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Immuno-capture PCR for detection of Aeromonas hydrophila
Journal of Microbiological Methods 49 (2002) 335 – 338 www.elsevier.com/locate/jmicmeth
Note
Immuno-capture PCR for detection of Aeromonas hydrophila Xuanxian Peng*, Jianying Zhang, Sanying Wang, Zhilin Lin, Weiyi Zhang Department of Biology and the Key Laboratory of Education Ministry for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen 361005, PR China Received 12 June 2001; received in revised form 6 November 2001; accepted 17 December 2001
Abstract In this report, we describe the use of universal primer PCR (UPPCR) for the detection of 16S ribosomal RNA (rRNA) genes from Aeromonas hydrophila captured by anti-A. hydrophila antibody coupled to a microplate. The approach combining immuno-capture with UPPCR provides a quick, sensitive, and reproducible way for the detection of bacterial cells. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Aeromonas hydrophila; Identification of bacteria; Immuno-capture; Universal primer PCR
PCR technologies in detection of bacteria may be divided into two types according to their primers. One is specific primer PCR (SPPCR) with a pair of specific primers for a target pathogen (Reischl et al., 2000); the other is universal primer PCR (UPPCR) with universal primers for almost all bacteria (Bernhard and Field, 2000; Osorio et al., 1999; Peng et al., 2000a,b). Determination of the identity of bacteria in a given sample by SPPCR could be very time consuming because numerous PCR reactions with different primers may be needed. The approach based on the utilization of UPPCR from conserved regions such as 16S ribosomal RNA (rRNA) gene can be used to amplify almost all of the bacteria (Osorio et al., 1999; Marchesi et al., 1998), but the organism being analyzed should be characterized further by subsequent steps including restriction fragment length polymor-
*
Corresponding author. Tel.: +86-592-2183805; fax: +86-5922181015. E-mail address: [email protected] (X. Peng).
phism (RFLP), single-strand conformation polymorphism (SSCP) or sequence analysis (Greisen et al., 1994; Peng et al., 2000a,b). Hence, pathogens detected by either SPPCR or UPPCR cannot usually be completed by a single PCR reaction. In this report, we have combined immuno-capture and UPPCR and developed a quick method of high sensitivity for detecting pathogenic bacteria. We termed this new method as immuno-capture UPPCR (iUPPCR). Four strains of Aeromonas hydrophila were used in this study. Strains 1 and 2 were purchased from the Institute of Hydrobiology (Chinese Academy of Sciences, Wuhan), and strain PPD 134/91 and strain PDD 35/85 were kindly provided by professor Sin Y.M., Biological Department, the National University of Singapore, Singapore. Cultures were grown for 18 h at 37 C in Luria – Bertani Medium (approximate 109 – 1010/ml) and harvested by centrifugation at 3000g/ min for 20 min. The bacteria preparation was diluted with distilled water to different concentrations as inicated in text before UPPCR analysis. Part of strain 1 cultures was diluted with 0.85% saline. Then, form-
0167-7012/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 7 0 1 2 ( 0 2 ) 0 0 0 1 0 - 6
336
X. Peng et al. / Journal of Microbiological Methods 49 (2002) 335–338
aldehyde was added to 1% of the final concentration to inactivate the bacteria. The inactive bacteria were diluted to 9108/ml after rinsing with sterile saline three times, and injected into mice dorsum at 0.1 ml/ animal by subcutaneous injection (four mice were immunized). One week later, the primed mice were boosted once a week till the agglutination titer reached over 1:2000. We collected sera, and purified the antibody using DEAE-32 column (Peng and Fang, 1988). iUPPCR was made up of immuno-capture and UPPCR procedures. In the former, microplate wells were coated overnight at 4 C with 50 ml of the purified mouse antibody (10 mg/ml) against A. hydrophila. Wells were then washed three times with PBS-T buffer (PBS with 0.1% Tween-20), prior to a 1-h blocking step with 10% BSA at 37 C. Twenty microliters of bacterial suspension was added to each well and incubated for 1 h at 37 C. Plates were washed five times as above, prior to adding 20 ml of sterile distilled water into each well, and were incubated in a boiling water bath for denaturation for 10 min. Eighteen microliters of the suspension was used as the template for PCR amplification. In the latter, the primers were designed against the conserved regions of 16S rRNA sequence to amplify the target sequences of expected sites of 909 –1410 bp. The primer sequences were 5V-AAACTCAAAGGAATTGAC-3V for the forward, and 5V-GACGGGCGGTGTGTA CAA-3Vfor the reverse. PCR was carried out in a 25 ml volume of reaction mixture containing 2.5 ml of 10PCR buffer, 3 ml of MgCl2 (25 mM), 0.5 ml of dNTPs (10 mM for each), 0.5 ml of each primer (24 mM), 0.1 ml of Taq polymerase (5 U/ml) and 18 ml of template to perform 40 cycles consisting of 95 C for 1 min, 55 C for 1 min and 70 C for 2.5 min. The amplified products were resolved on 2.5% agarose gels followed by staining with ethidium bromide. For comparative purposes, heat-denatured UPPCR was carried out in this study. In this approach, the cells were incubated in a boiling water bath for denaturation for 10 min. After boiling, 18 ml of the suspension was used as the template of UPPCR amplification. Analyses of differences between groups were tested for significance using Chi-square test with Yates’s continuity for small numbers, and two significant levels (0.05 and 0.01) were used. We validated the specificity of iUPPCR by three procedures. Firstly, bacterial suspension was pre-incu-
bated with the antibody before adding the cells into the wells that were coated with the same antibody. The pre-incubation was to compete for bacterial cells and with those might absorb into the coated antibody and to preclude cell trapping. We used the pair of primers to amplify the four strains of A. hydrophila. All of them showed the expected DNA fragments of about 500 bp. However, all of the positive samples became negative after their absorption with the three kinds of concentrations of antibodies (50, 100, 200 mg/ml of mouse IgG against A. hydrophila). Secondly, mouse anti-A. hydrophila was superseded with goat IgG against human g chain or pre-immune IgG as coated antibodies for controls, no specific products were detected. Thirdly, as the amplified DNA contains a readily recognizable pattern by HaeIII digestion, HaeIII was used to check specificity. Fig. 1 showed HeaIII restriction mapping and electrophoresis analy-
Fig. 1. Restriction mapping and electrophoresis analyses of representative iUP-PCR products. M, PGEM-7zf(+) HaeIII marker; lane 1, The restriction mapping (HaeIII) of iUP-PCR product; lane 2, iUP-PCR product (500bp).
X. Peng et al. / Journal of Microbiological Methods 49 (2002) 335–338 Table 1 Comparison of iUPPCR with heat-denatured PCR to the detection of 1, 5 and 10 CFU of A. hydrophila Positive samples/total samples (%)
Immuno-capture UPPCR Heat-denatured UPPCR
10 CFU
5 CFU
1 CFU
10/10 (100)
10/10 (100)*
4/10 (40)*
10/10 (100)
3/10 (100)
0/10 (0)
*P<0.01.
ses of representative iUPPCR products. M, lanes 1 and 2 represented PGEM-7zf(+) HaeIII marker, restriction mapping and iUPPCR product (500 bp), respectively. We evaluated the sensitivity of iUPPCR by two methods. First, the bacteria (strain 1) were diluted to 5, 10, 50, 500, 5000 CFU per 20 ml. Bacteria numbers were counted under a light microscope and then confirmed by growing on an agar plate. iUPPCR and heat-denatured UPPCR were carried out with the diluted bacteria samples. Both iUPPCR and heat-denatured UPPCR detected bacteria in the samples with bacterial concentration at and above 10 CFU. However, difference between iUPPCR and heat-denatured UPPCR appeared when five CFU samples were used. All five CFU samples were positive by iUPPCR but only 30% showed positive using heat-denatured UPPCR ( P<0.01). In the second experiment, 20 CFU in 360 ml were divided into 20 wells and then analyzed by the two methods. Four of the ten samples were positive by iUPPCR, while none of the 10 samples was positive by heat-denatured UPPCR. The results were summarized in Table 1. The reproducibility of iUPPCR was confirmed by six independent experiments (data not shown). Thus, iUPPCR is a high sensitive method in the detection of bacteria. To determine whether iUPPCR was suitable to the application of environmental bacteria, water sample with pH 6 was obtained from Furong Lake (a fishpond) in Xiamen University. Aliquots of the water were left unadjusted or adjusted to pH 7, 8, 9, or 10 before adding strain 1 to a final concentration at 10 CFU per 20 ml. iUPPCR detected bacteria in all samples that had bacteria, while control samples without cells all turned negative.
337
The iUPPCR developed in the present study is different from PCR-based detection method previously developed by others and ourselves. IC– PCR or iRT – PCR of virus detection requires gene specific primers (Harper et al., 1999; Rowhani et al., 1997; Peng et al., 1997, 2001; Rodoni et al., 1999), while iUPPCR utilizes universal primers. A single step immuno-capture in iUPPCR avoided complicated RFLP, SSCP or sequence analysis in the identification of pathogens. Most importantly, an affinity purification of bacteria enhances not only the sensitivity of the method but also the accuracy of PCR reaction because of the removal of interfering materials. In summary, we have developed a quick and sensitive method in detection pathogens.
Acknowledgements We are very grateful to Dr. Jiahuai Han and Dr. Shengcai Lin for critical comments on this manuscript. This work was sponsored by grants from the State Foundation of China for Natural Science (No. 39770585), the Foundation of Chinese Education Ministry for Excellent Young Teachers and IFS (A/ 2338-2).
References Bernhard, A.E., Field, K.G., 2000. Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes. Appl. Environ. Microbiol. 66, 1587 – 1594. Greisen, K., Loeffelholz, M., Purohit, A., Leong, D., 1994. PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacterial, including bacteria found in cerebrospinal fluid. J. Clin. Microbiol. 32, 335 – 351. Harper, G., Dahal, G., Thottappilly, G., Hull, R., 1999. Detection of episomal banana streak badnavirus by IC – PCR. J. Virol. Methods 79, 1 – 8. Marchesi, J.R., Sato, T., Weightman, A.J., Martin, T.A., Fry, J.C., Hiom, S.J., Dymock, D., Wade, W.G., 1998. Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA. Appl. Environ. Microbiol. 64, 795 – 799. Osorio, C.R., Collins, M.D., Toranzo, A.E., Barja, J.L., Romalde, J.L., 1999. 16S rRNA gene sequence analysis of photobacterium damselae and nested PCR method for rapid detection of the causative agent of fish pasteurellosis. Appl. Environ. Microbiol. 65, 2942 – 2946. Peng, X.X., Fang, L., 1988. HBsAg specific circulating immune
338
X. Peng et al. / Journal of Microbiological Methods 49 (2002) 335–338
complexes of IgA class in hepatitis B. J. Chi. Microbiol. Immunol. 8, 379 – 381. Peng, X.X., Zhou, Y.L., Wang, S.Y., 1997. Characterization of immunoglobulin classes in clearing hepatitis B virus using a new immunocapture PCR methods. Am. Soc. Cell Biol.: Thirty-seventh Annual Meeting Hot paper 97. Peng, X.X., Gao, H., Wang, S.Y., Zheng, W.Z., 2000a. Universal primer PCR with SSCP and RFLP for identification of fish disease pathogens. J. Fish. Chi. 24, 345 – 348. Peng, X.X., Zhou, Y., Wang, S.Y., Zhang, Y.L., 2000b. Quick identification for pathogenic vibrio of aquacultivated creatures by UPPCR – SSCP. J. Oceanogr. Taiwan Strait 19, 460 – 463. Peng, X.X., Zhu, H., Zhang, J.Y., Wang, S.Y., 2001. Analysis of HCV-Ig isotype complexes by a novel immuno-capture RT – PCR methods. Scand. J. Immunol. 54, 409 – 413.
Reischl, U., Linde, H.J., Metz, M., Leppmeier, B., Lehn, N., 2000. Rapid identification of methicillin resistant Staphylococcus aureus and simultaneous species confirmation using real-time fluorescence PCR. J. Clin. Microbiol. 38, 2429 – 2433. Rodoni, B.C., Dale, J.L., Harding, R.M., 1999. Characterization and expression of the coat protein-coding region of banana bract mosaic polyvirus, development of diagnostic assays and detection of the virus in banana plants from five countries in southeast Asia. Arch. Virol. 144, 1725 – 1737. Rowhani, A., Uyemoto, J.K., Golino, D.A., 1997. A comparison between serological and biological assays in detecting grapevine leafroll associated viruses. Plant Dis. 81, 799 – 801.
Note
Immuno-capture PCR for detection of Aeromonas hydrophila Xuanxian Peng*, Jianying Zhang, Sanying Wang, Zhilin Lin, Weiyi Zhang Department of Biology and the Key Laboratory of Education Ministry for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen 361005, PR China Received 12 June 2001; received in revised form 6 November 2001; accepted 17 December 2001
Abstract In this report, we describe the use of universal primer PCR (UPPCR) for the detection of 16S ribosomal RNA (rRNA) genes from Aeromonas hydrophila captured by anti-A. hydrophila antibody coupled to a microplate. The approach combining immuno-capture with UPPCR provides a quick, sensitive, and reproducible way for the detection of bacterial cells. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Aeromonas hydrophila; Identification of bacteria; Immuno-capture; Universal primer PCR
PCR technologies in detection of bacteria may be divided into two types according to their primers. One is specific primer PCR (SPPCR) with a pair of specific primers for a target pathogen (Reischl et al., 2000); the other is universal primer PCR (UPPCR) with universal primers for almost all bacteria (Bernhard and Field, 2000; Osorio et al., 1999; Peng et al., 2000a,b). Determination of the identity of bacteria in a given sample by SPPCR could be very time consuming because numerous PCR reactions with different primers may be needed. The approach based on the utilization of UPPCR from conserved regions such as 16S ribosomal RNA (rRNA) gene can be used to amplify almost all of the bacteria (Osorio et al., 1999; Marchesi et al., 1998), but the organism being analyzed should be characterized further by subsequent steps including restriction fragment length polymor-
*
Corresponding author. Tel.: +86-592-2183805; fax: +86-5922181015. E-mail address: [email protected] (X. Peng).
phism (RFLP), single-strand conformation polymorphism (SSCP) or sequence analysis (Greisen et al., 1994; Peng et al., 2000a,b). Hence, pathogens detected by either SPPCR or UPPCR cannot usually be completed by a single PCR reaction. In this report, we have combined immuno-capture and UPPCR and developed a quick method of high sensitivity for detecting pathogenic bacteria. We termed this new method as immuno-capture UPPCR (iUPPCR). Four strains of Aeromonas hydrophila were used in this study. Strains 1 and 2 were purchased from the Institute of Hydrobiology (Chinese Academy of Sciences, Wuhan), and strain PPD 134/91 and strain PDD 35/85 were kindly provided by professor Sin Y.M., Biological Department, the National University of Singapore, Singapore. Cultures were grown for 18 h at 37 C in Luria – Bertani Medium (approximate 109 – 1010/ml) and harvested by centrifugation at 3000g/ min for 20 min. The bacteria preparation was diluted with distilled water to different concentrations as inicated in text before UPPCR analysis. Part of strain 1 cultures was diluted with 0.85% saline. Then, form-
0167-7012/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 7 0 1 2 ( 0 2 ) 0 0 0 1 0 - 6
336
X. Peng et al. / Journal of Microbiological Methods 49 (2002) 335–338
aldehyde was added to 1% of the final concentration to inactivate the bacteria. The inactive bacteria were diluted to 9108/ml after rinsing with sterile saline three times, and injected into mice dorsum at 0.1 ml/ animal by subcutaneous injection (four mice were immunized). One week later, the primed mice were boosted once a week till the agglutination titer reached over 1:2000. We collected sera, and purified the antibody using DEAE-32 column (Peng and Fang, 1988). iUPPCR was made up of immuno-capture and UPPCR procedures. In the former, microplate wells were coated overnight at 4 C with 50 ml of the purified mouse antibody (10 mg/ml) against A. hydrophila. Wells were then washed three times with PBS-T buffer (PBS with 0.1% Tween-20), prior to a 1-h blocking step with 10% BSA at 37 C. Twenty microliters of bacterial suspension was added to each well and incubated for 1 h at 37 C. Plates were washed five times as above, prior to adding 20 ml of sterile distilled water into each well, and were incubated in a boiling water bath for denaturation for 10 min. Eighteen microliters of the suspension was used as the template for PCR amplification. In the latter, the primers were designed against the conserved regions of 16S rRNA sequence to amplify the target sequences of expected sites of 909 –1410 bp. The primer sequences were 5V-AAACTCAAAGGAATTGAC-3V for the forward, and 5V-GACGGGCGGTGTGTA CAA-3Vfor the reverse. PCR was carried out in a 25 ml volume of reaction mixture containing 2.5 ml of 10PCR buffer, 3 ml of MgCl2 (25 mM), 0.5 ml of dNTPs (10 mM for each), 0.5 ml of each primer (24 mM), 0.1 ml of Taq polymerase (5 U/ml) and 18 ml of template to perform 40 cycles consisting of 95 C for 1 min, 55 C for 1 min and 70 C for 2.5 min. The amplified products were resolved on 2.5% agarose gels followed by staining with ethidium bromide. For comparative purposes, heat-denatured UPPCR was carried out in this study. In this approach, the cells were incubated in a boiling water bath for denaturation for 10 min. After boiling, 18 ml of the suspension was used as the template of UPPCR amplification. Analyses of differences between groups were tested for significance using Chi-square test with Yates’s continuity for small numbers, and two significant levels (0.05 and 0.01) were used. We validated the specificity of iUPPCR by three procedures. Firstly, bacterial suspension was pre-incu-
bated with the antibody before adding the cells into the wells that were coated with the same antibody. The pre-incubation was to compete for bacterial cells and with those might absorb into the coated antibody and to preclude cell trapping. We used the pair of primers to amplify the four strains of A. hydrophila. All of them showed the expected DNA fragments of about 500 bp. However, all of the positive samples became negative after their absorption with the three kinds of concentrations of antibodies (50, 100, 200 mg/ml of mouse IgG against A. hydrophila). Secondly, mouse anti-A. hydrophila was superseded with goat IgG against human g chain or pre-immune IgG as coated antibodies for controls, no specific products were detected. Thirdly, as the amplified DNA contains a readily recognizable pattern by HaeIII digestion, HaeIII was used to check specificity. Fig. 1 showed HeaIII restriction mapping and electrophoresis analy-
Fig. 1. Restriction mapping and electrophoresis analyses of representative iUP-PCR products. M, PGEM-7zf(+) HaeIII marker; lane 1, The restriction mapping (HaeIII) of iUP-PCR product; lane 2, iUP-PCR product (500bp).
X. Peng et al. / Journal of Microbiological Methods 49 (2002) 335–338 Table 1 Comparison of iUPPCR with heat-denatured PCR to the detection of 1, 5 and 10 CFU of A. hydrophila Positive samples/total samples (%)
Immuno-capture UPPCR Heat-denatured UPPCR
10 CFU
5 CFU
1 CFU
10/10 (100)
10/10 (100)*
4/10 (40)*
10/10 (100)
3/10 (100)
0/10 (0)
*P<0.01.
ses of representative iUPPCR products. M, lanes 1 and 2 represented PGEM-7zf(+) HaeIII marker, restriction mapping and iUPPCR product (500 bp), respectively. We evaluated the sensitivity of iUPPCR by two methods. First, the bacteria (strain 1) were diluted to 5, 10, 50, 500, 5000 CFU per 20 ml. Bacteria numbers were counted under a light microscope and then confirmed by growing on an agar plate. iUPPCR and heat-denatured UPPCR were carried out with the diluted bacteria samples. Both iUPPCR and heat-denatured UPPCR detected bacteria in the samples with bacterial concentration at and above 10 CFU. However, difference between iUPPCR and heat-denatured UPPCR appeared when five CFU samples were used. All five CFU samples were positive by iUPPCR but only 30% showed positive using heat-denatured UPPCR ( P<0.01). In the second experiment, 20 CFU in 360 ml were divided into 20 wells and then analyzed by the two methods. Four of the ten samples were positive by iUPPCR, while none of the 10 samples was positive by heat-denatured UPPCR. The results were summarized in Table 1. The reproducibility of iUPPCR was confirmed by six independent experiments (data not shown). Thus, iUPPCR is a high sensitive method in the detection of bacteria. To determine whether iUPPCR was suitable to the application of environmental bacteria, water sample with pH 6 was obtained from Furong Lake (a fishpond) in Xiamen University. Aliquots of the water were left unadjusted or adjusted to pH 7, 8, 9, or 10 before adding strain 1 to a final concentration at 10 CFU per 20 ml. iUPPCR detected bacteria in all samples that had bacteria, while control samples without cells all turned negative.
337
The iUPPCR developed in the present study is different from PCR-based detection method previously developed by others and ourselves. IC– PCR or iRT – PCR of virus detection requires gene specific primers (Harper et al., 1999; Rowhani et al., 1997; Peng et al., 1997, 2001; Rodoni et al., 1999), while iUPPCR utilizes universal primers. A single step immuno-capture in iUPPCR avoided complicated RFLP, SSCP or sequence analysis in the identification of pathogens. Most importantly, an affinity purification of bacteria enhances not only the sensitivity of the method but also the accuracy of PCR reaction because of the removal of interfering materials. In summary, we have developed a quick and sensitive method in detection pathogens.
Acknowledgements We are very grateful to Dr. Jiahuai Han and Dr. Shengcai Lin for critical comments on this manuscript. This work was sponsored by grants from the State Foundation of China for Natural Science (No. 39770585), the Foundation of Chinese Education Ministry for Excellent Young Teachers and IFS (A/ 2338-2).
References Bernhard, A.E., Field, K.G., 2000. Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes. Appl. Environ. Microbiol. 66, 1587 – 1594. Greisen, K., Loeffelholz, M., Purohit, A., Leong, D., 1994. PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacterial, including bacteria found in cerebrospinal fluid. J. Clin. Microbiol. 32, 335 – 351. Harper, G., Dahal, G., Thottappilly, G., Hull, R., 1999. Detection of episomal banana streak badnavirus by IC – PCR. J. Virol. Methods 79, 1 – 8. Marchesi, J.R., Sato, T., Weightman, A.J., Martin, T.A., Fry, J.C., Hiom, S.J., Dymock, D., Wade, W.G., 1998. Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA. Appl. Environ. Microbiol. 64, 795 – 799. Osorio, C.R., Collins, M.D., Toranzo, A.E., Barja, J.L., Romalde, J.L., 1999. 16S rRNA gene sequence analysis of photobacterium damselae and nested PCR method for rapid detection of the causative agent of fish pasteurellosis. Appl. Environ. Microbiol. 65, 2942 – 2946. Peng, X.X., Fang, L., 1988. HBsAg specific circulating immune
338
X. Peng et al. / Journal of Microbiological Methods 49 (2002) 335–338
complexes of IgA class in hepatitis B. J. Chi. Microbiol. Immunol. 8, 379 – 381. Peng, X.X., Zhou, Y.L., Wang, S.Y., 1997. Characterization of immunoglobulin classes in clearing hepatitis B virus using a new immunocapture PCR methods. Am. Soc. Cell Biol.: Thirty-seventh Annual Meeting Hot paper 97. Peng, X.X., Gao, H., Wang, S.Y., Zheng, W.Z., 2000a. Universal primer PCR with SSCP and RFLP for identification of fish disease pathogens. J. Fish. Chi. 24, 345 – 348. Peng, X.X., Zhou, Y., Wang, S.Y., Zhang, Y.L., 2000b. Quick identification for pathogenic vibrio of aquacultivated creatures by UPPCR – SSCP. J. Oceanogr. Taiwan Strait 19, 460 – 463. Peng, X.X., Zhu, H., Zhang, J.Y., Wang, S.Y., 2001. Analysis of HCV-Ig isotype complexes by a novel immuno-capture RT – PCR methods. Scand. J. Immunol. 54, 409 – 413.
Reischl, U., Linde, H.J., Metz, M., Leppmeier, B., Lehn, N., 2000. Rapid identification of methicillin resistant Staphylococcus aureus and simultaneous species confirmation using real-time fluorescence PCR. J. Clin. Microbiol. 38, 2429 – 2433. Rodoni, B.C., Dale, J.L., Harding, R.M., 1999. Characterization and expression of the coat protein-coding region of banana bract mosaic polyvirus, development of diagnostic assays and detection of the virus in banana plants from five countries in southeast Asia. Arch. Virol. 144, 1725 – 1737. Rowhani, A., Uyemoto, J.K., Golino, D.A., 1997. A comparison between serological and biological assays in detecting grapevine leafroll associated viruses. Plant Dis. 81, 799 – 801.