Line Probe Assays (LPA)

Line Probe Assays (LPA) DNA Strip Technology Line Probe Assays / Reverse Hybridization • • • • • • • • Uses reverse hybridization to identify MTB while simultaneously detecting the most common SNPs a/w drug resistance. DNA extracted and target sequences are amplified by PCR using biotinylated primers. Hybridization to oligonucleotide probes immobilized on membrane strips. Colorimetric detection of captured amplicons on strip- as bands (Lines formed at amplicon –probe hybridization site) Pattern interpreted with key. WHO endorsed LPA (2008) -for rapid diagnosis of MDR-TB in high TB burden, resource poor settings. Used directly on smear positive sputum sample or positive culture Highly sensitive & specific -detection of MDR TB in smear+ sputum Commercially available kits 1. INNO LiPA Mycobacterium Assay (version2) - Innogenetics, Belgium - Tested & validated for culture isolates - Targets 16s-23s rRNA transcribed DNA spacer region - Identifies 16 different species of mycobacteria 2. Genotype Mycobacteria - Hain Lifesciences, Germany - Tested & validated for culture isolates as well as direct sputum specimen - Targets 23S rRNA gene A. Genotype MTBDR/ MTBDR Plus : Drug resistance gene for RIF (rpoB) & INH (inhA & katG) B. Genotype MTBDRsl : Drug resistance genes for XDR TB (embB, gyrA, rrs, eis) DNA Extraction Centrifuge the processed sample at 10,000 g for 15 mins Discard the supernatant using pipette Add 100 µl Genolyse Lysis Buffer-A while crushing the pellet Vortex vigorously for few secs Incubate at 95 ºC for 5 mins Add 100 µl Genolyse Neutralizing Buffer-B to the sample Vortex gently for few secs Centrifuge the sample at 13,000g for 5 mins Take out the supernatant as aliquot in separate Eppendorf Master Mix Preparation DNA Addition & Amplification Overview of reverse hybridization Separation of amplicons in single-stranded DNA Binding of an enzyme-conjugated protein Denaturation Hybridization Stringent wash Conjugate reaction Substrate reaction Binding of labelled amplicons to probes Removal of unspecifically bound DNA probes Enzymatic conversion of dye Instrument and reagent preparation      Turn on TwinCubator/ GT Blot, start the program to enable it to warm to 45 °C Pre-warm hybridization (HYB) buffer and stringent wash (STR) solution to 37–45 °C Bring rinse solution and sterile distilled water to room temperature. Dilute conjugate concentrate (CON-C) 1:100 with conjugate diluent (CON-D). Dilute the substrate concentrate (SUB-C) 1:100 with substrate diluent (SUB-D). Denaturation • Use clean/new tray • Dispense 20 μl denaturation (DEN) solution in a corner of each trough • Add 20 μl amplicon to DEN and carefully mix by pipetting up and down 5 times • Place numbered strips face side up using clean new plastic tweezers • Carefully add 1 mL of pre-warmed HYB buffer to each trough Hybridization by Twincubator Put the tray in twincubater & incubate for 30 mins Add 1 ml warm STR & incubate for 15 mins Add 1 ml RIN, incubate for 1 min than remove it Add 1 ml CON-D and remove after 30 min incubation Wash Twice with 1 ml of RIN for 1 min Wash once with 1ml of sterile distilled water for 1 min Add 1 ml of SUB-D and incubate for 3 - 20 min Cover tray with aluminum foil Wash Twice with 1 ml of sterile distilled water for 1 min Using clean tweezers, remove strips from the tray and dry them between two layers of absorbent paper Denaturation Hybridazation Stringent Wash-I Stringent Wash-II Conjugate Reaction Substrate Reaction GenoType MTBDRplus Molecular genetic assay for Identification to Resistance to Rifampicin and/or Isoniazid. • Identification of rifampicin resistance is enabled by the detection of the most significant mutations of the rpoB gene (coding for the α-subunit of the RNA polymerase). • For isoniazid resistance testing, the katG gene (coding for the catalase peroxidase) and the inhA gene (coding for the NADH enoyl ACP reductase) is examined. MTB DRplus • Each strip consist of 27 reaction zones(bands) • Including 6 controls( conjugate, amplification, TUB for M.tuberculosis complex, rpoB gene locus, inhA locus, katG locus control) • 8 rpoB WT and 4mutants(MUT) probes • 1 katG WT and 2 MUT probes • 2 inhA WT and 4MUT probes Reaction zones GenoType® MTBDRplus v2.0 Conjugate Control (CC) Amplification Control (AC) M. tuberculosis complex (TUB) rpoB Locus Control rpoB wild type probe 1 (rpoB WT1) rpoB wild type probe 2 (rpoB WT2) rpoB wild type probe 3 (rpoB WT3) rpoB wild type probe 4 (rpoB WT4) rpoB wild type probe 5 (rpoB WT5) rpoB wild type probe 6 (rpoB WT6) rpoB wild type probe 7 (rpoB WT7) rpoB wild type probe 8 (rpoB WT8) rpoB mutation probe 1 (rpoB MUT1) rpoB mutation probe 2A (rpoB MUT2A) rpoB mutation probe 2B (rpoB MUT2B) rpoB mutation probe 3 (rpoB MUT3) katG Locus Control katG wild type probe (katG WT) katG mutation probe 1 (katG MUT1) katG mutation probe 2 (katG MUT2) inhA Locus Control inhA wild type probe 1 (inhA WT1) inhA wild type probe 2 (inhA WT2) inhA mutation probe 1 (inhA MUT1) inhA mutation probe 2 (inhA MUT2) inhA mutation probe 3A (inhA MUT3A) inhA mutation probe 3B (inhA MUT3B) colored marker Conjugate Control (CC) A line must develop in this zone, documenting the efficiency of conjugate binding and substrate reaction. Amplification Control (AC) When the test is performed correctly, a control amplicon generated during amplification will bind to the Amplification Control zone on the strip. A missing band therefore indicates mistakes during amplification set-up or the carry-over of amplification inhibitors with the isolated DNA. In case of a positive test result, the signal of the Amplification Control zone can be weak. In this case, however, the amplification reaction was performed correctly and the test does not have to be repeated . M. tuberculosis complex (TUB) This zone hybridizes, as known, with amplicons generated from all known members of the Mycobacterium tuberculosis complex. If the TUB zone is negative, the tested bacterium does not belong to the M. tuberculosis complex and cannot be evaluated by this test system. rpoB , katG and inhA probes These reaction zones detect the respective gene regions (Uni probes) or they document, depending on their character, either by their absence (wild type probes) or their appearance (mutation probes) a resistance to rifampicin and/or isoniazid. Each pattern that deviates from the wild type pattern indicates resistance of the tested strain! INTERPRETATION All bands (except CC) should be compared to the AC control band. • If the TUB zone is negative, the tested bacteria does not belong to the M. tuberculosis complex and presence or absence of any other bands (except CC and AC) cannot be considered for evaluation. • If the locus control zones are negative, the respective mutationspecific positive bands cannot be considered for evaluation. • Absence of signal with wild type probes may predict resistance, while positive hybridization signal with a mutation-specific capture probe may predict resistance. • Presence of rare mutations may only be indicated by the lack of hybridization with one or more wild type probes. • INTERPRETATION GenoType® MTBDRsl v2.0 • • • • • • Detection of mutations involved in resistance to second-line drugs. Injectable drugs, fluoroquinolones. Presence of rare mutations that do not have mutation-specific capture probes may only be indicated by the lack of hybridization with one or more wild type probes “gyrA & gyr B” probes -resistance to fluoroquinolones i.e; Ofloxacin or Moxifloxacin “rrs”- resistance to injectable aminoglycosides/cyclic peptides i.e; amikacin, kanamycin, capreomycin, viomycin “eis”- probes detect low level resistance to Kanamycin. • gyr A locus has 3 wild type probes WT1, WT2 , WT3 and 5 mutant probes MUT 1, 2 , 3A , 3B , 3C & 3D • gyr B has - one WT probe & 2 mutant probes MUT 1 & 2 • rrs has 2 - WT1 , WT 2 & 2 mutant probes MUT 1 and MUT 2 • eis has 3 wild type probes WT 1, 2, 3 & 1 mutant probe MUT1 There are some common patterns of resistance and mutation seen 1) Fluoroquinolone resistance ( gyrA ) if WT3 probe band is absent , band for 3B or 3C mutant probe is m/c seen. 2) Injectable aminoglycosides ( amk/ kan/ capreo/ viomycin) resistance (rrs) if band for WT 1 absent , band for MUT 1 is present. 3) Resistance to low level kanamycin is very rare, for eis locus if band for WT1 absent, MUT 1 is seen Reaction zones of the GenoType® MTBDRsl v2.0 Conjugate Control (CC) A line must develop in this zone, documenting the efficiency of conjugate binding and substrate reaction. Amplification Control (AC) When the test is performed correctly, a control amplicon generated during amplification will bind to the Amplification Control zone on the strip. A missig band therefore indicates mistakes during amplification set-up or the carry-over of amplification inhibitors with the isolated DNA. In case of a positive test result, the signal of the Amplification Control zone can be weak. In this case, however, the amplification reaction was performed correctly and the test does not have to be repeated . M. tuberculosis complex (TUB) This zone hybridizes, as known, with amplicons generated from all known members of the Mycobacterium tuberculosis complex. If the TUB zone is negative, the tested bacterium does not belong to the M. tuberculosis complex and cannot be evaluated by this test system. gyrA, gyrB, rrs, and eis probes These reaction zones detect the respective gene regions (Uni probes) or they document, depending on their character, either by their absence (wild type probes) or their appearance (mutation probes) a resistance to rifampicin and/or isoniazid. Each pattern that deviates from the wild type pattern indicates resistance of the tested strain! INTERPRETATION • A positive result is reliable for detection of drug-resistant TB • Negative result not always rule out the presence of drug-resistant TB, • Should be confirmed by conventional culture and drug sensitivity test (DST). • USE : 1) Any smear positive patient or MDR culture positive patient who has positive MTBDR plus may undergo MTBDRsl • 2) Detection of XDR-TB in patients previously diagnosed with MDR-TB GenoType® Mycobacterium CM/AS Controls Ensure Valid Ensure Results Controls Valid Results • Conjugate control documents the efficiency of conjugate binding and substrate reaction • Universal control detects all known mycobacteria and members of the group of gram-positive bacteria with high G/C content. If this zone and the conjugate control zone stain positive but the remaining band pattern can not be assigned to a specific mycobacterium, additional methods have to be applied to identify the respective bacterial species • MTBC specific control hybridizes with amplicons generated from all members of the Mycobacterium tubercolosis complex GenoType® Mycobacterium CM The GenoType® Mycobacterium CM (Common Mycobacteria) permits the identification of the following mycobacterial species: M. avium ssp., M. chelonae, M. abscessus, M. fortuitum, M. gordonae, M. intracellulare, M. scrofulaceum, M. interjectum, M. kansasii, M. malmoense,M. peregrinum, M. marinum/ M. ulcerans, the M. tuberculosis complex, and M. xenopi. GenoType® Mycobacterium AS The GenoType® Mycobacterium AS (Additional Species) permits the identification of the following mycobacterial species: M. simiae, M. mucogenicum, M. goodii, M. celatum, M. smegmatis, M. genavense, M. lentiflavum, M. heckeshornense, M. szulgai/M. intermedium, M. phlei, M. haemophilum, M. kansasii, M. ulcerans, M. gastri, M. asiaticum, and M. shimoidei. Benefits of DNA Strip Technology • Fast, easy and cost-efficient to perform • Combines high specificity with maximum sensitivity • Internal controls ensure valid results • Low implementation costs • Easy to combine: Hybridization procedure equal for all • Can be automated • Extended validation data for each product Disadvantages of LPA • Expensive • Labour intensive • Require adequate lab infrastructure & highly trained personnel • Requires DNA extraction & amplification • Risk of contamination • Recommended for use only on smear-positive sputum specimens and culture isolates. • Does not eliminate need for conventional C & DST