| Yersinia pestis is the cause of the severe infectious disease, also named as plague, which has been responsible for three worldwide pandemics in the human history. Plague initially out-brook in the 6th century, which was collectively responsible for the loss of 200 million lives. Although plague pandemic has been controlled throughout the world, it is reported that there are still many endemic areas and some sporadic new endemic areas. The potential use of Yersinia pestis as an agent of biological weapon by bioterrorist means that plague still poses threat to human society. In order to fully control the plague pandemic's reoccurrence, the rapid diagnostic method and novel plague vaccine should be developed. It is indispensable to make a further diagnostic study to Yersinia pestis with DNA chip technology.DNA chip technology was first described in the late 1990s. The novel technique was originated from the idea of making integrated computer chip for parallel detection of biological agents. Based on principle of the Southern blot, the DNA fragments or oligonucleotide probes were deposited on the surface of substrate in parallel based on addressable approaches. The high density DNA microarray is formed by these probes fixed on the substrate. The DNA or RNA could be extracted from samples of various origins and labeled with fluorescent dyes. The labeled materials could hybridize with the DNA array according to the strict complementary nature of the base pairs, and the probes specifically bind with fluorescent-labeled samples in a form of double helix. The information of the double helix molecules in the DNA chips is acquired through the laser excitation and scanning. In this manner, the genetic information of the biological samples can be studied with high quantity, high throughput and in parallel.Yersinia pestis carries three plasmids associated with its pathogenicity: 9.5kb, 70kb and 110kb. The number of recognized ORF in plasmidssequence shows that the coding density is more than 70%, and most of the pathogenic genes specific to Yersinia pestis are located on these three plasmids. DNA probes of restricted fragments of the plasmids were prepared by nested PCR for the preparation of the Yersinia pestis DNA chips. The DNA samples were labeled fluorescent dye via the RD-PCR technique invented by Wenli Ma and Wenling Zheng.Improvement of hybridization protocol, such as cancelled purification of labeled samples, increased reaction temperature from 42 to 52and added rinsing steps with distilled water prior to low stringency washing at room temperature, have enhanced the sensitivities of plague diagnostic microarray and effectively kept the lower cross-hybridization and background of hybridized arrays. The modified hybridization protocol improved further the signal-to-noise ratio of hybridized arrays, yielding high quality hybridization signals. DNA chip in combination with RD-PCR technique, by which the samples can be amplified during the labeling process. In addition its high efficiency, high sensitivity, accuracy and expediency are achieved, while multiple genes can be tested simultaneously. Evidently, the DNA chip technology provides a new and scientific approach for the diagnosis of the severe infectious disease.Since the whole genome of Yersinia pestis has been sequenced, we chose the major known pathogenic genes and analyzed their specificity to Yersinia pestis with Blast program. Highly specific gene fragments were adopted to design probes with 60mers in length. All synthesized probes were printed on ploy-L-Lysine coated slides to prepare an oligo chip. RD-PCR technique was also used to label DNA sample as described above. The non-specific hybridization signals decreased with the improvement of hybridization efficiency. The results showed that oligo chip was able to obtain even higher signal-to-noise ratio.Furthermore, Oligo chip was applied to compare the expression profiles of the known pathogenic genes specific to Yersinia pestis between EV strainand wild strains isolated from Yunnan province, China...
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