Page 304 - The Veterinary Laboratory and Field Manual 3rd Edition

Microbiology 273

web tool is available for designing the primers gene of the influenza viral genome. In addition,
for LAMP. nucleotide sequencing can be used for tracking
the spread of infections based on constructing
a phylogenetic tree and this is an important
Sequencing
application since finding the source of infection
Nucleotide sequencing reveals the sequen- complements the control measures preventing
tial arrangement of four nucleotides (adenine, future disease outbreaks. Identifying genetic
guanine, cytosine and thiamine) in a stretch variation in immunodominant epitopes using
of genome (Figure 4.35). Sequencing is based sequencing technique also may explain vaccine
on identifying genetic variations in the nucleo- failures and facilitate use of appropriate vaccines
tide sequence of variable genome regions and for the control measures (Figure 4.36).
is therefore a highly discriminatory technique. Methodology can vary from the analysis of
For example, it is routinely used in diagnostic sequences from small regions of targeted genes
laboratories for differentiating highly patho- to comparing large segments or entire genomes.
genic avian influenza viral pathotypes from low Some examples of the different approaches are
pathogenic avian influenza pathotypes. In this given below.
particular case, sequencing targets the region
of cleavage site of the haemagglutinin (HA) MuLtI-LocuS SEquEncE tyPInG (MLSt)
MLST is a technique introduced by Maiden et al.
(1998) that uses sequence data from a number
of genes to identify isolates by their sequence
type (ST). There are genes that are considered
essential for the survival of the organism and
therefore are present in all isolates and are
strongly conserved. These are referred to as
housekeeping genes. MLST uses a selection of
these housekeeping genes that are distributed
around the genome for identification purposes.
For example, in Campylobacter jejuni, conserved
sequences within seven housekeeping genes are
amplified by PCR, sequenced and the sequence
data analysed. Each different ST for a given gene
(that is, allelic type) in the scheme is given a
unique number to identify it. The seven genes
used in this scheme are then concatenated to
form an allelic profile comprising seven num-
Figure 4.35 (a) Modern sequencing equipment bers. This combination of seven allelic numbers
used for nucleotide sequencing. Photo: Dr Davor denotes a unique strain type and has proven to
Ojkic, Animal Health Laboratory, University of be quite robust in that allelic profile strongly
Guelph, Canada. (b) A representative of sequenc- reflects the genetic relatedness in the case of
ing output. The blue, red, black and green peaks Campylobacter isolates and STs that differ by one
represent cytosine, thiamine, adenine, and guanine, allele can be grouped in clonal complexes (CCs)
respectively. See also Plate 20. Photo: Dr Rikia and are useful groupings for phylogenetic analy-
Dardari, University of Calgary, Canada. sis of populations.

Vet Lab.indb 273 26/03/2019 10:25