Fast simultaneous detection of K-RAS mutations in colorectal cancer

<p>Abstract</p> <p>Background</p> <p><it>RAS </it>genes acquire the most common somatic gain-of-function mutations in human cancer, and almost all of these mutations are located at codons 12, 13, 61, and 146.</p> <p>Methods</p> <p>We present a method for detecting these <it>K-RAS </it>hotspot mutations in 228 cases of colorectal cancer. The protocol is based on the multiplex amplification of exons 2, 3 and 4 in a single tube, followed by primer extension of the PCR products using various sizes of primers to detect base changes at codons 12, 13, 61 and 146. We compared the clinicopathological data of colorectal cancer patients with the <it>K-RAS </it>mutation status.</p> <p>Results</p> <p><it>K-RAS </it>mutation occurred in 36% (83/228) of our colorectal cancer cases. Univariate analysis revealed a significant association between <it>K-RAS </it>mutation at codon 12 of exon 2 and poor 5-year survival (p = 0.023) and lymph node involvement (p = 0.048). Also, <it>K-RAS </it>mutation at codon 13 of exon 2 correlates with the size of the tumor (p = 0.03). Multivariate analysis adjusted for tumor size, histologic grade, and lymph node metastasis also indicated <it>K-RAS </it>mutations at codon 12 and 13 of exon 2 correlate significantly with overall survival (p = 0.002 and 0.025). No association was observed between codon 61 and 146 and clinicopathological features.</p> <p>Conclusion</p> <p>We demonstrated a simple and fast way to identify <it>K-RAS </it>mutation.</p

Multiplex Amplification Refractory Mutation System Polymerase Chain Reaction (ARMS-PCR) for diagnosis of natural infection with canine distemper virus

<p>Abstract</p> <p>Background</p> <p>Canine distemper virus (CDV) is present worldwide and produces a lethal systemic infection of wild and domestic <it>Canidae</it>. Pre-existing antibodies acquired from vaccination or previous CDV infection might interfere the interpretation of a serologic diagnosis method. In addition, due to the high similarity of nucleic acid sequences between wild-type CDV and the new vaccine strain, current PCR derived methods cannot be applied for the definite confirmation of CD infection. Hence, it is worthy of developing a simple and rapid nucleotide-based assay for differentiation of wild-type CDV which is a cause of disease from attenuated CDVs after vaccination. High frequency variations have been found in the region spanning from the 3'-untranslated region (UTR) of the matrix (M) gene to the fusion (F) gene (designated M-F UTR) in a few CDV strains. To establish a differential diagnosis assay, an amplification refractory mutation analysis was established based on the highly variable region on M-F UTR and F regions.</p> <p>Results</p> <p>Sequences of frequent polymorphisms were found scattered throughout the M-F UTR region; the identity of nucleic acid between local strains and vaccine strains ranged from 82.5% to 93.8%. A track of AAA residue located 35 nucleotides downstream from F gene start codon highly conserved in three vaccine strains were replaced with TGC in the local strains; that severed as target sequences for deign of discrimination primers. The method established in the present study successfully differentiated seven Taiwanese CDV field isolates, all belonging to the Asia-1 lineage, from vaccine strains.</p> <p>Conclusions</p> <p>The method described herein would be useful for several clinical applications, such as confirmation of nature CDV infection, evaluation of vaccination status and verification of the circulating viral genotypes.</p

Study of Pseudorabies Virus Early Genes (IV)

假性狂犬病毒 (PRV)是豬重要傳染病-假性狂 犬病的病原.該病毒是一種阿爾發皰疹病毒.假 性狂犬病毒的基因表現是經由階段性的相互調 節控制而依序表現.一般而言,皰疹病毒的基因 可依其表現的階段性而分為三類:即發性早期 、早期、及晚期基因.即發性早期基因及許多 的晚期基因已被研究,但對假性狂犬病毒的早 期基因方面,除了胸腺核甘激鋂(TK)基因有被研 究外,尚乏其它有關早期基因的研究報告.早期 基因在假性狂犬病的複製及病毒的毒力等方面 扮演著相當重要的角色.筆者等已在今年度計 畫中建立了PRV cDNA的基因庫,下年度的計畫擬從 已建立的PRV cDNA 基因庫分析其為何基因,並擬 分下列四項工作進行:(一)PRV 早期轉錄體之定 性;(二)PRV早期轉錄體出現次序之探討;(三)PRV 轉錄體起動子之研究;(四)PRV 早期基因新基因 之確認.由於早期基因所表現的蛋白是負責調 控晚期蛋白(包括結構性蛋白)的合成的病毒蛋 白,故對於這些早期基因的確認及它對病毒複 製所扮演的角色的了解,將有助於對本病毒生 物學的了解,從而有助於研究者尋求控制本病 的良方.關鍵詞:假性狂犬病毒,早期基因,基因 選殖,核酸定序