PCR Lyme Disease (DNA extraction protocol from serum)

Edmond Godfroid godfroid at sga.ulb.ac.be
Thu Mar 28 05:55:39 EST 1996


In article <4j6124$7ek at morgana.mat.uc.pt>,
   psantos at gemini.ci.uc.pt (Paulo Santos) wrote:
>
>Does anyone use/know a good protocol for extraction of DNA from serum for PCR 
>in order to amplify Borrelia (Lyme disease)?
>
>A quick answer will be highly apreciated.
>
>Thank you in advance.
>
>
>
>Paulo Santos
>
>LUSOTRANSPLANTE                                Fax      +351-39-33674
>Centro de Histocompatibilidade do Centro       Telefone +351-39-33693
>Apartado 3004                                           +351-39-20527
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Dear Colleague,

We developed a PCR assay specific to B. burgdorferi sensu lato species. Oligonucleotide
primers based on Borrelia burgdorferi sensu lato ospA gene sequences was designed for use
in the PCR to type all (SL primers) or each (GI to GIII primers) of the B. burgdorferi sensu
lato genospecies involved in Lyme disease. These genospecies-specific primers were routinely
used in PCR assay on biological fluids (including serum) from Lyme patients. We prepared serum
as followed :

1. Add 1.5 ml of refrigerated biological fluid at 4 °C in microcentrifuge Eppendorf tube.

2. Centrifuge at 4°C for 10 min. at 12,000 g to pellet spirochetes.

3. Discard the supernatant by aspiration.

4. Add again 1.5 ml of clinical sample in the Eppendorf tube and repeat steps 2 and 3.

5. Wash the pellet with 1.5 ml of PBS.

6. Vortex and centrifuge for 10 min. at 12,000 g at 4 °C and discard the supernatant by aspiration.

7. Repeat steps 5 and 6 twice.

8. Add 30 µl of steril water, vortex to resuspend the pellet and add a drop of mineral oil.

9. Incubate for 10 min. at 100 °C in a water bath.

10. Store the prepared sample at 4°C until use.

This protocol gave us good results published in JCM vol. 33 (March 1995), p 602-608.

To improve the specificity and the sensitivity of our PCR assay and to simplify detection procedures of DNA
fragments from polymerase chain reaction , we have also developed a solid phase sandwich hybridization
system which is now manufactured by the Biocode company (Tel : 3241 / 52.26.36; Fax : 3241 / 52.51.96; Belgium). 
This format was used for the identification of Borrelia burgdorferi sensu lato, the causal agent of Lyme disease.
The system relied on the use of a specific capture probe covalently linked to polystyrene plates and
a specific polybiotinylated detection probe.  DNA fragments, resulting from polymerase chain reaction,
sandwiched between these two probes, were detected via enzymatic color development.

The new detection format outperformed agarose gel electrophoresis of PCR products in sensitivity and specificity.
Moreover, in view of its rapidity and simplicity, the system proved appropriate for the routine diagnostic analysis
of clinical specimens from Lyme disease patients.

Best regards,


Dr. Edmond GODFROID




Edmond Godfroid                godfroid at sga.ulb.ac.be

Universite Libre de Bruxelles   Secretariat Tel : +32-(0)67-88.94.61
Service de Genetique Appliquee                    +32-(0)2-650.94.61
Rue de l'industrie, 24                      Fax : +32-(0)67-88.94.77
B-1400 Nivelles                           Email : genapp at sga.ulb.ac.be



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