Molecular detection and serotyping of infectious bronchitis virus from FTA® filter paper
Molecular detection and serotyping of infectious bronchitis virus from FTA® filter paper
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DOI：
10.1637/7220
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发表时间：
2005-03-01
期刊：
影响因子：
1.4
通讯作者：
Hofacre, CL
中科院分区：
文献类型：
--
作者：
Moscoso, H;Raybon, EO;Hofacre, CL
我们研究了使用Flinders Technology Associates（FTA（R））滤卡储存含有传染性支气管炎病毒（IBV）的尿囊液的可行性，如阿肯色州DPI、康涅狄格州和马萨诸塞州，并通过逆转录酶（RT）-聚合酶链反应（PCR）进行鉴定，以及通过限制性片段长度多态性（RFLP）或核苷酸测序进行表征。FTA（R）纸是一种棉基纤维素膜，含有冻干的化学物质，可溶解多种类型的细菌和病毒。IBV在与FTA（R）接触时被灭活，如病毒不能在鸡胚中繁殖所示。SI基因的RT-PCR显示，尿囊液中的病毒RNA在FTA（R）过滤卡上储存后保持稳定，并且对于大的（1700碱基对[bp]）而不是小的（383 bp）PCR产物，稳定性是时间和温度敏感的。对扩增产物的分析表明，在不利环境条件（41 ℃）下储存于FTA（R）上至少15天的尿囊液中进行分子表征是可行的。使用FTA（R）卡采集、运输和储存含有IBV的样本是安全、廉价的，并且足以进行分子诊断。我们建议从海外进口的FTA（R）卡上的样本将首先通过RT-PCR进行分析，引物产生1700 bp的产物，然后对阳性病例进行RFLP。阴性病例将用产生383 bp产物的引物进行分析（以排除储存条件的不利影响），然后对阳性病例进行核苷酸测序。
We investigated the feasibility of using Flinders Technology Associates (FTA (R)) filter cards for the storage of allantoic fluid containing an infectious bronchitis virus (IBV), such as Arkansas-DPI, Connecticut, and Massachusetts, and for their identification by reverse transcriptase (RT)-polymerase chain reaction (PCR) and characterization by restriction fragment length polymorphism (RFLP) or nucleotide sequencing. FTA (R) paper is a cotton-based cellulose membrane containing lyophilized chemicals that lyses many types of bacteria and viruses. IBV was inactivated upon contact with the FTA (R), as shown by the inability of the virus to be propagated in embryonating chicken eggs. RT-PCR of the SI gene showed that viral RNA in allantoic fluid remained stable after storage on FTA (R) filter cards and that the stability was time and temperature sensitive for the large (1700 base pair [bp]) but not the small (383 bp) PCR products. Analysis of the amplified products showed that molecular characterization is feasible in allantoic fluid stored on FTA (R) under nonfavorable environmental conditions (41 C) for at least 15 days. The use of FTA (R) cards for the collection, transport, and storage of IBV-containing samples is safe, inexpensive, and adequate for molecular diagnosis. We propose that specimens coming from overseas on FTA (R) cards would be first analyzed by RT-PCR with primers yielding a 1700-bp product followed by RFLP of the positive cases. Negative cases would be analyzed with primers yielding a 383bp product (to exclude detrimental effect of the storage conditions) followed by nucleotide sequencing of the positive cases.