Introduction

Bacteria have existed long before mammalian evolution and infectious diseases have been present as long as there have been humans. Historically, treatment of infectious diseases was poor due to inadequate knowledge, a limitation that substantially contributed to disease spread and massive death. Approximately 150 years ago Louis Pasteur formulated the germ theory, explaining how contagious diseases could spread between humans and Robert Koch gave the first proof of the germ theory by demonstrating how Bacillus anthrax could infect sheep and later discovered the causative bacteria of tuberculosis and cholera. Until the discovery of the duplex DNA structure and the complementary rules in 1953 that the era of nucleic acid-based molecular diagnostics of infectious diseases began.

The cornerstone of most molecular biology technologies is the gene. To facilitate the study of genes, they can be isolated and amplified. Nucleic acid-based diagnostics of infectious diseases involves detection and characterization of both bacterial and viral infection using DNA/RNA methods. Today the major driving forces for developing new diagnostic techniques are reduced hands-on-time and faster methods, as well as increased sensitivity.Classical cultivation based identification and monitoring methods have dominated various clinical microbiology labs, but some bacterial pathogens like Neisseria gonorrhea, are difficult to be cultured in lab condition. There are two aspects of disease diagnosis with DNA technology. Those designed to identify specific pathogen responsible for causing disease and those test which are designed to identify genes coding for virulence factors of a bacteria. (Caskey, 1987)

Detection of Pathogens

1. Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) was developed approximately 25 years ago (Saiki et al., 1985 and1988). In PCR, DNA is amplified in a thermocycler by repeating the three major steps: denaturation of DNA template into single stranded DNA, annealing of oligonucleotide primers to their complementary targets, and extension of the primers by DNA polymerase to generate a copy of the target gene (Yang and Rothman, 2004). PCR has allowed a spectrum of advances ranging from the identification of novel genes and pathogens to the quantification of characterized nucleotide sequences (Erlich et al., 1991). PCR assays rapidly and precisely detects the presence of microorganisms, including those that are fastidious and slow growing, directly from clinical specimens. PCR is also used to test for the presence of antimicrobial resistance genes in samples. Numerous different PCR-based assays have been developed for diagnosis of infectious diseases.

1. Specific PCR

Specific PCR is the simplest PCR approach which is designed to amplify a gene specific to a target microbe. So the primers in specific PCR are designed such that they are strictly specific for the target microorganism. This is the most widely used method in the diagnosis of infectious disease. Many organism such as Mycobacterium, Streptococcus, Pneumococcus, Borrelia, Helicobacter, Rickettsia, Ehrlichia, Coxiella can be directly detected in clinical samples.Tuberculosis caused by Mycobacterium tuberculosis requires a fast, sensitive and cheap detection technique in order to initiate adequate control measures in order to contain the disease (Palomino, 2005). Multiplex PCR followed by hybridization to a DNA strip for detection of multidrug resistant M. tuberculosis is commercially available, for the simultaneous detection of rifampin (rpoB) and isoniazid (katG) resistance (Hillemann et al., 2005). The combined use of PCR and serological tests has improved the sensitivity of the diagnosis of leptospirosis in early phase of the disease (de Abreu et al., 2006).

1. Real time PCR

Real Time PCR is simple, specific, sensitive and fast. Real time PCR comprises both amplification and fluorescent detection of the sample in the same step. This also reduces the risk of contamination with amplified nucleic acids because the analysis is performed in a closed vessel. In general there are two different detection methods for real time PCR. One method relies on a fluorescent stain (e.g. SYBR Green), which is specific for double stranded DNA, and the other method relies on fluorescent resonance energy transfer (FRET) probes. SYBR Green provides a sensitive, but not specific, signal, whereas the FRET probes provides a sensitive and specific signal. Three different FRET probes exist: 5′ nuclease probes (TaqMan), molecular beacons, and FRET hybridization probes. TaqMan probes carry both a fluorescent dye and a quenching dye, and after the amplification step the 5′ nuclease activity of the Taq polymerase separates the fluorescent dye from the quencher, resulting in an increasing abundance of fluorescence after each PCR cycle (Espy et al., 2006).

Molecular beacons is a probe embedded within two complementary sequences, and carry both a fluorophore and a quencher at the ends of the probe, and in the absence of a target the probe exist as a hairpin structure, forcing the quencher near the fluorophore. When the probe hybridizes with a complementary target sequence, the fluorescent dye and the quencher is separated, so that a fluorescent signal is generated (Tyagi and Kramer, 1996). FRET hybridization probes consist of two DNA probes, each carrying a fluorophore. The upstream probe carries the fluorescent dye at the 3′ end, and a second probe designed to hybridize downstream, carries an acceptor dye at its 5′ end and is phosphorylated at its 3′ end to prevent it from being used by Taq polymerase during PCR amplification. The fluorescence from the upstream probe is absorbed by the adjacent acceptor dye at the downstream probe when the probes are hybridized next to each other and the dye is excited and emits light with a third wave length which is detected .

The limitations of real time PCR are the possibility of inhibition of the polymerase by the presence of certain compounds, and the risk of detecting contaminating DNA due to the high sensitivity of the method (Valasek and Repa, 2005). Staphylococcal methicillin resistance is mainly mediated by the mecA gene, which encodes a peptidoglycan transpeptidase (not inactivated by β-lactams) is detected by the above method(Livermoore, 2006).

1. Broad Range Ribosomal PCR

Broad range ribosomal PCR has played a key role in the identification of novel bacterial pathogens. The prokaryotic small subunit has RNA with a sedimentation coefficient of 16S has been most used for bacterial characterization as it contains alternating regions of sequence conservation and heterogenity. This procedure employed primers that recognize the conserved regions of 16S rRNA genes, amplification and sequence comparison with the known sequence in databases to characterize the bacteria. These characteristics inspired Woese in 1987 to deem rRNA the “ultimate molecular chronometer” (Woese, 1987). The first agent discovered by this technique was the agent of Bacillary angiomatosis in AIDS patients by Rochalimaea quintana. Often partial sequencing of the 16S rRNA gene is enough to identify bacteria, and it has been shown that sequencing of the 5′ end of 16S rRNA is sufficient for species level identification of most clinically relevant Mycobacterium isolates (Rogall et al 1990; Patel, 2001).

1. Rapid Primer PCR (RAPID) PCR

In RAPID PCR random range of nucleotide hexamers are used as primers. Being hexamers, these primers binds to multiple sites on bacterial chromosome, producing multiple sizes of amplicons. After running the PCR products on gel by electrophoresis a pattern of bands are obtained. RAPID-PCR can easily distinguish between different range of a bacterial species. Rapid PCR technology is used for identification of infectious organisms, determining antibiotic resistance eg, vanA and vanB genes for vancomycin-resistant Enterococci (VRE), detecting genes and gene mutations.Sensitivity equals and frequently exceed culture, enabling detection of <10 copies of target nucleic acid per reaction. It also eliminates contamination problems seen with PCR.

1. Nested PCR

In Nested PCR genomic DNA is amplified with two sets of primer. Firstly the target sequence is amplified using first set of primers, and a portion of amplification product is reamplified using another pair of PCR primers complimentary to the regions located beyond the 3′ end of first primers. As a result the second PCR product is shorter than first set. This method can be used to increase the sensitivity of detection and reduce contamination of nonspecific products. A nested PCR specific for the Mycoplasma pneumoniae P1 gene was used to diagnose mycoplasma infection in patients with severe pneumonia. (Talkington et al., 1998). Single-tube nested PCR in the diagnosis of tuberculosis for the repetitive IS6110 sequences was formulated by Chan et al.(Chan et al., 1996)

2. Ligase Chain Reaction

Ligase chain reaction (LCR) is a different type of amplification reaction in which the clinical sample is added to a reaction mixture containing a thermostable DNA ligase, a vast excess of two oligonucleotide primers specific to the pathogen to be detected and NAD (Nicotinamide adenine dinucleotide). If the sequence alignment is perfect, the ends of the primer will be close enough the each other to be covalently sealed by the DNA ligase. As in PCR, the denaturation step leads to separation of primers from the test DNA and then unused primer can bind to DNA and be linked. With the repeated cycles of denaturation, annealing and DNA ligase action, primer pair increases in concentration if they bind tightly enough to target DNA sequence for joining by DNA ligase. Finally, a wash step removes unhybridized primers and then a final denaturation step removes the bound primers and primer pairs from the test DNA. There will be two band on gel electrophoresis; one band corresponding to probe used for amplification, a second band equal to size to the sum of sizes of two probes, LCR is highly efficient as it can detect as few as 200-300 target molecule in a samples. LCR procedure allows an automated detection by employing flourescence or hapten labelled probe.LCR is used for detection of Borrelia burdgoferri (Hu et al.,1991), Listeria monocytogenes (Wiedmann et al., 1992), Neisseria gonorrhoeae (Birkenmeyer and Armstrong,1992), Mycobacterium tuberculosis (Iovannisci and Winn-Deen,1993) and Chalymdia trachomatis (Dille et al.,1993)

3. Nucleic Acid Hybridization

In Nucleic acid hybridization technique, small 15-30 bp long nucleotide sequence are used to detect complementary sequence in nucleic acid samples. Probes are prepared synthetically and can be radioactivity or non-radioactively labelled. Probes are being utilized in clinical diagnosis for detection of microorganism in various samples. First the DNA is isolated from the test sample and subjected to southern blot or dot blot hybridization with the probe. Some bacterial agents against which probe are used for diagnosis are Legionella, M.tuberculosis, Chlamydia spp. Campylobacter spp. etc.

4. Fluorescent In situ Hybridization

Fluorescent in situ hybridization (FISH) is a tool that today is widely used for identification, visualization and localization of microorganisms in many fields of microbiology (Peters et al., 2006; Poppert et al., 2002; Thurnheer et al., 2005). In 1989, De Long et al. reported the first in situ hybridization using fluorescently labeled ribosomal RNA-directed oligonucleotide probes. It allowed simultaneous monitoring of different species in the same sample through the use of multiple probes labeled with different fluorescent dyes. it is a safer method, gave better resolution and higher sensitivity. FISH is a rapid method for visualization and identification of bacteria directly in samples of interest and the technique is able to reveal non culturable species (Amann et al., 2001). Sogaard et al. used peptide nucleic acid (PNA) probes for FISH on blood samples in order to rapidly detect a series of infectious bacteria (Sogaard et al., 2005). In a study by Poppert et al. real time PCR and FISH were evaluated as rapid diagnostic techniques for determination of Neisseria meningitides directly in cerebrospinal fluid (Poppert et al., 2002).

5. DNA Microarrays

Bacterial DNA microarrays for clinical microbiology build upon the increasing amount of sequence information available on pathogenic bacteria. Basically DNA microarrays are dot blot hybridization experiments performed in a small and highly parallel format, allowing hybridization to multiple targets in the same assay. The amount of hybridized sample is quantified by signal intensity (Cassone et al., 2007). Korczak et al. in 2005 had surveyed pathotypes of different Escherichia coli strains and found a total of 32 probes that could distinguish the different pathotypes. Kang et al. in 2006 used DNA microarrays and suppression subtractive hybridization to determine the complexity of Salmonella enterica species and define genetic traits that are characteristics for epidemic strains of S. enterica.

6. Ribotyping Or Insertion Sequence Typing Using RFLP

In this method, DNA from the bacterial strain to be identified is isolated and is digested with one or more restriction enzyme. The fragments are separated on a gel by electrophoresis and is transferred to a membrane filters. The filter is incubated with a probe that hybridizes to rRNA genes. So the pattern of bands depending upon the type of restriction enzyme chosen, may be species or strain specific. Only disadvantage of this method is large amount of nucleic acid sample is required.

Identifying Virulence Factors

• Cloning

In this approach gene from the virulent strain of pathogen of interest is cloned into strain of E. coli that is avirulent and look for genes that increases the virulence of E. coli. Another way of identifying virulence factors is cloning and expression of genes responsible for virulence in E. coli and studying the immunological and immunodiagnostic potential of the expressed protein.

• Transposon Mutagenesis

Transposons can be used for insertional mutagenesis to generate a collection of mutants. These transposon also carry along with them a selectable marker, such as antibiotic resistance gene. A transposon integrating in a gene of Salmonella strain responsible for adhesion, so bacterial will no more be able to attach to tissue culture cells. Integrated transposon due to its selectable marker makes the identification of gene very easy. Only problem is that the transposon frequently carry transcriptional terminators and leads to polar mutations i.e., inhibition of expression of downstream genes also.

• Using Transcriptional Fusion Approach

In transcriptional fusion approach, promoter/operator (regulatory region of virulent gene) of a virulent gene is fused to reporter gene like lacZ, uidA, cat etc. Thus the reporter gene is expressed in the condition virulent gene would normally been expressed. It makes the identification of gene and also the conditions of cellular machinery and environmental factors (e.g. temperature dependence) for its expression.

• In vivo Expression Technology (IVET)

It is now possible to identify genes that are expressed by bacteria only in host species using in vivo expression technology, where virulence genes that are transcriptionally induced during infection can be identified (Mahon et al., 1995).It is observed that purine auxotrophs of serovar Salmonella typhimurium is unable to infect mice. Promoter less purine biosynthetic gene is introduced into chromosome of a strain randomly, the collection of fusions is inoculated into an animal and surviving clones are isolated. It is presumed that clones of bacteria which are surviving have expressed a gene particularly in vivo.

• Signature Tagged Mutagenesis

It is also an in vivo form of transposon mutagenesis involving cloning of random oligomers into a transposon. Thus a transposon has a signature tag on to it. These mixture of transposon are transformed into the target bacterial strain and transformations are selected by antibiotic resistance. Now these bacterial isolates are injected into animal for infection development. After development of infection, PCR based amplification is done based on primers which recognize the transposon portion as it is present in all the transformants, and tags are amplified. Now these mixture of tags are used to probe original collection of transposon generated mutants. So any loss of a mutant during the infection process can be known with this technology.

Future Perspectives

Detection of infectious bacteria by use of molecular diagnostics is an emerging technology but still it is not mature enough to have ripened fruits i.e., entire replacement of traditional culture methods. These methods will be automated so that they require little hands-on-time, are easy to use, and will allow detection of several different pathogens in the same analysis. Recombinant DNA technology has rapidly expanded our ability to diagnose disease.The wide application of recombinant DNA diagnostics will depend on simplicity, speed of results, and cost containment.

References

Amann R, Fuchs BM and Behrens S. 2001.The Identification of Microorganisms by Fluorescence In Situ Hybridisation. Current Opinions in Biotechnology. 12:231–236.

Birkenmeyer L and Armstrong AS.1992. Preliminary evaluation of the ligase chain reaction for specific detection of Neisseria gonorrhoeae. Journal of Clinical Microbiology. 3012:3089-3094.

Caskey CT. 1987. Disease Diagnosis by Recombinant DNA Methods. Science.236 4806: 1223-1229

Cassone M, Giordano A and Pozzi G. 2007. Bacterial DNA Microarrays for Clinical Microbiology. The Early Logarithmic Phase. Frontiers in Bioscience. 12:2658–69

Chan CM, Yuen KY, Chan KS, Yam WC, Yim KH. Ng WF and Ng MH. 1996. Single-tube nested PCR in the Diagnosis of Tuberculosis. Journal of Clinical Pathology. 49:290-294

de Abreu FC, Teixeira d FV and Calo RE. 2006. Polymerase Chain Reaction In Comparison with Serological Tests for Early Diagnosis of Human Leptospirosis. Tropical Medicine and International Health. 11:1699–1707

Dille BJ, Butzen C and Birkenmeyer LG. 1993. Amplification of Chlamydia trachomatis DNA by Ligase Chain Reaction. Journal of Clinical Microbiology. 31: 729-731.

Erlich HA, Gelfand D and Sninsky JJ. 1991. Recent advances in the polymerase chain reaction. Science. 252: 1643-1651

Espy MJ, Uhl JR and Sloan LM.2006. Real-time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing. Clinical Microbiology Reviews. 19:165–256

Hillemann D, Weizenegger M, Kubica T, Richter E and Niemann S. 2005. Use of the Genotype MTBDR Assay for Rapid Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Complex Isolates. Journal of Clinical Microbiology.43:3699–3703

Hu H, Elmore K, Facey I and Jenderzak D.1991. Detection of Borrelia burdgoferi by Ligase Chain Reaction. Abstracts of the General Meeting of the American Society for Microbiology. p.79

Iovannisci DM and Winn-Deen ES. 1993. Ligation Amplification and Fluorescence Detection of Mycobacterium tuberculosis DNA. Molecular Cell Probes. 71:35-43

Kang MS, Besser TE, Hancock DD, Porwollik S, McClelland M and Call DR. 2006. Identification of Specific Gene Sequences Conserved in Contemporary Epidemic Strains of Salmonella enterica. Applied Environmental Microbiology. 72:6938–6947

Korczak B, Frey J and Schrenzel J.2005. Use of Diagnostic Microarrays for Determination of Virulence Gene Patterns of Escherichia coli K1, a Major Cause of Neonatal Meningitis. Journal of Clinical Microbiology. 43:1024–1031.

Livermore DM.2006. Can Beta-Lactams be Re-Engineered to Beat MRSA? Clinical Microbiology and Infection.122:11–16.

Mahon MJ, Thones JW, Slauch JM, Hanna PC, Collies RJ and Mekalanos JJ.1995. Antibiotic Based Selection for Bacterial Genes that are Specifically Induced During Infection of Host. Proceedings of the National Academy of Sciences, USA . 12: 221-225

Neonakis IK, Gitti Z, Krambovitis E and Spandidos DA. 2008. Molecular Diagnostic Tools in Mycobacteriology. Journal of Microbiological Methods. 75: 1-11.

Palomino JC. 2005. Nonconventional and New Methods in the Diagnosis of Tuberculosis: Feasibility and Applicability in the Field. The European Respiratory Journal. 26:339–350.

Patel JB.2001. 16S rRNA Gene Sequencing for Bacterial Pathogen Identification in the Clinical Laboratory. Molecular Diagnostics. 6:313–321

Peters RP, Savelkoul PH, Simoons-Smit AM, Danner SA, Vanden-broucke-Grauls C M and van Agtmael M A. 2006. Faster Identification of Pathogens in Positive Blood Cultures by Fluorescence In Situ Hybridization in Routine Practice. Journal of Clinical Microbiology. 44:119–23.

Poppert S, Essig A, Marre R,Wagner M and Horn M.2002 Detection and Differentiation of Chlamydiae by Fluorescence In Situ Hybridization. Applied Environmental Microbiology. 68:4081–9.

Rogall T. Flohr T and Bottger E C.1990. Differentiation of Mycobacterium species by Direct Sequencing of Amplified DNA. Journal of General Microbiology.. 136:1915–20.

Saiki RK, Scharf S and Faloona F. 1985. Enzymatic Amplification of Betaglobin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia. Science.230:1350–4.

Saiki RK, Gelfand DH and Stoffel S.1988. Primer Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase. Science. 239: 481-491.

Sogaard M, Stender H and Schonheyder H C. 2005. Direct Identification of Major Blood Culture Pathogens, Including Pseudomonas aeruginosa and Escherichia coli, By a Panel of Fluorescence In Situ Hybridization Assays Using Peptide Nucleic Acid Probes. Journal of Clinical Microbiology. 43:1947–1949.

Talkington DF, Thacker WL, Keller DW and Jensen J S. 1998. Diagnosis of Mycoplasma pneumoniae Infection in Autopsy and Open-Lung Biopsy Tissues by Nested PCR. Journal of Clinical Microbiology.. 364: 1151-1153

Thurnheer T, Gmur R and Guggenheim B. 2005. Multiplex FISH Analysis of a Six species Bacterial Biofilm. Journal of Microbiological Methods.56:37–47.

Tyagi S and Kramer FR.1996. Molecular beacons: probes that fluoresce upon hybridization. Nature Biotechnology. 14:303–308.

Valasek MA and Repa JJ. 2005. The power of Real-Time PCR. Advances in Physiology Education. 29:151–159.

Wiedmann M, Czajka J, Barany F and Batt C A. 1992. Discrimination of Listeria monocytogenes from other Listeria species by Ligase Chain Reaction. Applied Environmental Microbiology. 58:3443-3447

Woese CR. 1987. Bacterial evolution. Microbiological Reviews. 51: 221-271.

Yang S and Rothman RE. 2004. PCR-based Diagnostics for Infectious Diseases: Uses, Limitations, and Future Applications in Acute-Care Settings. Lancet Infectious Diseases.4:337–348