For Research Use Only. Not for Use in Diagnostic Procedures.

Rapid identificationof M. tuberculosis

and 60 related speciesin same analysis

Less than $6 USD pertest in consumables

Environmentally-friendly solvents

Automated and intuitiveuser operation

Fluorescence detection;200x more sensitive

than traditionalUV-HPLC detection

No HPLC experiencerequired.

DescriptionThe Sherlock

®Mycobacteria Identification System (MYCO-LCS) was

launched in 2003 and represents the only commercially availablemycobacterial identification system that analyzes cellular mycolic acids byHigh Performance Liquid Chromatography (HPLC). The Sherlock MYCO-LCS represents a rapid, accurate and comprehensive solution for theidentification of over 60 mycobacteria and related species within 2 hoursfrom pure culture.

InstrumentationThe Sherlock MYCO-LCS is composed of a Windows

®XP/2000-based

computer loaded with the MIDI Sherlock software. The computer isinterfaced with an Agilent 1100/1200 Series HPLC. The Agilent HPLC hasseveral unique features that ensure high quality analysis. The Agilent needleand autosampler are in the flow path, resulting in no carryover and fewermisidentifications resulting from carryover.

Sherlock OperationAfter harvesting an extremely small cell mass, a 1.5-hour sample preparationconverts the mycolic acids into a form suitable for HPLC analysis. Samplesare loaded into the HPLC autosampler for analysis, information about eachsample is logged into the Sherlock Sample Table and the batch is processed.The Sherlock software automatically sets the operating parameters of theHPLC, recalibrates within a preset interval and makes adjustments forvariances in sample concentration.

When the HPLC run is complete, the Sherlock’s automated patternrecognition software matches the unknown mycolic acid composition with“stored” patterns of mycobacteria species/groups and related species. Eachlibrary entry is a computer-generated composite of reference strains for thespecies. Finally, the computer prints the Composition Report, which includesthe peak naming and library classification results. A reference comparisonchromatogram is also generated with each analysis for classic visualconfirmation of results.

Sherlock® Mycobacteria Identification SystemOverview

Figure 1- Visual Confirmation.The user can perform a visualcomparison of the sample HPLCchromatogram (blue) to a known“reference” HPLC chromatogram(red). This feature was added tothe Sherlock MYCO-LCS to helplabs using a “manual” UV-HPLCprotocol more easily make thetransition to the Sherlock MYCO-LCS’s automated namingsystem.

M. tuberculosisSample Chromatogram

M. tuberculosisReference Chromatogram

Mycobacterium-abscessus/chelonaeMycobacterium-asiaticumMycobacterium-aurum/vaccaeMycobacterium-bovis BCG (not 35737)Mycobacterium-celatumMycobacterium-chelonae/abscessusMycobacterium-flavescensMycobacterium-fortuitum/peregrinumMycobacterium-gordonaeMycobacterium-haemophilum (30C, chocolate)Mycobacterium-interjectumMycobacterium-intermediumMycobacterium-kansasiiMycobacterium-lentiflavum/triplexMycobacterium-MAC A (avium/intracellulare)Mycobacterium-MAC B(avium/intracellulare)Mycobacterium-MAC C (intracellulare/avium)Mycobacterium-MAIS complex(scrofulaceum/avium/intracellulare)Mycobacterium-malmoenseMycobacterium-marinum(30C)Mycobacterium-mucogenicumMycobacterium-neoaurumMycobacterium-nonchromogenicum/terraeMycobacterium-peregrinum /fortuitumMycobacterium-simiaeMycobacterium-szulgaiMycobacterium-terrae/nonchromogenicumMycobacterium-thermoresistibleMycobacterium-trivialeMycobacterium-tuberculosis complex(TB,bovis,africanum,microti)Mycobacterium-xenopi

Sherlock® Mycobacteria LibrarySpecies List

Genera include:Corynebacterium, Dietzia,

Gordonia, Nocardia,Rhodococcus and

Tsukamurella

Separate entries for MAIand M. scrofulaceum

Library containsadditional rapid growing

Mycobacteria spp.

Same extractionprocedure and

HPLC column asMIDI’s standard

Mycobacteria library.

Sherlock Mycolic Acid Bacteria (MAB) Library DescriptionThe Sherlock Mycobacteria Library is optimized for analysis ofMycobacteria spp. which typically contain the longer chain-length mycolicacids. In addition to Mycobacterium, a number of other bacterial generapossess shorter chain-length mycolic acids of varying sizes. They includeCorynebacterium, Dietzia, Gordonia, Nocardia, Rhodococcus andTsukamurella and are best analyzed using the Sherlock Mycolic AcidBacteria Library.

Sherlock System BackgroundThe Sherlock Mycobacteria Identification System (MYCO-LCS) analyzesand identifies mycobacteria and related species based on the production ofmycolic acids. The system is used, along with other identification methods, toidentify mycobacteria and related species that have been isolated fromclinical specimens by traditional culturing techniques. Sherlock uses aprepared sample and high performance liquid chromatography (HPLC) toyield qualitatively and quantitatively reproducible mycolic acid compositionprofiles. The mycolic acid profile is then compared to a “library” stored in thecomputer to determine identity of the unknown.

The Sherlock Mycobacteria Identification System is composed of aWindows

®XP/2000-based computer loaded with the MIDI Sherlock

software and an Agilent ChemStation. The computer is interfaced with anAgilent 1100/1200 Series HPLC. The Agilent HPLC has several uniquefeatures that ensure high quality analysis. The Agilent needle andautosampler are in the flow path, resulting in no carryover.

Fact Sheet

min5 10 15 20 25 30

LU

0

50

100

150

200

250

FLD1 A, Ex=345, Em=425 (E07419.694\A0101024.D)

2.7

66

3.2

10 3.4

05

4.1

44

4.7

24

4.9

32

14

.71

1

15

.33

51

5.9

19

16

.47

21

6.9

90

17

.49

21

7.9

81

18

.45

91

8.9

26

19

.38

61

9.8

28

20

.25

92

0.6

84

21

.09

4

29

.31

7

HPLC Chromatogram of Nocardia otitidiscaviarum

Sherlock® Mycolic Acid Bacteria Library

Corynebacterium-diphtheriae Mycobacterium-mucogenicumCorynebacterium-glucuronolyticum Mycobacterium-neoaurumCorynebacterium-jeikeium Mycobacterium-neworleansenseCorynebacterium-kutcheri Mycobacterium-scrofulaceumCorynebacterium-macginleyi Mycobacterium-smegmatisCorynebacterium-minutissimum Mycobacterium-vaccaeCorynebacterium-pseudodiphtheriticum Nocardia-abscessusCorynebacterium-riegelii Nocardia-africanaCorynebacterium-striatum Nocardia-asteroidesCorynebacterium-ulcerans Nocardia-brasiliensisCorynebacterium-xerosis Nocardia-brevicatenaDietzia-maris Nocardia-carneaGordonia-aichiensis Nocardia-farcinicaGordonia-bronchialis Nocardia-novaGordonia-rubropertincta Nocardia-otitidiscaviarumGordonia-sputi Nocardia-paucivoransGordonia-terrae Nocardia-pseudobrasiliensisMycobacterium-abscessus Nocardia-transvalensisMycobacterium-aurum Nocardia-veteranaMycobacterium-avium complex Rhodococcus-equiMycobacterium-boenickei Rhodococcus-erythropolisMycobacterium-brisbanense Rhodococcus-rhodniiMycobacterium-canariasense Rhodococcus-rhodochrousMycobacterium-chelonae Tsukamurella-inchonensisMycobacterium-cosmeticum Tsukamurella-paurometabolaMycobacterium-flavescens Tsukamurella-tyrosinosolvensMycobacterium-fortuitum-acetamidolyticum Tsukamurella-wratislaviensisMycobacterium-fortuitum-fortuitumMycobacterium-immunogenum

Sherlock® Mycolic Acid Bacteria LibrarySpecies List

Sherlock® Mycobacteria Identification System

Specification Sheet

General Description

The Sherlock® MycobacteriaIdentification System was developed andis marketed by MIDI, Inc., Newark, DE,USA. The system is intended to aid inthe identification of M. tuberculosis anddifferentiation from other mycobacteriaand related species through the analysisof mycolic acids derived from culturedbacterial samples.

Sherlock uses a sample preparationprocedure and high performance liquidchromatography (HPLC) to yieldqualitatively and quantitativelyreproducible mycolic acid compositionprofiles. Automated pattern recognitionsoftware matches the sample’scomposition with stored patterns ofmycobacteria species/groups and relatedspecies. The Sherlock software alsocalibrates and monitors the system toensure proper functioning.

The Sherlock MycobacteriaIdentification System is designed forease of use. As a result, no HPLCexperience is required for operation.

Technology

Mycolic acid analysis by HPLC is beingused by laboratories for diagnosis oftuberculosis and other mycobacterialinfections. The technique was developedby the U.S. Centers for Disease Control,working with the “HPLC Users Group.”

The CDC technique was used as thestarting point for the SherlockMycobacteria Identification System.The extraction protocol was adaptedfrom the methodology established at theTexas Department of Health, Bureau ofLaboratories, Mycobacteriology/Mycology Branch.

Species Libraries

Mycobacteria Library

The Sherlock Mycobacteria Librarycontains over 25 species/groups, createdfrom well-characterized mycobacterialstrains.

Mycolic Acid Bacteria (MAB) Library

In addition to Mycobacterium, a numberof other bacterial genera possess shorterchain-length mycolic acids of varyingsizes. They include Corynebacterium,Dietzia, Gordonia, Nocardia,Rhodococcus and Tsukamurella.The Sherlock MAB Library contains38 species, created fromwell-characterized strains.

Low Per Sample Costs

It costs less than $6 USD per sample forall consumables. This includes reagents,internal standards, calibration standards,glassware and culture media.

Instrument Throughput

Following a short preparation procedure(typically done in batches), the samplesare loaded into the instrument’sautosampler. The automated systemtakes over and analyzes each sample. The Sherlock system can analyze up

to 4 samples per hour.

Sample Preparation

Each sample is prepared for analysisusing a liquid-liquid extraction. Harvesting a small quantity of cells

from the culture plate is the first step.It typically takes less than30 minutes to harvest cells from30 plates into 30 test tubes.

The four-step sample preparationprocess requires about 1.5 to 2.0 hoursfor a batch of 30 samples. During thistime, approximately 45 minutes offree time is available for thetechnician to do other tasks.

Culturing

Sherlock requires pure cultures. Themycobacteria cultures typically aregrown on solid medium such asMiddlebrook 7H10 (or 7H11) at 35-37C,until visible growth is noted. Sensitivefluorescent detection often allows foranalysis from a single colony. Otherspecies may normally be grown underdifferent conditions and the librarieshave been constructed to reflect this.Examples would be the culturing ofM. marinum and M. haemophilum at30C, with the latter organism beinggrown on chocolate agar rather than onMiddlebrook.

Bio-Safety

Warning: Observe Biosafety Level IIIpractices while working with viablecultures. An approved biological safetycabinet must be used!

Laboratories that handle dangerouspathogens will typically perform thesample extraction in a BSL-3 lab andtransfer decontaminated extracts out toanother lab for instrument analysis. Thisallows the instrument to be maintainedand serviced by technicians outside theBSL-3 lab.

With the MIDI System, Biosafety isenhanced because live organisms are notintroduced into the instrument. The firststeps of the sample procedure kill thecells (addition of potassium hydroxidesolution followed by autoclaving for 30minutes). After this, the technician is nolonger working with live organisms.

The MIDI protocol uses a benignmixture of methanol and ispopropanol asits solvent system. This eliminatespotential deleterious health effects orenvironmental contamination that mightresult from exposure to or disposal oftoxic solvents (such as methylenechloride), which are often used incomparable HPLC separations.

InstrumentationThe Sherlock MycobacteriaIdentification System is composed of aWindows® XP/2000-based computerloaded with the MIDI Sherlock softwareand an Agilent ChemStation (A.10 orabove). The computer is interfaced withan Agilent 1100/1200 HPLC.

Many of the hardware components of theMIDI System are designed to meetspecifications required for robust andreproducible performance. Thequaternary pump employs low pressuremixing to maintain a constant rate ofsolvent delivery. The autosampler, withflow-through design, eliminates carryover contamination and the Peltiertemperature control is capable ofmaintaining column temperatures towithin 0.1C. A long-lifechromatographic column is able toprovide consistent performance overthousands of runs.

In addition, the MIDI system employsfluorescence detection, which enhancessensitivity more than 200-fold comparedwith UV detection.

Markets Using Sherlock Diagnostic Laboratories Large Hospitals Pharmaceutical Companies Public Health Departments

The information in this publicationis subject to change without notice.

Copyright 2009 MIDI, Inc.All Rights Reserved

Date: 14 September 2009

MIDI, Inc.125 Sandy DriveNewark, Delaware 19713Phone: 302-737-4297Fax: 302-737-7781Email: sales@midi-inc.comWeb: www.midi-inc.com

Sherlock Mycobacteria Identification System