SPADA Meeting Book

laboratory origin of the culture and the method used to propagate the traceable culture from that 136 origin. 137

138

5.1.2 Orthogonal Test Results

It is also important that the culture verification statement describe how available orthogonal 139 test results address the relatedness of the test and index cultures. Orthogonal testing relies on 140 multiple analytic techniques to compare one culture to another. Cultures can be compared with 141 respect to morphology; genotypic and phenotypic properties; metabolic, immunological, and 142 molecular features; molecular functions; and virulence. However, the quality of microbial 143 verification and confidence associated with it ultimately depends on the number, type, and 144 diversity of applied assays. Culture producers should strive for more comprehensive approaches 145 to orthogonal testing. Minimally, orthogonal testing should include a mix of genotypic and 146 phenotypic assays. Table 1 provides examples of various tests and the largely orthogonal 147 categories to which they apply.

148 149

Table 1. Examples of orthogonal assays

150

Category

Example assays

Morphology

Colony plating, Gram stain

Genotypic properties Phenotypic properties

Next generation sequencing, RFLP a , MLVA b , MLST c Fatty acid-based microbial identification, Mass spectrometry

Metabolism

Biochemical arrays

Immunological assay response Molecular assay response

ELISA d , Bead-based multiplex assays, DFA e , IFA f

Real-time PCR g

Molecular function

Phage sensitivity, motility, hemolysis In vivo studies using animal models

Virulence

151 152 153 154 155 156 157

a RFLP = Restriction fragment length polymorphism b MLVA = Multilocus variable number tandem repeat analysis

c MLST = Multilocus sequence typing

d ELISA = Enzyme-linked immunosorbent assay e DFA = Direct fluorescent antibody assay f IFA = Indirect immunofluorescence assay

g PCR = Polymerase chain reaction

7