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Abstract
Background
Xenotransplantation is thought to be one of the alternative methods to overcome the shortage of human organs for transplantation. Recipients should be immunosuppressed for graft survival, and thus, there is a need for developing diagnostic modality that can detect diverse infections originating from animals and recipients rapidly, in the early stage, and with high sensitivity using small volume of samples. This study was carried out to develop a fast, simple, and robust technique for the preparation of HCMV DNA and PERV RNA using small volume of samples.
Materials and Methods
Nucleic acids were extracted from serially diluted samples with one step extraction method as well as with Qiagen kit. The presence of genomic DNA of human cytomegalovirus (HCMV) and porcine endogenous retrovirus (PERV) was detected by PCR and specific primer set, respectively. RNA of HCMV and PERV was extracted and then detected by RT-PCR and specific primer set, respectively. For absolute quantification of HCMV, standard curve was established by real time PCR.
Results
HCMV DNA and PERV RNA were prepared from culture supernatant and cells for PCR or RT-PCR with one step extraction method. It was possible to extract both the DNA and RNA from the samples in about 20 minutes with one step extraction method in a single tube. HCMV and PERV could also be detected by PCR and one step extraction method, respectively. It was also good with small quantity samples.
Conclusions
One step extraction method is simpler and faster method than other extraction methods when there are two types of DNA and RNA viruses in one sample. From these results, we could see that the one step extraction method could be very useful in detecting HCMV and PERV rapidly from the pig cells or organ transplanted recipients with a small amount of sample.
References
1. Sachs DH, Sykes M, Robson SC, Cooper DK. Xenotransplantation. Adv Immunol. 2001; 79:129–223.
2. Denner J. Recombinant porcine endogenous retroviruses (PERV-A/C): a new risk for xenotransplantation? Arch Virol. 2008; 153:1421–1426.
3. Le Tissier P, Stoye JP, Takeuchi Y, Patience C, Weiss RA. Two sets of human-tropic pig retrovirus. Nature. 1997; 389:681–682.
4. Patience C, Switzer WM, Takeuchi Y, Griffiths DJ, Goward ME, Heneine W, Stoye JP, Weiss RA. Multiple groups of novel retroviral genomes in pigs and related species. J Virol. 2001; 75:2771–2775.
5. Ericsson T, Oldmixon B, Blomberg J, Rosa M, Patience C, Andersson G. Identification of novel porcine endogenous betaretrovirus sequences in miniature swine. J Virol. 2001; 75:2765–2770.
6. Mang R, Maas J, Chen X, Goudsmit J, van Der Kuyl AC. Identification of a novel type C porcine endogenous retrovirus: evidence that copy number of endogenous retroviruses increases during host inbreeding. J Gen Virol. 2001; 82:1829–1834.
7. Denner J. Immunosuppression by retroviruses: implications for xenotransplantation. Ann N Y Acad Sci. 1998; 862:75–86.
8. Aoe M, Tao H, Yamane M, Toyooka S, Sano Y, Okazaki M, Date H. Cytomegalovirus infection in lung transplantation; current status, detection, prophylactic treatment. Kyobu Geka. 2007; 60:988–992.
9. Jeoseph S, David WR. In: Molecular Cloning: A Laboratory Manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press; 2001.
10. Gault E, Michel Y, Dehée A, Belabani C, Nicolas JC, Garbarg-Chenon A. Quantification of human cytomegalovirus DNA by real-time PCR. J Clin Microbiol. 2001; 39:772–775.
11. Machida U, Kami M, Fukui T, Kazuyama Y, Kinoshita M, Tanaka Y, Kanda Y, Ogawa S, Honda H, Chiba S, Mitani K, Muto Y, Osumi K, Kimura S, Hirai H. Real-time automated PCR for early diagnosis and monitoring of cytomegalovirus infection after bone marrow transplantation. J Clin Microbiol. 2000; 38:2536–2542.
12. Ahumada-Ruiz S, Taylor-Castillo L, Visoná K, Luftig RB, Herrero-Uribe L. Determination of human cytomegalovirus genetic diversity in different patient populations in Costa Rica. Rev Inst Med Trop Sao Paulo. 2004; 46:87–92.
13. Kubar A, Saygun I, Yapar M, Ozdemir A, Slots J. Real-time PCR quantification of cytomegalovirus in aggressive periodontitis lesions using TaqMan technology. J Periodontal Res. 2004; 39:81–86.
14. Mengelle C, Sandres-Sauné K, Pasquier C, Rostaing L, Mansuy JM, Marty M, Da Silva I, Attal M, Massip P, Izopet J. Automated extraction and quantification of human cytomegalovirus DNA in whole blood by real-time PCR assay. J Clin Microbiol. 2003; 41:3840–3845.
15. Rhinow K, Schmidt-Westhausen AM, Ellerbrok H, Pauli G, Schetelig J, Siegert W. Quantitative determination of CMV-DNA in saliva of patients with bone marrow and stem cell transplantation using TaqMan-PCR. Mund Kiefer Gesichtschir. 2003; 7:361–364.
Figure 1.
Detection of HCMV DNA for US17 from supernatant of HCMV-infected FLF. The samples were diluted with 10 fold from 1 to 10-3 and extracted with phenol and one step extraction methods, and used as templates of PCR. Lane M represents the molecular marker. Lane N represents the PCR results in the absence of templates. The other lanes represent the diluted samples.
Figure 2.
Detection of PERV DNA for gag sequence from PK-15. The samples were diluted with 10 fold from 1 to 10-5, extracted with phenol and one step extraction methods, and used as templates of PCR. Lane M represents the molecular marker. Lane P represents the positive DNA control. Lane N represents the PCR results in the absence of templates. The other lanes represent .the various diluted samples.
Figure 3.
Detection of HCMV DNA for US17 from HCMV-infected fibroblast, diluted with 10 fold from 1 to 10-4, extracted with one step and Qiagen methods, and used as templates of PCR. Lane M represents the molecular marker. Lane N represents the PCR results in the absence of templates. The other lanes represent the various diluted samples.
Figure 4.
RT-PCR analysis of HCMV infected FLF for HCMV RNA IE1 sequences. RNA of HCMV-infected fibroblast diluted with 10 fold from 1 to 10-4, extracted with one step and Qiagen methods and used as templates of RT-PCR. Then RT-PCR products were used as templates of PCR. Lane M represents the molecular marker. Lane N represents the RT-PCR results in the absence of templates. The other lanes represent the various diluted samples.
Figure 5.
RT-PCR analysis of culture supernatant from PK15 for PERV RNA gag sequences. RNA of PK15 supernatant diluted with 10 fold from 1 to 10-4, extracted with one step and Qiagen methods and used as templates of RT-PCR. Then RT-PCR products were used as templates of PCR. Lane M represents the molecular marker. Lane N represents the RT-PCR results in the absence of templates. The other lanes represent the various diluted samples.
Figure 6.
Establishment of quantitative real time PCR. (A) represent a standard curve of serial diluted DNA from HCMV-infected FLF. CT values were expressed in arbitrary units. Logarithms (base 10) were plotted against crossing points. (B) represent a amplification plot.
Table 1.
Comparison of the Spectrophotometry Results between One Step and Phenol Extraction*
| Method | Phenol extraction | One step extraction |
|---|---|---|
| Sample volume | 20 μL | 20 μL |
| 260/280 nm | 1.815±0.092 | 1.802±0.011 |
| Total amount of DNA (μg) | 1.705±0.115 | 1.868±0.031 |
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