ARROW Automated system for Nucleic Acid isolation and cell preparation

NORDIAGMycobacteriaAutomated DNA/RNA extraction from clinical samples using the NorDiag Arrow with the Arrow BUGS’n BEADS™ kit for diagnosis and obtaining drug susceptibility information using the GenoType MTBDRplus, GenoType MTBDRsI, GenoQuick MTB from Hain Life SciencesIntroductionMore than two billion people – one third of the world’s total population are infected with TB bacilli. A total of 1.77 million people died from TB in 2007 (including 456 000 people with HIV), equal to about 4800 deaths a day. TB is a leading killer among people living with HIV, who have weakened immune systems. Multidrug-resistant TB (MDR-TB) is a form of TB that does not respond to the standard treatments using first-line drugs. MDR-TB is present in virtually all countries surveyed by the World Health Organization (WHO) and its partners. Extensively drug-resistant TB (XDR-TB) occurs when resistance to second-line drugs develops. It is extremely difficult to treat and cases have been confirmed in more than 50 countries. The WHO has estimated that 5 % of all TB cases harbour a multi-drug resistant strain.Definitive diagnosis of mycobacterial infection is by identification of the causative organism in a clinical sample. However, difficulties in culturing these slow-growing organisms in the laboratory present a diagnostic challenge, and PCR-based methods that are able to identify not only presence of MTBC, but also provide information on drug susceptibility, can be invaluable. Rapid, reliable techniques for drug-susceptibility testing (DST) has been identified as an essential need, not only for surveillance, but also for development of adequate treatment regimens for individual cases.Clinical samples for molecular mycobacteria detection are one of the most difficult and time consuming samples for nucleic acid purification. Most automated platforms do not accommodate these samples without extensive pre-treatment. Even with pre-treatment they are often crude nucleic acid preparation.Use of NorDiag’s Arrow Instrument (figures 1-3) and standard DNA/RNA extraction protocol (Arrow BUGS’n BEADS™) is a sensitive and convenient approach to avoiding this bottleneck in obtaining information on the occurrence and drug susceptibility of mycobacteria in clinical samples.NorDiag’s fully automated procedure for DNA/RNA isolation from mycobacteria in decontaminated clinical samples is highly efficient. The extracted DNA/RNA can be used for identifying mycobacteria and for DST. Results are comparable to those obtained by gold standard culture methods.Application NotesAN-22-10Figure 2. Unique new closed tip/pump assembly on the NorDiag Arrow instrument. Patent pending.Disposable pumpPipette tipFigure 1. NorDiag Arrow NORDIAGwww.nordiag.comNorDiag ASA Frysjaveien 400884 Oslo, NorwayTel: +47 22 02 65 65 Fax: +47 22 02 65 66E-mail: info@nordiag.comNorDiag ABInstrumentvägen 19SE-126 53, Hägersten, SwedenTel: +46 (0) 8 440 04 85Fax: +46 (0) 8 411 18 50E-mail: info@nordiag.comNorDiag Inc901 S. Bolmar, Suite R,West Chester, PA19382, USATel: +1610 344 7987 Fax: +1610 344 7989E-mail: info.us@nordiag.comAN-22-10 Automated Solutions For DiagnosticsStudy samples• 50 clinical samples from routine diagnostics included in study (samples either previously culture-positive for MTBC or from patients with known MTBC infection). - 46 pulmonary specimens (sputum, pleural fluids, bronchial washings, bronchoalveolar lavage). - 4 extrapulmonary specimens (urine, gastric lavage, stool, biopsies).• Smear results (staining on microscope slide) of specimens: - 35 smear negative - 14 smear positive - 1 no smear result• Samples stored for a maximum of 3 days at 2-8° C before NALC-decontamination by standard operating procedures.• Following decontamination, samples were resuspended in 1-1.5mL phosphate buffer and stored for a maximum of 2 months at -20°C.Study analyses• 500 µL sample added to liquid culture, and two 100 µL to solid media. All incubated for 8 weeks if negative.• DNA/RNA isolation on Arrow Instrument and amplification assays: - Decontaminated specimen mixed by careful up-and- down pipetting. - 700 µL transferred to 2mL screw-cap tube. Heat inactivation at 80° C for 20 min. - Protocol on Arrow Instrument selected. - Required number of sealed Arrow BUGS’n BEADS reagent cartridges loaded. - Sample tubes loaded. - Start protocol. - 5µL eluted DNA/RNA used in 3 different amplification assays from Hain LifeScience (GenoType MTBDRplus, GenoType MTBDRsI, GenoQuick MTB) following kit protocols provided by manufacturer, and using the primers provided. Taq-buffers and 1 U HotStart-Taq were from Qiagen (Hilden, Germany). Table 1. Specificity, sensitivity, and predictive values of RNA amplification assay following automated mycobacterial RNA isolation on NorDiag’s Arrow Instrument: • Sensitivity: 100 % • Specificity: 94.1 % • PPV: 88.9 % • NPV: 100 %Study results (table 1)• 32 specimens culture-negative - Of these, 31 negative by the 4 amplification assays and 1 positive by all 4 assays (this sample was smear-positive and had previous MTBC results recorded)• 16 specimens culture-positive for MTBC - All these samples were MTBC-positive by the 4 different amplification assays.• 2 specimens culture-positive for Mycobacterium intracellulare - One of these samples was MTBC-positive by the 4 different amplification assays and the other was MTBC- negative by the 4 assays.• Culture DST results were not available, but for 3 of the assays a wild-type band pattern was obtained for the DST investigated.Figure 3. NorDiag Arrow in use.Ordering Information8.31.01 NorDiag Arrow Instrument 1 piece12.01.02 Arrow BUGS’n BEADS™ (CE, IVD) 96 preps12.06.02 Arrow Stool DNA (CE, IVD) 96 preps12.07.02 Arrow Blood DNA 200 (CE, IVD) 96 preps12.17.02 Arrow Blood DNA 500 (CE, IVD) 96 preps12.08.02 Arrow Viral NA (CE, IVD) 96 preps8.32.01 Extra Box of 96 Tips for Arrow 96 tips RESULTS:Positive for MTBC by all 4amplification assaysNegative for MTBC by all 4amplification assaysMTBC-positive by culture 16 0M. intracellulare by culture1 1Negative by culture1 31