Genetic identification of microorganisms WORLD OF MICROBIOLOGY AND IMMUNOLOGY240•
read in a sequential manner starting from a fixed point in the
gene. The insertion or deletion of a nucleotide shifted the read-
ing frame in which succeeding nucleotides were read as
codons, and was thus termed a frameshift mutation. It was also
found that whereas two closely spaced deletions, or two closely
spaced insertions, could not suppress each other, three closely
spaced deletions or insertions could do so. Consequently, these
observations established the triplet nature of the genetic code.
The reading frame of a sequence is the way in which the
sequence is divided into the triplets and is determined by the
precise point at which translation is initiated. For example, the
sequence CATCATCAT can be read CAT CAT CAT or C ATC
ATC AT or CA TCA TCA T in the three possible reading
frames. Sometimes, as in particular bacterial viruses, genes
have been found that are contained within other genes. These
are translated in different reading frames so the amino acid
sequences of the proteins encoded by them are different. Such
economy of genetic material is, however, quite rare
The same genetic code appears to operate in all living
things, but exceptions to this universality are known. In
human mitochondrial mRNA, AGA and AGG are termination
or stop codons. Other differences also exist in the correspon-
dences between certain codon sequences and amino acids. In
ciliates, there are also unusual features in that UAA and UAG
code for glutamine (CAA and CAG in other eukaryotes) and
the only termination codon appears to be UGA.See also Bacteriophage and bacteriophage typing; Gene
amplification; Genetic identification of microorganisms;
Genetic mapping; Genetic regulation of eukaryotic cells;
Genetic regulation of prokaryotic cells; Genotype and pheno-
type; ImmunogeneticsGENETIC IDENTIFICATION OF
MICROORGANISMSGenetic identification of microorganismsThe genetic identification of microorganismsutilizes molec-
ular technologies to evaluate specific regions of the genome
and uniquely determine to which genus, species, or strain a
microorganism belongs. This work grew out of the similar,
highly successful applications in human identification using
the same basic techniques. Thus, the genetic identification
of microorganisms has also been referred to a microbial
fingerprinting.
Genetic identification of microorganisms is basically a
comparison study. To identify an unknown organism, appro-
priate sequences from the unknown are compared to docu-
mented sequences from known organisms. Homology
between the sequences results in a positive test. An exact
match will occur when the two organisms are the same.
Related individuals have genetic material that is identical for
some regions and dissimilar for others. Unrelated individuals
will have significant differences in the sequences being evalu-
ated. Developing a database of key sequences that are unique
to and characteristic of a series of known organisms facilitates
this type of analysis. The sequences utilized fall into two dif-ferent categories, 1) fragments derived from the transcription-
ally active, coding regions of the genome, and, 2) fragments
present in inactive, noncoding regions. Of the two, the non-
coding genomic material is more susceptible to mutation and
will therefore show a higher degree of variability.
Depending on the level of specificity required, an assay
can provide information on the genus, species, and/or strain of
a microorganism. The most basic type of identification is clas-
sification to a genus. Although this general identification does
not discriminate between the related species that comprise the
genus, it can be useful in a variety of situations. For example,
if a person is thought to have tuberculosis, a test to determine
if Mycobacteriumcells (the genus that includes the tuberculo-
sis causing organism) are present in a sputum sample will
most likely confirm the diagnosis. However, if there are sev-
eral species within a genus that cause similar diseases but that
respond to entirely different drugs, it would then be critical to
know exactly which species is present for proper treatment. A
more specific test using genomic sequences unique to each
species would be needed for this type of discrimination. In
some instances, it is important to take the analysis one step
further to detect genetically distinct subspecies or strains.
Variant strains usually arise as a result of physical separation
and evolutionof the genome. If one homogeneous sample of
cells is split and sent to two different locations, over time,
changes (mutations) may occur that will distinguish the two
populations as unique entities. The importance of this issue
can be appreciated when considering tuberculosis. Since the
late 1980s, there has been a resurgence of this disease accom-
panied by the appearance of several new strains with antimi-
crobial resistance. The use of genetic identification for rapid
determination of which strain is present has been essential to
protect health care workers and provide appropriate therapy
for affected individuals.
The tools used for genetic studies include standard
molecular technologies. Total sequencing of an organism’s
genome is one approach, but this method is time consuming
and expensive. Southern blot analysis can be used, but has
been replaced by newer technologies in most laboratories.
Solution-phase hybridization using DNAprobes has proven
effective for many organisms. In this procedure, probes
labeled with a reporter molecule are combined with cells in
solution and upon hybridization with target cells, a chemilu-
minescent signal that can be quantitated by a luminometer is
emitted. A variation of this scheme is to capture the target cells
by hybridization to a probe followed by a second hybridiza-
tion that results in precipitation of the cells for quantitation.
These assays are rapid, relatively inexpensive and highly sen-
sitive. However, they require the presence of a relatively large
number of organisms to be effective. Amplification tech
nologies such as PCR(polymerase chain reaction) and LCR
(ligase change reaction) allow detection of very low concen-
trations of organisms from cultures or patient specimens such
as blood or body tissues. Primers are designed to selectively
amplify genomic sequences unique to each species, and, by
screening unknowns for the presence or absence these regions,
the unknown is identified. Multiplex PCR has made it possi-
ble to discriminate between a number of different species in awomi_G 5/6/03 3:18 PM Page 240