Genotyping Cryptococcus neoformans var. neoformans with specific primers designed from PCR-fingerprinting bands sequenced using a modified PCR-based strategy
Genotyping Cryptococcus neoformans var. neoformans with specific primers designed from PCR-fingerprinting bands sequenced using a modified PCR-based strategy
复制标题
DOI：
10.1080/714030933
复制
发表时间：
2000-04-01
期刊：
影响因子：
2.9
通讯作者：
Viviani, MA
中科院分区：
文献类型：
--
作者：
Cogliati, M;Allaria, M;Viviani, MA
基于使用（GACA）（4）引物通过聚合酶链反应（PCR）指纹图谱获得的四条主要条带（420、475、540和800 bp）的不同组合，新型隐球菌变种可被细分为六种基因型（VN 1-VN 6）。本研究的目的是确定能够扩增这些条带的特异性引物。采用改进的基于PCR的测序策略来克服使用（GACA）（4）作为单循环测序引物的局限性。用五种限制性内切酶消化由具有两个末端（GACA）（4）序列的扩增子组成的原始条带。选择产生两个或三个片段的消化产物。预期每个片段具有不超过一个末端（GACA）（4）序列，使得循环测序成为可能。将片段纯化并用（GACA）（4）引物测序。然后设计针对四个主要条带中的每一个的新引物，并使用纯化的条带和PCR指纹产物作为模板对剩余区域进行测序。利用这些引物对12 C. neoformans菌株和五种其它酵母菌菌株。新的引物，作为单独的对或在所有对的混合物中使用，仅在C中扩增预期的条带。neoformans var，neoformans菌株，证实了选择用于该酵母分子分型的条带的种属特异性。
Cryptocuccus neoformans var, neoformans can be subdivided into six genotypes (VN1-VN6) based on different combinations of four major bands (420, 475, 540 and 800 bp) obtained by polymerase chain reaction (PCR) fingerprinting using the (GACA)(4) primer. The aim of this study was to identify specific primers able to amplify these bands. A modified PCR-based sequencing strategy was adopted to overcome the limitations of using (GACA)(4) as a single cycle sequencing primer. The original bands, made up of amplicons with two terminal (GACA)(4) sequences, were digested with five restriction enzymes. Digestion products yielding two or three fragments were selected. Each fragment was expected to have no more than one terminal (GACA)(4) sequence, making cycle sequencing possible. Fragments were purified and sequenced with the (GACA)(4) primer. New primers specific for each of the four major bands were then designed and the remaining regions were sequenced using both purified bands and PCR-fingerprinting products as template. These primers were used to amplify the genomic DNA of 12 C. neoformans strains and five strains of other yeast species. The new primers, used as separate pairs or in a mixture of all pairs, amplified the expected bands only in C. neoformans var, neoformans strains, confirming the species specificity of the bands selected for molecular typing of this yeast.