DNA DIAGNOSTICS

DNA DIAGNOSTICS

DNA diagnostics means qualitative and quantitative analysis of DNA, RNA and proteins. The common techniques used are, for DNA — PCR, RFLP (restriction fragment length polymorphism), electrophoresis, Southern hybridization, cloning, etc; for RNA – reverse transcription, real-time PCR, Northern-blotting, etc; for proteins-PAGE electrophoresis, Western-blotting, monoclonal antibodies, immuno-precipitation, ELISA, liquid chromatography, (HPLC), mass spectroscopy, etc.

The main aims of DNA diagnostics are:

  • Detection of the presence of nucleic acid of specific sequence, e.g. identification of animal species
  • Structural analysis of nucleic acid
  • Determination of genotype, e.g. clinically significant mutations and polymorphisms
  • Quantification of nucleic acid (RNA) of specific sequence. To describe intensity and changes in gene expression in particular tissue (e.g., tumors)
  • Quantification of proteins and types of their post-translational modification

DNA diagnostics helps in the detection of:

  1. Monogenic and polygenic inherited diseases
  2. Tumors
  3. HLA-typization in cases of transplantation
  4. Identification of people in forensic medicine
  5. Disease progress during the therapy

The principle of DNA diagnostics involves the isolation of DNA, then amplification of a DNA region by PCR (other analyses) followed by visualization of results in the gel. The characteristics of DNA diagnostics for detection of polymorphism of the predisposition gene are:

  • Target analyses — In target analyses, the localization and the whole sequence of a gene is known and the mutation of the gene is known. An examination of family members is not needed.
  • Complete analyses — In complete analyses, the localization and the whole sequence of a gene are known but the mutations of the gene are unknown. An examination of family members is necessary.

The main characteristics of a detection system in DNA diagnostics are:

  • Sensitivity—Sensitivity means that the test must be able to detect very small amounts of target even in the presence of other molecules.
  • Specificity—Specificity: the test yields a positive result for the target molecule only
  • Simplicity —Simplicity: the test must be able to run efficiently and inexpensively on a routine basis

DNA diagnostics includes the following systems:

  1. DNA Hybridization
  2. PCR
  3. Restriction endonuclease analysis
  4. RAPD (random amplified polymorphic DNA)
  5. DNA fingerprinting

DIAGNOSIS OF MYCOBACTERIUM TUBERCULOSIS

The genus Mycobacterium is made up of 30 species and many subspecies and it has two groups fast growing and slow growing species. In this genus, all the members are non-motile, non spore forming chemo-heterotrophs that posse’s genome with high G + C contents (55-70%). Mycobacterium tuberculosis causes the disease tuberculosis. Tuberculosis usually attacks the lungs and also affect other parts of the body. It is spread through the air, when people who have the disease cough, sneeze, or spit. It has waxy walls and is slow growing

Symptoms of Tuberculosis

This disease cause fever, chills, night-time sweating, persistant chronic cough, constant tiredness, blood tinged sputum, chest pains, pallor, loss of appetite and weight.

Methods of Diagnosis

  1. Evaluation of clinical symptoms
  2. Microscopic examination of sputum or other body fluid
  3. X- ray detection of chest
  4. Culturing of sputum and other biological fluids
  5. Serological examination
  6. Molecular diagnostics
  7. Detection of significant number of acid-fast bacilli (using Zeihl-Neelsen acid fast stain method) in sputum or tissue samples.
  8. 156110-based diagnostic technique for mycobacterium tuberculosis complex.

Zeihl-Neelsen Acid Fast Stain Method

This is a commonly used diagnostic method for mycobacterium tuberculosis. The procedure is as follows:

  • Place the cells on glass microscopic slide and heat them
  • Flood the slide with carbal fuchsin stain
  • Again heat the slides until it steams
  • Pour off the carbal fuchsin stain
  • Wash the slide thoroughly with water
  • Decolorize with acid-alcohol (5 minutes)
  • Wash the slide again thororughly with water
  • Flood the slide with methylene blue counterstain for one minute.
  • Wash with water
  • Blot excess water and dry in handover Bunsen flame

1S6110-based Diagnostic Technique

The discovery of polymorphic DNA sequence in mycobacterium tuberculosis and the subsequent use of methods based on the insertion sequence IS6110 both for amplification and for typing of isolates of mycobacterium tuberculosis complex have facilitated early diagnosis of tuberculosis and differentiation of strains, respectively.

Restriction fragment length polymorphism (RFLP) and hybridization of DNA extracted from mycobacterium tuberculosis, non-tuberculous mycobacteria and non-bacterial species with a probe derived from IS6110 confirmed that IS6110 was specific for mycobacterium tuberculosis complex. In addition, DNA amplification with IS6110 specific primers yielded 181 bp fragment only in DNA from mycobacterium tuberculosis complex isolates.

Evaluation of Clinical Symptoms

Light microscopy: This is used to examine acid fast-stained sputum or smear.

Culture method: This is the most reliable method for diagnosis of tuberculosis. In this method, primary sputum (culture of Mycobacterium from clinical specimen) is decontaminated by sodium hydroxide and N-acetyl- L- cystein and then plated on to Lowenstein-Jensen medium or Middlebrook 7H11 suplemented with specific antibiotic.

DIAGNOSIS OF TRYPANOSOMA CRUZI

Trypanosoma is a protozoan and belongs to Trypanosomatidae family. This family is made up of large number of widely distributed species in nature which have complex life cycle involving both vertebrate and invertebrate hosts. T. cruzi causes American trypanosomiasis (also called chagas disease) which is spread by insects.

Symptoms of Chagas Disease

Chagas disease produce non specific symptoms and the parasites invade spleen, liver, lymph nodes and central nervous system (CNS) where they multiply and destroy infected cells.

Methods for Diagnosis

  1. Microscopic examination: This method involves the detection of parasite in the fresh blood or stained smear under the microscope.

Advantage—It is easy to perform and require less time.

Disadvantage —It is not accurate always sometimes fail to detect the parasite in infected patient.

  1. Xeno-diagnosis: This is also called gold standard detection method. In this method, uninfected insects are fed upon patient’s blood. Then the intestinal contents of this insect are examined microscopically after 30/F_24-640 days for the presence of parasite.

Advantage—It gives accurate detection.

Disadvantage—It is very laborious, time consuming and costly.

  1. Serological detection

(a) Indirect Immuno-fluorescence Test: It is used for the detection of chronic Chagas disease. Infected patient have detectable level of specific antibodies that bind to the parasitic antigens.

Advantage- It is easy and can detect low level of parasitic antigen.

Disadvantage- Occurrence of false positive reaction.

(b) Anti-T.cruzi IgM Assay: This assay detects the presence of IgM antibodies produced against T. cruzi infection.

Advantage- It is easy to perform and require less time.

Disadvantage:

  1. It is not widely available.
  2. It fails to discriminate between current and. past infection.
  3. PCR based diagnosis: PCR amplification method can be used as one of the best detection method for T. cruzi because it has highly repetitive nuclear and minicircle DNA sequences.
  4. 188 bp DNA fragment present in the multiple copies in the genome of T.cruzi that is usually absent from the genome of other related parasites.
  5. The relative hybridization locations of TCZ1 and TCZ primers which gives an amplification product of 188 bp are shown in Fig.1.
  6. T. cruzi contain a giant mitochondrion (also called kinetoplast) in its cytoplasm which is located adjacent to the basal body of flagellum. All species of T. cruzi contain 14 copies of a highly conserved 12 nucleotide sequence of KDNA. This kinetoplast contain interwined circular DNA molecules called maxicircles and minicircles which make 510% of the total DNA (Kinetoplast DNA or KDNA).

(a) Maxicircles:

  • Homogenous in size
  • 20 to 40 Kb in length
  • 30-50 copies are present in each organism
  • Function is to encode mitochondrial proteins

(b) Minicircles

  • Heterogenous in size
  • Vary in length 1-3 Kb
  • 10000-30000 copies are present in each organism
  • Function to encode guide RNAs participate in RNA editing of maxicircle transcripts
  • Minicrcle sequence is flanked by species-specific conserved regions

Questions

1. Define diagnostic immunology.
2. Which tests are used in immunodiagnostics?
3. Define probe. What are the functions of a probe?
4. Give some examples of immunological techniques used in immunodiagnostics.
5. Infection by trypanosome cruzi causes which disease?
6. What are the symptoms of Chagas disease?
7. Discuss the different methods for the diagnosis of trypanosome cruzi.
8. What are the advantages of microscopic examination of trypanosome cruzi?
9. Which serological test is used for the detection of Chagas disesase and what are its advantages?
10. Discuss the anti- trypanosome cruzi lgM assay and its advantages.
11. Explain the PCR based diagnosis of trypanosome cruzi.
12. What are maxicircles and minicircles?