Molecular Diagnosis PDF

VMA 5114 Principles of Infectious Diseases Molecular Diagnostic Techniques  Learning Objectives Be aware of electrophoresis Understand restriction fragment length polymorphism (RFLP) Define hybridization & probe Comprehend nucleic acid amplification Describ...

VMA 5114 Principles of Infectious Diseases Molecular Diagnostic Techniques  Learning Objectives Be aware of electrophoresis Understand restriction fragment length polymorphism (RFLP) Define hybridization & probe Comprehend nucleic acid amplification Describe protein detection  Diagnostic techniques  Direct examination  Culture  Immunologic methods  Molecular analysis  Molecular diagnosis DNA, RNA & proteins can be used to help identify the pathogenic agents Sensitivity – true positive rate Specificity – true negative rate Reduction in dependency on culture Safe  Molecular diagnosis  Electrophoresis  Restriction fragment length polymorphism (RFLP)  Hybridization  Nuclear acid amplification – target  Protein detection  Electrophoresis Separated in an electrophoretic field Nega vely charged molecules → posi ve end Mobility – size: the smaller, the faster – structure: supercoiled>linear>nicked circles (DNA) - http://arbl.cvmbs.colostate.edu/hbooks/genetics /biotech/gels/agardna.html + Rotavirus strains detected by SDS-PAGE L 1 & 2, long-profile strains L 3, short-profile strain (SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis) Urbina D et al., 2004 Int Microbiol 7:113  Restriction fragment length polymorphism (RFLP) Analyzing differences among homologous DNA sequences by restriction enzymes Restriction enzymes: cut DNA at the specific recognition nucleotide sequences Sequence specific  Restriction enzymes EcoRI – sticky end 5’ GAATTC 3’ 5’G AATTC 3’ 3’ CTTAAG 5’ 3’CTTAA G 5’ 5' overhang Over 3000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially  Restriction enzymes SmaI – blunt end CCCGGG CCC GGG GGGCCC GGG CCC Mixed Cryptosporidium spp. infections Cama V et al., 2006 EID 12:1025  Forensic analysis VSS E E An example of a RFLP analysis performed for forensic purposes. Lanes 1,2,7,11 and 15 have molecular weight markers. Evidence is in lanes 9 and 12. The victim's DNA is in lane 4 and suspects' in 5 and 6 (Stokely 1997). MM M M M http://www.bio.davidson.edu/courses/molbio/molstudents/01anford/molecularpaper1.html Police used consumer genealogical websites to identify Golden State Killer suspect Parenthood identification http://www.cas.miamioh.edu/~wilsonkg/old/gene2005/group1/wholednaproject.htm  Hybridization Hybridization Denatured, single-stranded DNA, i.e., probe binds to a complementary single-stranded sequences – Dot, in situ, Southern, Northern, microarray  Hybridization Probe – fragment of nucleic acids – labeled using radioisotope, enzyme, or chemiluminescence – detecting complementary sequences in the samples – high degree of specificity – various in size Chikungunya virus RNA detected in the human patient acute phase serum and CSF samples Kumar JS et al., Indian J Med Res 138, 2013, pp 117  Nucleic acid amplification  Target amplification – enzyme-mediated process to synthesize copies of targeted nucleic acid Polymerase chain reaction (PCR) Isothermal amplification, such as LAMP Highly sensitive False positive  Products: 2n; n – cycles 10 cycles: 1,024 20 cycles: 1,048,576 30 cycles: 1,073,741,824 http://www.aaranyak.org/pcr.html  PCR Primers single-stranded DNA fragments, complementary to sequences flanking the region to be amplified. The distance between the primer binding sites determines the size of the PCR product. Determine the specificity.  Features of Primers Types of primers – Random – Specific Primers – Product size – Annealing temperature – Specificity Nucleotide composition Thermostable Polymerases – FYI Polymerase T ½, Extension Type of Source 95oC Rate (nt/sec) ends Taq pol 40 min 75 3’A T. aquaticus Amplitaq 80 min >50 3’A T. aquaticus (Stoffel fragment) Vent* 400 min >80 95% Thermococcus blunt litoralis Deep Vent* 1380 min ? 95% Pyrococcus blunt GB-D Pfu >120 min 60 Blunt Pyrococcus furiosus Tth* 20 min >33 3’A T. (RT activity) thermophilus *Have proof-reading functions and can generate products over 30 kbp PCR diagnosis of T. foetus in WY cattle  PCR variations Reverse-transcriptase PCR Nested PCR Multiplex PCR Quantitative or real-time PCR More  Real-Time or Quantitative PCR (qPCR) Probe or dye to generate a fluorescent signal from the product. Signal in real time allows quantification of starting material. Signal – an exponential curve with a lag phase, a log phase, and a stationary phase. Lag phase is inversely proportional to the amount of starting material. Quantitative PCR (qPCR) Threshold fluorescence level Threshold cycles for each sample  LAMP Pros: No thermal cycler needed Quick – 1h Sensitivity  PCR Visible results Cons Design of primer sets complicate Diagnosis of T. tenax by LAMP & PCR Matthew MA Microorganisms 2022, 10:594  Protein detection Western blot diagnosis of equine protozoal myeloencephalitis (EPM) Hoane JS et al., 2005 10.1128/CDLI.12.9.1050-1056.2005