| To ensure the safety of clinical blood use, blood should go through strict testing before entering the blood banks. As projects of blood routine examination, the detection of HIV(human Acquired imunodeficiency syndrome), HBV(hepatitis B), HCV(hepatitis C)and syphilis need rigorous testing and secondary screening with two different reagent manufacturers. As the four diseases have developed serious threat to transfusion safety, how to control and prevent the spread of the diseases effectively in the population is urgent and of great importance.At present, pathogens detection depends on the immunological diagnosis (ELISA) and molecular biology testing. ELISA is unable to detect the pathogens during the window phase and from blood samples newly infected, therefore, might result in false negative results. Although the molecular biology methods can shorten the window phase, it works only for a single pathogen detection at a time, which is time-consuming and labor-intensive.Microarray technology, characterized as high-throughput, high-speed and enhanced accuracy, has become a powerful tool for gene expression profiling, genotyping, mutation detection, pathogen detection and molecular diagnosis. Multiple pathogens can be detected at the same time, which can not only be used for large-scale screening, but also be more efficient. Nevertheless, the application of gene chips are still faced with some problems, including quality control and sample labeling procedures, etc. Based on the two issues, this study can be divided into three parts,In the first part, we designed universal sequence-tagged oligonucleotide probes and introduced an universal sequence tag (UST) into one or both ends of the UST. Thus the fluorescence-labeled oligomers complementary to UST could be used in quality control of the oligonucleotide microarrays spots.The design was as follows:we selected two probes of HIV as core target sequences and linked with USTs to the ends of probes. UST sequences could be classified into three types:USTs of probes distributed at the 5'-end of the complementary sequence of UP, USTs in the middle, USTs at the 3'-end. Then we printed them on the surface of epoxy-coated chips. To explore the appropriate UST sequence and linkage with core sequence, hybridization was performed by using Cy5-UP. The five probes (No.1,8,9,10,12) exhibited strong fluorescent signals as well as high value of SNR, which can beregarded as positive probes.The selected probes were printed onto slides. Cy5-labeled universal primer (Cy5-UP) and random primer (Cy5-RP) were diluted into 20μmol/L,2umol/, and 0.2μmol/L, respectively,, and then were hybridized with the slides. After being washed and dried, microarrays were used for scanning and analyzing. The hybridization with Cy5-UP in concentration of 20μmol/L and 2μmol/L showed stronger signals than that with Cy5-RP at the same concentration. As the concentration went down to 0.2μmol/L, probe 8 still showed strong signals. The negative control (probe19 without UST) and blank control showed no signal. When the array was hybridized with Cy5-RP, probe 19 showed fluorescent signals. There were nearly no signal in hybridization with 0.2μmol/L Cy5-RP and week signals in that with 1μmol/L Cy5-RP. Therefore, Cy5-UP was proved to be more sensitive and effective than Cy5-RP in spot QC of universal-tagged microarrays. As a result, probe 8 could be regarded as the best UST, the hybridization of which was 10mer probes near both of 5'end and 3'end complementary sequences, followed by probe 1, the hybridization of which was only near the 5'end with the same UST as probe 8. Considering the cost and efficiency, probe 1 was superiority to the else probes. In order to verify the stability of the UST, we linked it to the ends of HBV oligonucleotide probes. Hybridization results with Cy5-UP illustrated that the selected UST remained stable fluorescence signal, which proved to be practicable for quality control.In the second part of this study, we explored the universal primer labeling (UPL) in the labeling of DNA samples. Sample labeling is one of the key processes during the microarray detection, which require faster and more efficient labeling technology.Restriction display(RD) technique, as a patent in our laboratory, has proved to be an effective method for genome-wide labeling. To begin with, the genes were digested with SAU3AI in order to isolate restriction fragments, then the adapters were linked to both ends of the fragments. After that, the universal primers were designed according to the sequence of the enzyme restriction sites and the adapters. As the process involved lots of steps including digestion, linkage and so on, it was less suitable for clinical laboratory use.UPL was originally used to amplify RNA sample. It introduced a primer USN2 (two random bases added in the end of universal primer).After the first round of amplification of 10 cycles, the reaction mixture would be linked with the universal primer sequence in the 5'end, followed by fluorescently labeled universal primers (Cy3-US) after the second round of amplification. Because of its rapidness and efficiency, UPL has been widely adopted in sample labelings. However, this technology has been rarely mentioned in the application of DNA samples. If UPL could be practicable in DNA samples, then it can label both the DNA and RNA samples at one time. Thus this part aims to explore the technology in the DNA sample labeling,with RD as a control.At first, we labeled gene fragments of TP(treponema pallidum) with RD and UPL, respectively. After hybridization with 11 probes screened, the results showed that there was no significant difference with the two labeling methods, which showed clearly that UPL technology was feasible to amplify DNA samples. Since UPL technology eliminated the steps such as digestion and linkage, its application could be promoted in the subsequent research.A number of fragments displayed through the agarose gel electrophoresis after the second amplification of UPL labeling. To further verify whether the amplified fragments had correspondence with the probes, we finished a round of T cloning and transformation experiments,100 clones were selected. We found the DNA fragments ranged at 200bp-1000bp. Since USN2 had the same restriction site GATC as the enzyme Sau3AI and the site was 256 bases for each site, the amplified DNA fragments was basically inside the range, which meant the result were correspondent with the expectation. As can be seen, UPL technology is able to amplify small amounts of DNA samples effectively and be used for subsequent experiments.Based on the above two parts, we applied a small-scale of clinical samples for detection, in order to testify the feasibility of the UST and UPL technology. Serum samples were collected, including 30 HBV cases,8 HCV cases,11 HIV simulated samples,16 cases of syphilis samples and 30 cases of normal human serum (as negative controls). In this part, we completed the testing of clinical samples. Firstly, we took HBV as a target and screened probes from the first 13 probes mentioned in the first part. Probes were hybridized with both the HBV plasmid Bj58 and extract from serum samples and 6 probes were screened out. Secondly, UST sequence was linked to the ends of HIV, HCV and syphilis probes.6 probes of syphilis were screened during the second par. HIV and HCV probes were based on the pre-screened probes, which were also 6 probes, respectively. All the probes were printed on the surface of a combined detection chip. Nucleic acid DNA or RNA were extracted from serum samples and labeled with UPL technology. The hybridization results illustrated that the probe signals were correspondent to pathogens. As can be seen, the new design of oligonucleotide probes and quality control method are practicable, and the use of UPL labeling in clinical samples is also feasible.In summary, we have established an effective method of chip quality control, and explored the UPL technology in the labeling of DNA samples. The detection of pathogens through clinical serum samples has been verified that the feasibility of both quality control methods and labeling methods, which has laid the experimental basis for future clinical applications. |
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