. Male specimens of each family will be selected for taxonomical ID.
SAMPLING ALTERNATE HOSTS
1. Plant species will be collected from coconut farms heavily infected with CILY exhibiting
symptoms associated with phytoplasma: stunting, yellowing, withering, little leaves, leaf curl,
chlorosis, flower or fruit malformation, and symptomless plants.
2. Cut the tender parts of the plants (stem upwards) with a sterilized kitchen knife.
3. Cut the vines and leaves for creeping plants.
4. Put samples in a labelled polythene bag before being transported to the lab.
5. Separate representative samples for botanical ID.
6. Once in the lab, swab each plant with a paper towel soaked in 70% ethanol, to remove dust
particles, insects and microorganism from the plant surfaces.
7. Remove lamina, leaving a small strip around the midribs and veinlets.
8. Pool two individual plants of each species to form a sample
9. Cut into small pieces (about 0.2 x 1 cm) with a sterile scalpel.
10. Place into plastic bags labeled accordingly.
11. Use immediately or store at -20°C until ready for use.
QUANTITATIVE REAL-TIME PCR (Q-PCR)
Method
A. Obtaining plasmid gene DNA for standards
1. Amplify a segment of CSPWD phytoplasma 23S rDNA using primers RTA
(GGCACTAAGAGCCGATGAAG) and RTB (TCCTCCCATCTTCCAACAAG).
2. Similarly, amplify a segment of coconut 18S rDNA using primers CnA
(AACGAGACCTCAGCCTGCTA) AND CnB (GCGATCCGAACACTTCACC).
3. Clone each fragment (phytoplasma DNA and coconut DNA) using the Promega pGEM®-T Easy
Vector system (Promega, USA). Prepare glycerol stocks of the transformed E. coli and store at 80oC for long term storage.
4. Extract plasmid gene DNA using GenEluteTM Plasmid Miniprep Kit (Sigma, USA).
5. Quantify DNA extracts using a spectrophotometer.
6. Dilute extracts using a two-fold serial dilution ranging from 2-1 to 2-17, with a starting
concentration of 10 ng µL-1.
B. Using cleaned-up PCR products for standards
1.
2.
3.
4.
C.
1.
2.
3.
Amplify phytoplasma and coconut DNA using primers described in steps 1&2 of section A.
Clean-up PCR products using GenEluteTM PCR clean-up kit (Sigma, USA)
Quantify DNA extracts using a spectrophotometer.
Dilute extracts using a two-fold serial dilution ranging from 2-1 to 2-17, with a starting
concentration of 10 ng µL-1.
Setting up & running reactions
Make an outline of your reaction plate in your notebook to act as a guide.
Make two master mixes for n+1 samples (one for phytoplasma & the other for plant DNA)
Prepare master mix by adding 12.5 µl of SYBR mix; 8.5 µl of ultra-pure deionized water; 0.75 µl
each of the forward and reverse primers and 2.5 µl of DNA template (For a 25µl reaction mix).
For a 20µl reaction mix, use the following: 10 µl of SYBR mix; 7 µl of ultra-pure deionized water;
0.75 µl each of the forward and reverse primers and 1.5 µl of DNA template. Primers
RTribF1/RTribr1 will be used to for the phytoplasma reaction and Cn18SF3/Cn18SR3 will be used
for the plant DNA reaction.
Apart from the standards, adjust concentration of DNA templates to approximately 10 ng/µL.
4. Run a two-step amplification reaction with an initial incubation at 50 OC for 2 min; an initial
denaturation at 94oC for 3 min followed by 40 cycles of 94oC for 15 s and 60oC for 1 min.
5. Perform a melt analyses by ramping from 55-95oC, rising by 0.5oC per cycle.
6. Export data to excel and calculate amount of phytoplasma DNA. Normalise data by dividing
amount of phytoplasma DNA by amount of plant DNA in each sample.
Loop mediated isothermal amplification (LAMP)
Preparing LAMP primer mix
 Make up all primer stocks to 100µm
 Make a mix of:
152µl water
4µl F3; 4µl B3
20µl each of the other 4 primers (FIP, BIP, BL, FL)
Total volume of mix will be 240µl
Running the LAMP reactions
 For 8 LAMP reactions, make a master mix of 46µl water, 23µl primer mix and 115 µl enzyme
master mix.
 Aliquot 20µl of this mix into each of 8 tubes and add 1µl DNA.
 Run the reactions on the Genie at 63oC for 30 min. Perform melt analyses by raising
temperature to 90oC and dropping to 70oC to determine the melting temperature of amplicons.
 Include positive and negative (water) controls. Positive test samples will have same/similar
annealing temperature to the positive control. Amplification graphs of positive test samples will
also have characteristic sigmoid shapes.
 To check for phytoplasma, run 23S assay and COX assay for plant DNA.
 Install software on personal laptop and export data after each day’s work.
NB: For in-field use,
1. Prepare mixes and aliquot into reaction tubes in the lab early morning / previous evening and
keep at 4OC.
2. Put on ice/PCR eppendorf coolers and transport to field (they do well at ambient temperature).
3. Take along enough LFD buffer Cs and a pipette and tips to add template DNA.
4. In the field, collect about 0.5 g coconut stem borings into bottles containing LFD buffer C with
steel ball bearings.
5. Shake mixture vigorously for about 2 min and use 2µl of the mix directly in the LAMP reaction.
6. Bottles can be stored long term and/or DNA extracted back in the lab should further tests be
required.