Dot-blot hybridization was applied in this paper to detect five viruses infecting cucurbitaceous crops, Zuccini yellow mosaic virus （ZYMV）, Watermelon mosaic virus （WMV）, Cucumber mosaic virus （CMV）, Papaya ring...Dot-blot hybridization was applied in this paper to detect five viruses infecting cucurbitaceous crops, Zuccini yellow mosaic virus （ZYMV）, Watermelon mosaic virus （WMV）, Cucumber mosaic virus （CMV）, Papaya ringspot viruswatermelon strain （PRSV-W） and Squash mosaic virus （SqMV）, as a good alternative assay in seed health test and epidemiological and transgenic research. Digoxigenin-labelled cDNA probes of the five viruses were synthesized by PCR with the specific primers and applied in dot-blot hybridization to detect five viruses in crude extraction of the infected leaves. And three SqMV probes of different lengths （0.55, 1.6, and 2.7 kb, respectively） were designed to investigate the effect of hybridization. The results showed that the sensitivity for detecting the crude extraction of infected leaves by ZYMV, WMV, CMV, PRSV-W, and SqMV was down to 1：160, 1：160, 1：320, 1：160, and 1：320, respectively. Three SqMV probes of different length showed no differences on the sensitivity and specificity. The digoxigenin-labelled probes prepared by PCR could be used for accurate and rapid identification of 5 viruses infecting cucurbitaceous crops with good stabilities, sensitivities, specificity, and reproducibilifies.展开更多
Using Kuala pear leaves and cortexes as materials, the total RNA was extracted using two methods and these two methods were compared. The most suited methods for Kuala pear were screened; and biotin-labeled cDNA probe...Using Kuala pear leaves and cortexes as materials, the total RNA was extracted using two methods and these two methods were compared. The most suited methods for Kuala pear were screened; and biotin-labeled cDNA probe was synthesized using RT-PCR. The main factors that affected the sensitivity of hybridization were studied. Studies indicated that the highest sensitivity was obtained under the following conditions： probe concentration 400 ng mL^-1, formamide concentration 45%, temperature 42℃, hybridization time 6 hours. The best hybridization results were obtained when the nitrocellulose membrane purchased from Gelman was used. Better blocking of hybridization was obtained using Tween 20 compared with albumin in bovine. The detection of the total RNA using different tissues and different extraction methods was compared. This study indicates that the total RNA of fresh leaf, old leaf, cortexes, and frozen leaf showed signs of hybridization using the two extraction methods.展开更多
Objective: To explore the usefulness of long distance reverse transcript combining linear amplification (LD-RTPCR) in labeling slight trace probe used for cDNA microarray. Methods: Total RNA from BEP2D cells was extra...Objective: To explore the usefulness of long distance reverse transcript combining linear amplification (LD-RTPCR) in labeling slight trace probe used for cDNA microarray. Methods: Total RNA from BEP2D cells was extracted and labeled by two different methods, LD-RTPCR with Cy3-dCTP as fluorescent dye and traditionally used RNA reverse transcript (RT) with Cy5-dCTP as fluorescent dye. Then, the probes labeled by two methods were mixed equally and hybridized with the cDNA microarray. Results: Scan and analysis of the microarray showed that the two methods labeled probes had consistent results. Conclusion: LD-RTPCR was proved useful for labeling cDNA microarray probe, especially for limited RNA material.展开更多
文摘Dot-blot hybridization was applied in this paper to detect five viruses infecting cucurbitaceous crops, Zuccini yellow mosaic virus （ZYMV）, Watermelon mosaic virus （WMV）, Cucumber mosaic virus （CMV）, Papaya ringspot viruswatermelon strain （PRSV-W） and Squash mosaic virus （SqMV）, as a good alternative assay in seed health test and epidemiological and transgenic research. Digoxigenin-labelled cDNA probes of the five viruses were synthesized by PCR with the specific primers and applied in dot-blot hybridization to detect five viruses in crude extraction of the infected leaves. And three SqMV probes of different lengths （0.55, 1.6, and 2.7 kb, respectively） were designed to investigate the effect of hybridization. The results showed that the sensitivity for detecting the crude extraction of infected leaves by ZYMV, WMV, CMV, PRSV-W, and SqMV was down to 1：160, 1：160, 1：320, 1：160, and 1：320, respectively. Three SqMV probes of different length showed no differences on the sensitivity and specificity. The digoxigenin-labelled probes prepared by PCR could be used for accurate and rapid identification of 5 viruses infecting cucurbitaceous crops with good stabilities, sensitivities, specificity, and reproducibilifies.
基金supported by the National Natural Science Foundation of China(30060053).
文摘Using Kuala pear leaves and cortexes as materials, the total RNA was extracted using two methods and these two methods were compared. The most suited methods for Kuala pear were screened; and biotin-labeled cDNA probe was synthesized using RT-PCR. The main factors that affected the sensitivity of hybridization were studied. Studies indicated that the highest sensitivity was obtained under the following conditions： probe concentration 400 ng mL^-1, formamide concentration 45%, temperature 42℃, hybridization time 6 hours. The best hybridization results were obtained when the nitrocellulose membrane purchased from Gelman was used. Better blocking of hybridization was obtained using Tween 20 compared with albumin in bovine. The detection of the total RNA using different tissues and different extraction methods was compared. This study indicates that the total RNA of fresh leaf, old leaf, cortexes, and frozen leaf showed signs of hybridization using the two extraction methods.
基金supported by the National“973”Key Basic Research Program of China(No.G1998051207)
文摘Objective: To explore the usefulness of long distance reverse transcript combining linear amplification (LD-RTPCR) in labeling slight trace probe used for cDNA microarray. Methods: Total RNA from BEP2D cells was extracted and labeled by two different methods, LD-RTPCR with Cy3-dCTP as fluorescent dye and traditionally used RNA reverse transcript (RT) with Cy5-dCTP as fluorescent dye. Then, the probes labeled by two methods were mixed equally and hybridized with the cDNA microarray. Results: Scan and analysis of the microarray showed that the two methods labeled probes had consistent results. Conclusion: LD-RTPCR was proved useful for labeling cDNA microarray probe, especially for limited RNA material.
