| Virus is one strict type of cell parasites which is mainly composed of coat protein and nucleic acid. Some of them can infect humans or plants, causing serious human diseases or plant diseases and then harming human health or crop production. Thus, a rapid, efficient and sensitive detecting method of virus is important to control the spread of virus. The aims of this study are to synthesize different surface-modified iron oxide magnetic particles, analyze the adsorption and desorption between nucleic acid (DNA and RNA) and as-prepared magnetic particles, develop the method for extracting viral nucleic acid, and achieve the detection of virus by mean of real-time PCR/RT-PCR assay. The main contents and as-obtained results of the work are summarized as follows:(1) Two types of iron oxide magnetic particles were synthesized using two different methods, thermal decomposition method and solvothermal method, whose sizes were 10 nm and 200 nm, respectively. Firstly, the 10-nm nanoparticles were modified by the hydrolysis of tetraethoxysilane (TEOS), which can enhance the dispensability of particles in aqueous solution and obtain the negative-charged particles. Secondly,200-nm iron oxide magnetic particles were modified with TEOS and γ-aminopropyltriethoxysilane (APTS), respectively, in order to obtain aminosilane-modified magnetic particles (AMPs). Thirdly,10-nm iron oxide nanoparticles were functionalized with 4-carboxyphenylboronic acid, and subsequently carboxyphenylboronic acid-functionalized magnetic particles (CPBA-MNPs) were otained. The results of characterization indicated that the magnetic particles above mentioned were successfully synthesized with attachment of silicon-hydroxyl, amino or phenylboronic acid group.(2) The adsorption and desorption between silica-coated magnetic particles (SMPs) and nucleic acid (salmon sperm DNA or E.coli RNA) was analyzed. The capacity of nucleic acid adsorption to silica-coated magnetic particles was analyzed in the presence of guanidinium thiocyanate (GTC) with different concentration, indicating that the amount of nucleic acid adsorbed onto silica-coated magnetic particles was highest at 1.0 M GTC. The maximum of adsorption is up to 10.6 mg DNA or 7.7 mg RNA per 1 g of SMPs with 4 M GTC at pH 5.5 without the addition of ethanol. The influencing factors were analyzed in term of the adsorption of nucleic acids onto SMPs. The adsorption capacity in acidic condition was found to be higher than that in alkaline condition. In addition, the addition of ethanol and isopropanol could decrease the polarity of mixture and enhance the adsorption capacity of nucleic acid. The adsorption and desorption between nucleic acid and 4-carboxyphenylboronic acid-functionalized magnetic particles (CPBA-MNPs) were analyzed. The results indicated that divalent cations (such as Mg2+, Ca2+) and ethanol could strongly improve the adsorption of nucleic acid. In acidic solution, the adsorption capacity of RNA was higher than that in alkaline solution. The competitive analogues which bear cis-diol group could be used in the desorption of RNA because of reverse binding between phenylboronic acid and RNA. By analysis of elution efficiency of different elution buffers, Tris and EDTA solution (50 mM Tris,5 mM EDTA, pH 8.8) was used to elute the nucleic acid with a high elution efficiency of 75.6 ± 7.6%(DNA) and 60.1 ± 5.9%(RNA).(3) Under the study of the adsorption and desorption between nucleic acid and SMPs, the feasibility of the extraction of hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA from sera using SMPs was analyzed, followed by detection of HBV and HCV using real-time PCR and RT-PCR. The results suggested that the viral nucleic acid could be effectively released by addition of protein K and 1.0 M GTC. In the use of one volume of ethanol, the nucleic acid was successfully extracted using SMPs. Furthermore, real-time PCR or RT-PCR assay was developed to detect HBV or HCV, and then the standard curves of HBV and HCV were set up to determine the viral load. To assess the recovery of SMPs method, the known-copy HBV plasmids or HCV transcripts were added into the negative sera. The results from real-time PCR and RT-PCR indicated that the recovery of SMPs method was 30 ± 11.1%(HBV plasmid) and 34 ± 7.3%(HCV transcript), which was higher than that of commercial kits. Additionally, these methods were used for rapid detection of HBV and HCV in the entry-exit traveller of Beijing port, indicated that SMPs and real-time PCR/RT-PCR is effective for extracting viral nucleic acid and for detecting virus with good performance.(4) A method based on SMPs for extracting the nucleic acid of plant virus/viroid was established and applied to detect Arabis mosaic virus (ArMV), Lily symptomless virus (LSV), Hop stunt viroid (HSVd) and grape yellow speckle viroid 1 (GYSVd-1). The RNA standards which covered the amplification sequences of real-time RT-PCR were obtained from reverse transcription in vitro, and were used to plot the standard curves, which covered six or seven orders of magnitude with a detection limit of 100 copies per each assay. To evaluate the extraction efficiency of SMPs, the standards were spiked into the lysates of plant samples and recoverd using SMPs. Results showed that the recovery rate of SMPs was comparable to the commercial kits (TRIzol and RNeasy Plant mini kit) when the standards were extracted from lily leaves, whereas it was two or three times higher than commercial kits when the standards were extracted from grapevine leaves. The nucleic acids were extracted from 15 ArMV-positive lily leaf samples and 15 LSV-positive lily leaf samples using SMPs. The results did not show a statistically significant difference from other methods on detecting ArMV, but LSV. Compared to TRIzol, the SMPs method has the same level of virus load. But its mean virus load of was 0.5log10 lower than the RNeasy Plant mini kit. Nucleic acid was extracted from 19 grapevine-leaf samples with SMPs and the two commercial kits and subsequently screened for HSVd and GYSVd-1 by real-time RT-PCR. Regardless of HSVd or GYSVd-1, SMPs method outperforms other methods on both positive rate and the viroid load. Therefore, SMPs method was able to efficiently extract the nucleic acid of RNA viruses or viroids, so as to realize the rapid detection for plant virus/viroid.(5) Based on the adsorption and desorption of nucleic acid and CPBA-MNPs, a novel method were developed for extracting nucleic acid from complex samples. The results of the feasibility of CPBA-MNPs for extracting nucleic acid indicated that Tris and EDTA showed an adverse effect on nucleic acid adsorption onto CPBA-MNPs, and total nucleic acids, genomic DNA, and RNA could be separately extracted from maize and soybean seeds with high qualities using CPBA-MNPs under different conditions. Furthermore, genomic DNA and RNA extracted from the genetically modified maize seeds and unmodified soybean seeds infected by Bean pod mottle virus and Tobacco ringspot virus were detected by real-time PCR and RT-PCR. Results showed that the nucleic acids extracted from these seeds using CPBA-MNPs were suitable for the detection of genetically modified seeds and seed-borne viruses. |
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