E-SPACE WORKSHOP. Isothermal methods : LAMP and RPA. Delphine Massé & Isabelle Robène. E-space mid-term meeting Montpellier January 25-26, 2018

Transcription

1 E-SPACE WORKSHOP Isothermal methods : LAMP and RPA Delphine Massé & Isabelle Robène E-space mid-term meeting Montpellier January 25-26, 2018

2 LAMP assay Loop-mediated isothermal amplification Notomi et al., 2000 (Japon) Developed for detection of many different pathogens Nematodes Insects Virus Bacteria Fungi But also Herbal medicine identification Forbidden vegetables in strict vegetarian diets Quality assessment of meat products

3 LAMP assay Loop-mediated isothermalamplification 1200 articles using LAMP on PubMed 200 publications per year 20% plant diseases pathogens 50 plant viruses 20 bacterial plant diseases 7 fungal plant diseases Several phytoplasmas

4 Principe One step amplification for RNA or DNA target Enzyme = Bst DNA polymerase Bacillus stearothermophilus High-strand deplacement Exponential amplification target : folds Isothermal amplification 60 à 65 C within 30 à 60 min

5 Principe 2 pairs of primers (Notomi et al., 2000) FIP : forward inner primer BIP : backward inner primer F3: forward outer primer B3: backward outer primer Loop primers (Nagamine et al. 2002) : optional LF: loop forward primer LB: loop backward primer High specificity because 4 primers recognizing 6 distinct regions Speed up the reaction and the specificity (Eiken Chemical Co., Ltd)

6 2 mains steps Principe Product a structure with stem-loops at each end FP, RP, complementarity of sequences => matrix Cyclic amplification Inner primer, amplicon with size more and more important + matrix (Eiken Chemical Co., Ltd)

7 Principe

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9 Detection strategies Turbidity New composant generated during amplification Magnesium pyrophosphate Stable only a short time Indicators into the reaction after amplification BET Fluorescence under UV light

10 Detection strategies Metal-ion indicators into reaction before amplification Hydroxynaphthol (HNB) ph change Calcein + manganese Visible light Fluorescence under UV light Berberine Visible light Fluorescence under UV light Positive reaction

11 Detection strategies Indicators into the reaction before amplification Evagreen dye, SYBR Green, Pico Green, Malachite green Visual detection Fluorescence signal collection Time positive values SYBR Green Malachite green Fluorescence signal collection Positive reaction is green

12 Detection strategies Real-time fluorescence measurement Different Lamp Instruments : to simplify the procedure, amplification and detection can be combined in a single system Genie II/III - 16/8-well device with touch screen to follow DNA amplification in real-time - Stand-alone operation without PC - Internal rechargeable Li-Po battery - 2 dyes (Genie III)

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14 Detection strategies Master Mixs and Enzymes available Optigene ISO-001 standard mastermix (GspSSD) GspM and GspM2.0 polymerases TIN-001 DNA polymerase Lyse & LAMP (Plant leaves used directly) NEB Bst 1 WarmStart. Lucigen OmniAmp polymerase LavaLAMP DNA Master Mix

15 Cost Euros Q/reaction Cost/reaction Isothermal Master Mix-Dried reagents 300 runs 500 1,7 Genie tubes large pack ,2 TOTAL 1,9 Euros Q/reaction Cost/reaction Bst DNA polymerase 8000 unit 260 8U 0,3 Genie tubes large pack ,2 Sybergreen I nucleic A 1ml 630 1µl 0,6 TOTAL 1,1 Genie II

16 Performance Sensitivity : equal or times more PCR : pg Bacteria Xanthomonas oryzae pv. oryzae : 10 5 CFU/ml Ralstonia solanacearum : CFU/ml Xanthomonas axonopodis pv. dieffenbachiae : 10 4 CFU/ml Virus Mosaic virus on wheat : 100 times greater than PCR Bean pod mottle virus : RT LAMP times greater than RT PCR 9 rice viruses : ten fold more sensitive than RT PCR

17 Example : Sugarcane viruses LAMP SYBER Green I SCMV 1ng SrMV 1ng LAMP gel 1ng 1ng RT PCR 100pg 100pg qrt PCR 10fg 10pg Keizerweerd, 2015

18 Example : Grapevine fanlealf virus Dilution series ELISA 10 4 RT PCR 10 5 IC RT PCR 10 4 RT LAMP Gel 10 3 IC RT LAMP Gel 10 2 RT LAMP GeneFinder or Phenol red 1:10 IC RT LAMP GeneFinder or Phenol red 1:10 RT LAMP Almasi, 2016 IC RT LAMP GeneFinder Phenol red

19 Example : Xanthomonas citri pv. citri Rigano, 2010

20 Example : Xylella fastidiosa Bst DNA Polymérase (New England Biolabs) Concentration en bactéries /ml macérat Harper et al., 2010

21 Response time 5 20 min Performance

22 Advantages Multiplex and simplex amplification Easy Outside field test Fast Sensitive Specific Applicable for DNA and RNA Robust Less affected by non target DNA and inhibitory molecules

23 Drawbacks Reaction products are series of DNA fragment different sizes : not be used for other assays Technical constraints for the primers design An excess concentration of indicator may inhibit polymerase or change indicator color Too long incubation time : false positives Multiplexing : the larger number of primers per target in LAMP increases the primer-primer interactions CONTAMINATION : false positives : NOT OPEN TUBES

24 Combination of LAMP with other techniques LAMP with lateral flow dipstick (LFD) More easily applied for field diagnosis Combination between biotinylated primers with FITC-labeled DNA probes IC LAMP Reducing time and cost (DNA/RNA extraction)

25 Improvements Lyophilized LAMP Bst activity preserved at -20 C Low temperature transport and storage is not possible Faster enzyme Ex : GspSSD DNA polymerase Run : 20 min

26 True life : Banana viruses BBTV, BSV Revelation Calcein HNB SYBER Green Enzymes Bst NEB Mastermix OptiGene Results Detection BBTV and BSV only with Mastermix OptiGene on Genie II Detection BSV with calcein (UV only)

27 True life : Ralstonia solanacearum Unsuccessful tests with SYBR Green, SYBRSafe added before amplification : inhibition Unsuccessful test of commercial kit based on ph shift False positive when adding SYBR Green in post amplification (even with oil) => Conclusion : we are now focusing on real-time fluorescence monitoring

28 Key factors Lamp primer design Tm Stability at the end of the primers GC content Secondary structure Distance between primers

29 Lamp primer design

30 Others isothermal amplification technologies More than 15 different strand displacement amplification (SDA); cross priming amplification (CPA); rolling circle amplification (RCA); helicase-dependent amplification (HDA); recombinase polymerase amplification (RPA); nucleic acid sequence-based amplification (NASBA) transcription mediated amplification (TMA).

31 RPA Recombinase polymerase amplification First publication in 2006* A remarkable output of publications (pub med 178, 62 in 2017) Mainly in the field of human and animal diseases (bacteria, viruses, fungi, parasites) and GMO, a few for plant viruses *Piepenburg O, Williams CH, Stemple DL, Armes NA. DNA detection using recombination proteins. PLoS Biol Jul;4(7):e204

32 How Does RPA Work? + om/watch?v=tv8dvdqco ZE very fast amplification (detection capability in minutes in most cases) amplicon length of under 500bp optimal temperature of 37 C - 42 C Copies

33 Different RPA strategies Supplier TwistDx ( Agdia (AmplifyRP ) RNA/DNA amplification Detection agarose gel lateral flow strips real-time fluorescent probes

34 Commercial kits : Twist Dx

35 Twist Amp nfo kit + LF probe Twist Amp exo kit + exo probe Chao CC. et al., PLoS Neglected Tropical Diseases 9(7)

36 Commercial kits : Agdia + specific kits for Cmm, Xf, rt or end-point detection

37 TwistAmp exo assay According to Daher et al., Clinical Chemistry 62: (2016)

38 Twista TM 4300 euros T8 Iso instrument 4978 euros portable instruments measurement of two fluorophores: FAM and ROX or equivalent 8 standard 0.2 ml tubes + magnetic mixing of RPA reactions (requires micro balls in RPA reaction mix)

39 Design of RPA primers and probe No sofware : Manual design RPA primers : bases long Amplicon : pb (optimal pb) Exo Probe : bp

40 Primer design Avoid G in 5 Inversely, C in 5 G and C in 3 are beneficial Minimize hairpins, dimer formations 30 % <GC<70%

41 Post-RPA detection with LF probe

42 LF probe

43 ARN pathogens

44 ADN pathogens

45 Plant pathogens Pathogens Plum pox virus Little cherry virus 2 Begomovirus Tomato chlorotic dwarf viroid biological samples Analytical sensitivity Plant crude extracts Plant crude extracts crude extracts Plant crude extracts time to Detection method result (min) reference 1 fg purified RNA rt/lf Zhang et al ND (< RT-PCR) 25 LF Mekuria et al g cloned TYLCV genome (< PCR) 1pg purified RNA ( = RT-PCR) 60 AG Londono et al., rt Hammond et al., 2016 Rose rosette virus Total plant RNA 1 fg/µl (= RT-PCR) 60 AG Babu et al., 2017 Hop stunt viroid banana bunchy top virus Phytophtora spp. Phytophtora infestans Fresh crude extracts, FTA cards both purified DNA and crude leaf sap template 10 ng/μl (= RT-PCR) LF ND (> PCR in crude sape ) Kappagantu et al., AG Kapoor et al., 2017 Plant crude fg ADN (=qpcr extracts Total plant ) RNA 5-17 rt/lf Miles et al., 2015 Simplifed DNA extraction from 50fg/μL (= Lamp) 10 rt Ammour et al., 2017 plant material Phytophtora sojae crude plant extract 10 pg/μl (<qpcr) rt Rojas et al., 2017 Candidatus Phytoplasma mali crude Tris DNA extracts <10 copies (=qpcr) 11 rt/lf Valasevich et al., 2017

46 Advantages Crude sample Less affected by inhibitory molecules than PCR About amplicons in minutes Sensitive <10 copies DNA/RNA can be tailored to work in the laboratory or in the field Commercial kits with lyophilised reagents

47 Drawbacks design of probe/primers not intuitive Amplicons 500bp Risk of contamination if opening tubes after amplification Cost 4-10 $/reaction

48 Cost Real -time euros lateral flow euros lateral flow ++ euros Exo kit 4.03 nfo kit 4.03 nfo kit 4.03 probe 0.17 probe 0.17 probe 0.17 primers primers primers LF 3.12 LF +chamber Agdia HLB 300 $ for 8 tests GRBaV : 240 $ for 8 tests

49 First tests using an exo probe designed for Ralstonia solanacearum Positive results with Exo probe tested with nfo and exo kits Tests on DNA extracts (20 ng/ul) of target, non target strains and negative controls : only target strains are tested positive Tests on target bacterial suspensions ranging from 10 8 to 10 2 CFU/ml

50 First tests using an exo probe designed for Ralstonia solanacearum Kit positive control (10 3 cp/reaction) DNA of target strains 60 ng /reaction (= 10 7 copies /reaction ) 10 8 CFU/ml Under optimization Test of different primers pairs Different primer concentrations 10 7 CFU/ml Different reaction temperatures Negative control Limit of detection < 90 copies / reaction

51 Lamp vs. RPA Technology Target Initial denaturation Incubation (ºC) Reaction time (min) Multiplex Primers required Commercial source sensitivity to inhibitors sensitivity cost ( ) RPA DNA/RNA N Y 2 Y TwistDx, Agdia N <10 copies LAMP DNA/RNA Y Y 4-6 Y optigene, NEB, Lucigen N <10 copies 4-10

52 TP LAMP Detection Ralstonia solanacearum on bacteria and FOC 4 on banana Master mix OptiGene with their technical conditions 15µl + 5µl DNA + 5µl primers 65 C 30 min Bacterial suspensions 10 2 to 10 8 CFU/ml

53 TP RPA Real-time detection of Ralstonia solanacearum using TwistAmp exo assay 1 positive control (kit) Bacterial suspensions from 10 7 to 10 2 CFU/ml + negative control TwistAmp exo kit Rs specific probe (eurogentec) and primers 3 µl template in 50 µl mix final Add to freeze-dried reaction pellets + acetate magnesium 39 C, 20 min

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55 1)LAMP UY031 6 primers Condition OptiGene : 15µl mastermix, 5µl ADN, Tm65 C

56 LAMP UY031 6 primers

57 2)UY031 5 primers

58 UY031 5 primers Réactifs N livraison [Sol mère] [puits] Vol. d'un puits (µl) Eau 7,8 FIP 50 µm 0,8 µm 0,4 BIP 50 µm 0,8 µm 0,4 Floop 50 µm 0,4 µm 0,2 Rloop 50 µm 0,4 µm 0,2 F3 10 µm 0,2 µm 0,5 B3 10 µm 0,2 µm 0,5 mastermix optigen 1 x 12,5 2,5µl solution bact 65 C