PCR and Diagnostics

1. PCR and Diagnostics • Unique sequences of nucleotides if detectable can be used as definitive diagnostic determinants • NA hybridisation is the basis for rapid reliable assays • Used to use Southerns • PCR provides mechanism to obtain sufficient DNA and a mechanism to “pull” specific DNA if present

2. Points of Hybridisation Scheme • Sufficient single stranded DNA • Appropriate unique single stranded probe • Known conditions for binding of the probe to unique complimentary sequence in template • Method to detect

3. PCR provides a method to get sufficient DNA • PCR can be used to generate probe or can be used instead of SB to “pull “ specific sequence • Like SBlot need to know conditions of binding • With PCR can do end point detection or RT-PCR • PCR is faster and simpler than SB

4. Effective Diagnostic Test Criteria • Sensitive • Specific • Rapid • Technically simple

5. PCR has made major contributions in • Diagnosis of microbes • Diagnosis of genetic disease • Prenatal diagnosis

6. PCR in Microbial diagnostics • PCR used to amplify DNA of microbe so more easily detected • Viruses commonly detected this way • Hospitals slow to adapt but beginning to, Public health labs already doing it • Methods employed include • 1. Southern blot • 2. Elisa style • 3. PCR presence or absence

7. PCR in Microbial diagnostics • Careful primer design can allow different species to be distinguished • Sensitive- can have very little DNA present and still detect • Rapid • Bench work involved is technically simple • Diagnostic kits available for common infections, already in doctors office

8. Standard procedure 1. Collect sample 2. Cultivate org. 3. Identify Time consuming. May not be able to cultivate PCR based procedure Collect sample Isolate DNA and PCR using path specific primers Analyse Fast; can be perfomed in one day or less, very sensitive Infection with pathogenic microbe

9. PCR, using primers specific for a 142 bp fragment of the mycobacterium 65 kDa antigen gene. Following PCR, the reaction was cleaned up and digested with HhaI (H) or BstUI (B) restriction enzymes. The restriction pattern is indicative of the species M. tuberculosis. M: markers.

10. PCR based diagnostics lend themselves to multiplexing- search for several different organisms at the same time Useful for detecting organisms that are difficult to grow Can get information concerning viral loading using quantification methods Can only detect antibiotic resistance if sequence is known unlike on culture plates where can test organism using multiple antibiotic discs.