Technical Notes: 1 The Specificity of the microCompass ... - Lonza

Technical Notes: 1
The Specificity of the microCompass TVC Assay
Introduction
The microCompass System is a rapid microbial detection system
that can provide quantitative results in four hours. Both
filterable and non-filterable samples can be tested for a variety
of applications. The technology is based upon amplification of
ribosomal RNA using real-time, reverse transcriptase PCR. The
microCompass TVC Detection Kit for total viable count (TVC) is
designed to detect all viable bacteria present in a test sample.
The kit contains primers and a probe that match ribosomal 16S
RNA sequences universally found in all bacteria (Gram positive,
Gram negative, spores, aerobic and anaerobic bacteria).
versus log (RNA concentration) for bacteria shown in bold in Table
1 .
3.4
4.4
microCompass TVC RT-PCR Inclusivity
Aim
The main objective of this study is to provide data on the specificity
and sensitivity of the microCompass TVC Detection Kit.
Experimental Protocol
Inclusivity
Inclusivity (sensitivity) is a measure of the ability of a test method
to detect all of the desired target organisms. Since the microCompass
TVC Detection Kit should detect all bacteria, it is necessary
to demonstrate the detection of a wide range of bacterial strains.
The inclusivity test should also confirm that the efficiency of the
RT-PCR reaction is approximately the same for all bacteria, including
both laboratory and environmental isolates. Using bioinformatics,
more than 100,000 sequences of bacterial 16S rRNA were
first aligned and used to design the TVC primers and probe.
The inclusivity tests were performed on pure culture samples as
follows:
1. rRNA was extracted from more than 45 bacterial strains
(pharmacopeial and environmental strains - Table 1) representing
a wide range of genus and species, using the microCompass
RNA Extraction Kit.
2. The extracted RNA was measured using spectrophotometric
analysis, after staining with the Ribogreen Stain (Fisher
Bioblock Scientific).
3. Three concentration levels of each RNA (250,000, 25,000
and 2,500 fg) were analyzed using the microCompass TVC
Detection Kit.
Results
All 45 bacteria tested were positive (Table 1) for all three levels of
RNA tested. Diagram 1 shows the Cycle Threshold (Ct) obtained
5.4
16.00 19.00 22.00 25.00 28.00
Figure 1 : Ct obtained vs. log[RNA] for bacteria shown in bold in Table 1.
Each bacterial strain (pharmacopeial and environmental isolates) tested was
detected by the microCompass TVC Detection Kit.
Exclusivity
Exclusivity (specificity) is a measure of a test method to not report
a positive result for those organisms that are not the target of
the assay. Exclusivity is demonstrated by an absence of interference
in the alternative method from a relevant range of non-target
strains, which are potentially cross-reactive. Since the TVC Assay
is designed to detect all bacteria, a relevant range of non-targets
would be yeast and mold strains that might be found in similar
samples. For this method, samples were prepared as follows:
1. RNA was extracted from 30 different yeast and mold strains
(pharmacopeial and environmental strains - Table 2) using
the microCompass RNA Extraction Kit.
2. The RNA concentration was measured using spectrophtometric
analysis after staining with the Ribogreen Stain.
3. Each RNA sample was analyzed using the microCompass
TVC Detection Kit.
Results
All 45 inclusivity strains of bacteria tested were positive
(Table 1) for all three concentration levels of RNA tested.
Figure 1 shows the Ct obtained versus log (RNA concentration)
for bacteria shown in bold in Table 1. All yeast and
mold samples tested (Table 2) were negative with the
microCompass TVC Detection Kit.

microCompass Technical Notes 1
Table 1 : Bacteria tested strains list (Data shown in Diagram 1 for bacteria in bold)
Organism Group Organism Species Source
Endospore-forming Gram
positive Bacillus
Gram positive Coccus
Gram negative
Bacillus
Non–spore-forming
Gram positive Bacillus
Contact Information
Bacillus circulans,
(ES)
Bacillus licheniformis,
(ES)
Bacillus cereus, (CIP 78.3)
Bacillus pumilus,
(ES)
Bacillus marocanus,
(ES)
Bacillus subtilis, (ATCC 6633)
Paenibacillus pabuli,
(Unknown)
Clostridium sporogenes (CIP 79.39)
Staphylococcus aureus, (ATCC 6538)
Micrococcus luteus, (CIP A 270 T)
Micrococcus lylae,
(ES)
Staphylococcus hominis,
(ES)
Staphylococcus epidermidis,
(ES)
Staphylococcus saprophyticus,
(ES)
Staphylococcus warneri,
(ES)
Staphylococcus arlettae,
(ES)
Staphylococcus carnosus,
(ES)
Macrococcus bovicus,
(ES)
Peptostreptococcus anaerobius (CIP 104411)
Escherichia coli, (ATCC 8739)
Salmonella abony, (NCTC 6017/CIP 80.39)
Serratia marcescens, (NCTC 10211)
Pantoea agglomerans,
(ES)
Klebsiella spp, (SD 54.19)
Enterobacter gergoviae (SD 53.21)
Pseudomonas aeruginosa, (ATCC 9027)
Pseudomonas fluorescens, (ATCC 13525)
Burkholderia cepacia, (CIP 80.24)
Stenotrophomonas maltophilia,
(ES)
Pseudomonas stuzeri,
(ES)
Acidovorax delafieldii,
(ES)
Burkholderia multivorans,
(ES)
Bordetella hinzii,
(ES)
Brevundimonas vesicularis, (CIP 101035)
Flavobacterium jonhsoniae,
(ES)
Delftia acidovorans,
(ES)
Ralstonia picketii
(ES)
Flavimonas oryzihabitans,
(ES)
Fusobacterium nucleatum, (CIP 1011303)
Bacteroides fragilis,
(Unknown)
Prevotella denticola,
(Unknown)
Porphyromonas gingivalis, (CIP 103683)
Methylobacterium spp
(ES)
Rothia dentocariosa,
(ES)
Corynebacterium afermentans,
(ES)
Tsukamurella inchonensis,
(ES)
Mycobacterium spp,
(ES)
Tsukamurella paurometabola,
(ES)
Propionibacterium acnes,
(ES)
Actinomyces bovis, (CIP 103258)
Bifidobacterium bifidum, (CIP 56.7)
Bifidobacterium longun (CIP 64.62)
Discussion and Conclusion
The microCompass TVC Detection Kit allows detection of a broad
range of bacterial strains, including aerobic and anaerobic bacteria,
endospore-forming and non–spore-forming Gram positive Bacillus,
Gram positive Coccus, Gram negative Bacillus, Enterobacteriaceae
and Non-Enterobacteriaceae. In this study, the microCompass
TVC Detection Kit was found to have 100% inclusivity/sensitivity
for detecting a wide range of bacterial strains and 100% exclusivity/specificity
for non-bacterial strains. Regardless of bacterial
source-species the relative efficiency of the RT-PCR results were
the same levels of detection. It can therefore be concluded that the
microCompass TVC Detection Kit is a rapid, sensitive and specific
test for the quantitative detection of bacteria in a wide variety of
samples, providing results in hours, versus several days for conventional
reference methods.
Table 2 : Yeasts and Molds tested strains list
Organism
Group
Yeasts
Molds
Organism
Species
Source
Candida albicans, ATCC 10231
Candida tropicalis, ATCC 201380
Candida kefyr, ATCC 2512
Candida krusei, ATCC 6258
Debaryomyces polymorphus, ATCC 18577
Pichia fermentans, ATCC 28789
Rhodotorula glutinis, ATCC 16740
Aspergillus oryzae, ATCC 10124
Aspergillus niger, CIP 1431-83
Aspergillus fumigatus,
ES
Botrytis cinerea, ATCC 11542
Cladosporium cladosporioides,
UMIP 1232.80
Cladosporium sphaerospermum
Penz,
ES
Aspergillus repens, ATCC 48521
Organism
Species
Source
Saccharomyces cerevisiae, ES
Torulaspora delbrueckii, ATCC 204289
Zygosaccharomyces rouxii, UMIP 2021.92
Kluyveromyces yarrowii, ATCC 200789
Cryptococcus terreus, ATCC 11799
Pichia pastoris, ATCC 204414
Kluyveromyces lactis ATCC 8563
Fusarium oxysporum, ATCC 16322
Geotrichum candidum, ATCC 34614
Monascus purpureus, ATCC 16365
Penicillium roquefortii, UMIP 1404.82
Penicillium chrysogenum, ES
Rhizopus stolonifer, UMIP 1583.85
Trichoderma viride, ATCC 26802
Trichothecium roseum, ATCC 8685
Verticillium dahliae ATCC 16535
1.) Ct/Cp (Cycle Threshold/Crossing Point) is the number of RT-PCR cycles required to detect a
flourescent signal above the background. Ct/Cp is inversely proportional to the starting concentration
of target in the sample, i.e. the lower the Ct/Cp, the higher the concentration of the sample.
2.) RT-PCR (Reverse Transcription -Polymerase Chain Reaction) RT converts RNA to amplifiable DNA.
Once the DNA strand is built, it is then amplified by PCR.
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TB-TVC 05/08