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1 Dream or reality? 1!"# $$%&' &# ( ) **+**,' 2 1

2 Assays based on specific recognition of nucleic acids, e.g. RNA or DNA Detection = qualitative tests e.g. Chlamydia trachomatis, Neisseria gonorrhoeae etc. Quantitation = quantitative tests e.g. HIV, HCV, HBV viral load 3 & NAD Clinical Quality control? Assay development? Standardization? Commercial Kits. Virology (HIV, HBV, HCV,..) Bacteriology (CT, MTB, NG,...) Homebrew Kits. research, Enterovirus oncology, Respiratory genetics, clinical viruses diagnostics Arboviruses (ASRs) bacterial... lung panel 4 Clinical Pathogens for which no commercial NAD kit is available 2

3 -./0! 1 *. - 2 *&.&#! # # * 3 *&/# * *5 lipid bilayer #*6& RNA proteincore $% $$#*. $ 6 3

4 $ ## # < 77= 777 Licensing-in key technologies System development > ## 5 $$9:;!'" 5 9:;!'" % ;**,< ) 9:;!' 6 88 " 5 %&' 9:;!/!# 1 7 $ ## # %&' % - % mini $4 %&' ) 88( easy $4? %&' ;$? %&' &* ** 88, %&'. ::A. ; %&' :.B$ %&' 9; 8 4

5 " %&' &# Clinical sample Nucleic acid Isolation BOOM Real Amplification time amplification NASBA NASBA & detection Detection molecular ECL beacons

6 # Sample + Lysis buffer Nucleic acid Silica Release of nucleic acid Stabilization of nucleic acid Washes Centrifugation Purified Nucleic acid Proteins Lipids etc. Elution buffer Binding of nucleic acid Purification of nucleic acid, removal of inhibitors Recover nucleic acid in small volume 11 Boom technology (the reference method for nucleic acid extraction) "3# $4 # - $4 # 12 6

7 % ) Sample + Lysis buffer Nucleic acid pre-steps Silica Washes Proteins Lipids etc. Elution buffer Magnets on the instrument Magnet Purified Nucleic acid 13 $# % Sample + Lysis buffer Nucleic acid pre-steps : manual Silica Washes Magnets Proteins Lipids etc. Elution buffer on the : automated instrument Purified Nucleic acid 14 7

8 NucliSens Magnetic Extraction Reagents SSCAs (Significant Sustainable Competitive Advantage) One protocol for all o DNA and RNA o multiple sample types and volumes High quality eluate next generation technology from the company that invented BOOM o high purity o high yield / concentration factor User-convenient o easy (user-friendly) and robust procedure High throughput o up to 24 extraction per hour Customer value Easy daily workflow planning Eluate can be used for several different applications One extraction platform for all molecular testing High cost-efficiency Optimal downstream testing o less re-testing o high sensitive testing Better service to lab s customers High cost-efficiency High user appreciation High cost-efficiency High cost-efficiency 15 EMMD 2003 P71c Nucleic acid isolation using magnetic silica particles Peter van Deursen, Patrick de Bie, André Verhoeven, Anke van Grinsven, Marcel Jacobs, Govaert Borst, and Paul van de Wiel. biomérieux, R&D Molecular Diagnostics Department, Boseind 15, 5281 RM Boxtel, The Netherlands For more information please mail to peter.vandeursen@eu.biomerieux.com 16 spiking 1 ml of normal EDTA plasma with 400 ng of total E.Coli RNA recovery OD260/OD % 80% 60% 40% 20% 0% % 85% RNACONCLUSIONS DNA direct extracted extracted extracted M: Marker B: Blank D: Direct (not extracted) E: Extracted RNA DNA extraction procedure for broad range of sample types direct extracted extracted extracted integrity of the isolated nucleic acid M D E E M M B D E E 2.9 kb 1.5 kb 0.1 kb Intact RNA and DNA recovered with high efficiency (83% and 85%, respectively) and high purity (OD260/O280 ratio of 2.1 and 1.8, respectively) Simple, easy, fast (24 samples within 90 minutes) nucleic acid Target Sample type HIV-1 RNA Plasma (EMMD poster 71a) HBV DNA Serum Enterovirus RNA CSF, Stool, Throat swap (EMMD poster 74b ) CMV mrna Whole blood Human mrna (U1A) Sputum (EMMD poster 38c), Whole blood Mycobactrium tuberculosis RNA Sputum (EMMD poster 71b) Mycobacterium tuberculosis DNA Sputum (EMMD poster 71b) Legionella pneumophila RNA Lung biopsy (EMMD poster 38c) Bordetella pertussis RNA Gargle (EMMD poster 38c) Mycoplasma pneumoniae RNA Gargle, Throat swap (EMMD poster 38c) 4.3 kb 8

9 $4 $#!# *. # A.. C high throughput (24 samples in an hour) automation of the washing steps (manual addition/aspiration) of buffers high run size flexibility different sample types, one protocol * very small footprint easy working procedures and no maintenance minimal reagent and consumable &$4 $!.. *. # A.. C high throughput high level of automation high run size flexibility different sample types, one protocol * small footprint easy working procedures and low maintenance minimal reagent and consumable usage 18 9

10 $$ $ 2 $#*. 19 NASBA RNA as principle target DNA, when using tricks Isothermal (41 C) Continuous ssrna amplicons PCR DNA as principle target RNA, when using tricks Different temperatures Cyclic dsdna amplicons 20 10

11 ; &*.. $ >.. $#*./* 8 *,8#1 %&A. * "#. #*.. $- # $ 21 9# # $> &# # $ $#*. *- $- /9:;!A9;A :.BA% - A@- AE AA A CCC1 # # $&3 - - F /;1 #, $.. # &

12 !"# $$ sense RNA oligo P1 Q F 5 3 F Q Target specific molecular beacons RNase H oligo P1 Reverse Transcriptase RT RT RNase H & oligo P2 Reverse Transcriptase T7 RNA polymerase oligo P2 anti-sense RNA 3 5 T7 RNAP F Q F Q Molecular beacon hybridization 23 E # G F G Q Fluorescent Dye Loop Stem Quencher DNA probes with a stem-loop structure and two modified ends loop sequence complementary to the target sequence for hybridization 6-7 base pair stem sequences Different labels for different targets (wild type and calibrator) 24 12

13 13 25 #* 26.$ "# $$ Time (minutes) Normalized fluorescence Fluorescence signal Features isothermal (41C) continuous process (1 hr) multiple labels data points = 120 for each curve closed system

14 !# 3 NASBA reaction 20 ul Normalized fluorescence F Q Fluorescent signal (real-time) Q F F Q F Q Time (minutes) 27 %&' /!"# $$1 Advantages of a single step for Amplification and Detection: H #. # H6 H 9 * H*# Additional advantage: I.3 #*. 14

15 $$ The perfect match: 1 $ $# $$ $#* 2 3 #* # JK.#*# & 2 $$ #* L 29 '-M # N $$ Internal control (IC; system control) RNA Wild-type P2 WT 6-FAM P1 IC/Calibrator P2 IC ROX P

16 $" '. "3 # /E"1 "3. &/>$ O1 : /&# 1 Molecular beacon NASBA target: Sample (WT) Control (SC) FAM 6-ROX 31 %&' ) %O" $": %$6":%$@6:>:$": $%"%": 32 easymag minimag Desktop computer Strip centrifuge NucliSens EasyQ Incubator NucliSens EasyQ Analyzer 16

17 Basic Kit as Building-block 33 )* $#*. # B $ #*.. standardized primer and probe sets optimized and standardized procedures all reagents provided complete kit quality controlled and CE marked minimizes QC work 34 17

18 %&' ) Open platform for home brew RNA Amplification Assays Standardized Reagents Standardized Procedures Standardized Instrumentation Online Helpdesk Support Lysis Buffer Boom Technology Amplification Detection Assay set-up Software Protocols Extraction minimag/easymag EasyQ Analyzer User defined Primers and Probes 2 options biomérieux s Target Specific Reagents 35 %:;&' %$**3 : Meningitis: enterovirus, HSV 1/2 Resp. viral panel: RSV A/B, hmpv, Influenza Resp. bact. panel: Legionella, Chlamydia Pneumoniae, Mycoplasma Pneumoniae Trombosis: FactorV, factor II 36 18

19 %:;&' % Bacterial cause (25% mortality) Haemophilus influenza type B (7%) Meningo coccus (25%) Pneumo coccus (47%) 70-90% viral cause (very low mortality) 80-90% enterovirus Today: suspision meningitis CSF Bacterial culture WBC count ( bact.) Glucose test ( bact.) Protein test ( bact.) Gram coloring Viral Culture (if possible) AB Treatment + hospitalisation 37 %:;&' %&' % - Result in 3,5 hours for exclusion of bacterial meningitis Internal control (homologue) Analytical specificity major Enterovirus serotype groups, including - Poliovirus 1-3, Coxsackievirus A2-12, A15-18, A20, 21, 24,,Coxsackie B1-6, Echovirus 1-9, 11-15, 17-21, 24-27, Enterovirus Analytical sensitivity - 93 copies per nucleic acid extract (50% hit rate) copies per nucleic acid extract (95% hit rate) No cross reactivity with Herpes Simplex Virus, Varicella Zoster Virus, Measles, Cytomegalo virus and Mumps 38 19

20 %:;&' Bronchiolitis 50-90% RSV positives Pneumonia 5-40% RSV positives Respiratory diseases : 10-15% hmpv Infections RSV + hmpv => 6 # * & * # /# 1 Today suspision bronchiolitis: Rapid Test Ag RSV = sensitivity 80% Test IF = difficult interpretation Culture hmpv= No test available 39 %:;&' %&' ;+@; Positive Identification virus after 3 hours Internal control (homologue) To rule out bacterial infection Isolation child or not Reduce contamination risk Treatment or not Analytical specificity RSV: Major RSV A+B serotype groups No cross reactivity with Influenza A&B, Measles, Adenovirus 1&5, hmpv A1, A2, B1 and B2 Analytical specificity hmpv: All subtypes of hmpv No cross reactivity with RSV A&B, Sars, Influenza A&B 40 20

21 %:;&' #5 Linearity : IU Sensitivity: 175 IU (95% hitrate) Subtypes: A-J Results in 4 hours Internal control (homologue) -:"4$3 * 41 %:;&' " # * 9. # -/ 1 Low concentration of anticoagulants Normaly APC inhibits Factor V: in APC resistant persons the factor V is not inhibited due to factor V mutation High concentration of coagulants Factor II (protrombine) mutation gives as a result a higher factor II concentration; protrombine is activated into trombine 5# 3 # & 42 21

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23 %&' ) The Network ) 3 we as company share our extensive NASBA knowledge with all users on this site application database containing exhaustive number of customer-developed tests ** -*# direct customer support via web-hosted helpdesk for the development of NEW customer-driven assays 45 ; 333!** ** # #5- *