- Genotyping of chlamydial species by Omp-1 gene restriction analysis:

  C. trachomatis is classified in 18 serovars: A, B, Ba, C, D, E, F, G, H, I; Ia, J, K, L1, L2, L2a y L3. This classification is based on immunoepitope analysis of the major outer membrane protein (MOMP), with polyclonal and monoclonal antibodies. The MOMP is the immunodominat antigen and contains four variable domains (VDs) that are flanked and interespaced by five constant domains. Three of the VD (VD1, VD2 and VD4) are surface exposed and contain antigenic epitopes that are going to be serotyping targets.
  The need of multiple passages in cell culture and a large panel of monoclonal antibodies are major drawbacks of MOMP serotyping, therefore, the RFLP (Restriction Fragment Length Polymorphism) of the previously amplified Omp-1 gene, where the MOMP are encoded, is simple, fast  and a powerful tool in epidemiological studies. This method enables the differentiation of not only all known serovars and serovariants but also genovariants.
  First of all, the Omp-1 gene is amplified by PCR (polymerase chain reaction). Then, the obtained product is digested by restriction enzymes. Each kind of Chlamydia will give a characteristic band pattern when the electroforetic migration is made.
  Here you can see a typical protocol of chlamydial genotyping:

- Materials y Methods:

     Chlamydia strains and clinical isolates.

     PCR pretreatment methods: simple ten minutes boiling or transfer the samples to 1,5ml Eppendorf tubes. Complete to 1,5ml with sterile PBS. Include a negative control sample. Centrifuge at 13000 RPM, 4°C, 1h 30 on a microcentrifuge. Remove the supernatants. Add 130ml of lysis buffer ( Tris-HCl: pH8 10mM, MgCl2 2,5 mM, KCl 50 mM, Tween 20  0,5% ) /tube: 125ml of buffer + 5ml of proteinase K. Vortex to homogenize the pellets. Incubate at 55-60°C, 1h on a thermostated water-bath then at 100°C, 10 min. to denature the proteinase K. Keep lysates at -20°C.

     DNA amplification ( PCR ): place 34ml H2O, 5ml buffer 10X, 1ml dNTP 10mM, 1,5ml of each primer, 2ml MgCl2 ( 25mM ), 0,4ml taq polimerase ( 5U/ml ) per tube. Then add 10ml of the sample to amplificate. The primers SERO 1A ( sense 5'-atgaaaaaactcttgaaatcgg- 3' ) and SERO 2A ( antisense 5'-tttctagat/cttcatt/cttgtt- 3' ) are used. The obtained PCR products ( 10ml each one ) are analysed by electroforesis on 1,5% agarose gel. Migration conditions: 40mV, 1h, buffer TBE 0,5X. After the migration, stain the agarose gel in ethidium bromide and observe it on a UV transilluminator.

       RFLP ( Restriction Fragment Length Polymorphism ): place 2ml buffer 10X ( buffer TBE 10X: Tris 162gr., boric acid 50gr., EDTA Na2.2H2O  9,5gr., H2O csp ), 12,5 ml H2O and 0,5 ml of Alu I per tube. Then add 5ml of the PCR product and incubate 1,30 h. 37°C. Centrifuge the tubes rapidly and add 3ml of Blue. Place 22ml of the digested samples in the well os a 12% vertical polyacrylamide gel. Migration conditions: 10mA for 1 gel, 20mA for 2 gels, 3h, in TBE 1X. At the end of the migration, stain the gel with 4,5ml of ethidium bromide in water 10 minutes. Observate it on UV transilluminator.