Detection of Human Cytomegalovirus (HCMV) and Porcine Endogenous Retrovirus (PERV) with One Step Extraction Method

Jung Heon Kim; Eun-Suk Jung; Eung-Soo Hwang

doi:10.3947/ic.2010.42.1.12

Original Article

Infect Chemother 2010;42(1):12-16.

Published online: 28 February 2010

DOI: https://doi.org/10.3947/ic.2010.42.1.12

Detection of Human Cytomegalovirus (HCMV) and Porcine Endogenous Retrovirus (PERV) with One Step Extraction Method

Jung Heon Kim^1,³, Eun-Suk Jung^1,³, Eung-Soo Hwang^1,^2,³

¹Departments of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, Korea

²BK21 Division of Human Life Science, Seoul National University College of Medicine, Seoul, Korea

³Institute of Endemic Disease, Seoul National University Medical Research Center, Seoul, Korea

Corresponding Author: Eung-Soo Hwang, M.D. Department of Microbiology and Immunology, Seoul National University College of Medicine, 28 Yeongeon-dong, Jongro-gu, Seoul 110-799, Korea Tel: +82-2-740-8307, Fax: +82-2-743-0881 E-mail: hesss@snu.ac.kr

Received 3 September 2009 Accepted 8 January 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

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Keywords: Porcine endogenous retrovirus, Human Cytomegalovirus, Transplantation, One step extraction method, GeneReleaser

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

^* Extraction of DNA from 2x105 FLF infected with HCMV.

The value was expressed as mean±standard deviation.

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 2.

Standard Curve Equation of Serially Diluted FLF Infected with HCMV

Slope	Y-Intercept	r²
-3.68265±0.195772	39.47974±1.336752	0.993185±0.008655

The value was expressed as mean±standard deviation.

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