4       Molecular diagnostic methods

 

Most of the characteristics of living bacteria are associated with the genes present on their bacterial chromosome. This structure consists of a double-stranded DNA helix, with all the properties required to control replication of the bacterium, store its genetic information and express some characteristics unique to the organism. All these properties are controlled by specific enzymes and the genetic message is, in some instances, subsequently decoded in a process involving other enzymes, leading to the synthesis of a bacterial protein (see Chapter 3).

The properties of DNA used for analytical purposes derive from its chemical structure. These have been used to develop many of the modern protocols for detection of bacterial pathogens in clinical specimens. A DNA molecule has three important analytical features that facilitate its utilization as a diagnostic target:

• Recognition properties: base-pairing rules in DNA underpin the analytical approach of molecular techniques including DNA probe hybridization, DNA sequencing, the polymerase chain reaction (PCR) and, more recently, microarrays.
• Stability and robust flexibility: the DNA molecule is inherently stable, which facilitates its recovery from degraded material.
• Sequence features: when the DNA sequence of any cell is closely examined, open reading frames (ORFs) encoding genes and other features can be determined.

Molecular hybridization

Any labelled DNA probe, under suitable experimental conditions, would be capable of binding or hybridizing to its complementary strand in solution (based on the DNA base-pairing rules). It is this binding event that is subsequently detected. Examples of molecular hybridization techniques include Southern and northern blotting.

DNA sequencing is the most powerful analytical/diagnostic approach that exists in the molecular armoury. Insight into the understanding of any DNA molecule derives from its nucleotide sequence. The nucleotide sequence can be used to deduce the primary protein structure of the corresponding protein which can subsequently be compared with similar sequences from other organisms. DNA-binding sites and other regulatory features of genes can also be identified.

The DNA sequence of a gene, or the ORF, can be determined using either a chemical- or an enzyme-based approach. The technical principles of the latter method on which modern dideoxy DNA sequencing protocols are based involves the partial replication of a short DNA sequence using all four deoxyribonucleotides (dNTP) and a chemically modified dideoxyribonucleotide (ddNTP) lacking a hydroxyl group at the 2′-carbon on the ribose sugar ring. Like hybridization, this method is based on sequence recognition according to the base-pairing rules and accurate enzymatic synthesis, all of which are features of the naturally occurring replication event. To sequence a DNA molecule the following steps are usually required: primer hybridization, sequence reaction, detection and data analysis.

In a later development of this technology, fluorescent-based automated DNA sequencing was designed to reduce the manual manipulations involved whilst increasing sample throughput. More recent advances in DNA sequencing technology have produced instrumentation capable of sequencing a bacterial genome in a few hours (see Chapter 6).

The PCR assay was developed out of the strategies used for DNA sequencing. Typically a PCR protocol consists of three repeated steps, resulting in the amplification of a discrete segment of DNA (or RNA, after the addition of a reverse-transcription step, see following paragraphs). In the first of these, the template DNA, which has been recovered from a crude preparation of genomic DNA isolated from a microbial pathogen of veterinary interest or from blood or other tissue samples, is denatured, separating the two DNA strands. This is followed by an annealing step, wherein the reaction temperature is lowered, allowing two synthetic DNA primers or oligonucleotides to bind (hybridize) to the template. These primers are located on opposite DNA strands. Finally, the temperature is increased again (typically, 74°C) and a thermostable DNA polymerase enzyme begins a round of synthesis. These steps constitute one cycle and in a conventional PCR reaction up to 30 such cycles are carried out. This repetitive cycling between temperatures facilitates the amplification of a specific DNA target by up to one million-fold.

A programmable thermal cycler controls the rate of temperature change, length of incubation at each temperature and the number of times each cycle is repeated. Multiple cycles produce an amplified PCR product, or amplicon, that can be detected by conventional agarose gel electrophoresis, stained with ethidium bromide and visualized using ultraviolet light.

Conventional PCR-based assays have been developed to detect a broad range of target genes in pathogenic bacteria associated with animals, including food-borne zoonotic pathogens. Commercial kits are also available for these and other pathogenic organisms.

A potential limitation of DNA-based diagnostic methods is that they detect both viable and non-viable bacterial cells. This limitation can be overcome either by using an enrichment step before nucleic acid extraction or by performing an RNA-based detection method using reverse transcriptase (RT)-mediated PCR, in a protocol known as RT-PCR. These assays can also be used to detect RNA deriving from viruses such as rotavirus, coronavirus and norovirus.

Detection and simultaneous quantification of amplicons in real time is an important enabling technology in molecular diagnostics. The method facilitates the determination of the absolute number of a specific DNA target, such as a virulence gene of veterinary importance, relative to a housekeeping gene, such as 16S rRNA, within a living cell. Quantitative real-time PCR (qPCR) can be used to quantify bacteria, other microorganisms and individual genes. Real-time PCR uses fluorescence to detect the presence or absence of a particular DNA or RNA target. It is this detection process that differentiates real-time from conventional PCR.

Expression of any gene in a microorganism or other cell can be determined by measuring the mRNA transcription, using RT-PCR. This technique is referred to as quantitative RT-PCR (qRT-PCR). Based on this protocol, commercial kits are now available to detect and quantify a range of pathogenic organisms relevant to veterinary medicine.

The development of DNA microarrays is based on the use of a solid support to which a series of genes or chemically synthesized segments of those genes can be attached. DNA microarrays can be used in several ways. The arrays can provide useful information for identifying those genes controlling growth of an organism under defined culturing conditions, including aerobic versus anaerobic environmental conditions. In environmental microbiology, DNA microarrays containing 16S rDNA sequences can be used to identify bacteria and other microorganisms present in a particular environment. This DNA microarray is termed a phylochip. Comparative genome analysis makes use of DNA microarrays to compare the gene index of different serovars of Salmonella. DNA chips have been developed to aid in the simultaneous identification of a number of important pathogens including bacteria and viruses that may share similar environmental niches.