“双甲酸夹心”快速样本前处理方法的建立及在丝状真菌质谱鉴定中的初步应用

Establishment of Fast Pretreatment Method for Sample with Double Formate Sandwichmethod and Preliminary Application in Identification of Filamentous Fungiby the Bruker Microflex LT MALDI-TOF Mass Spectrometer

目的解决布鲁克microflex LT MALDI-TOF质谱仪在临床微生物学实验室丝状真菌鉴定中样本处理技术操作繁琐,耗时较长的弊端,建立一种快速、简便的丝状真菌质谱鉴定样本前处理方法。方法以2017年1月~2018年3月陕西省人民医院门诊及住院患者临床标本分离到的各种丝状真菌为研究对象,采用"双甲酸夹心"法进行上机前处理,即于96孔不锈钢靶板上用无菌微量吸头滴加1μl甲酸,再选取培养基上生长的丝状真菌菌落(幼龄菌丝体最好)用蘸取少许甲酸溶液的无菌牙签挑取菌丝体与甲酸溶液混合并进行充分研磨,待其自然干燥后在其表面再次滴加1μl的甲酸溶液,干燥后再加1μl基质液(仪器配套),待干燥后即可上机。对检测结果采用形态学和基因测序方法进行确证。结果新建方法前处理鉴定的丝状真菌有曲霉菌、青霉菌、拟青霉、镰刀菌、皮肤癣菌、接合菌、暗色真菌、双向真菌,并且对一些形态不典型或少见丝状真菌都能正确鉴定到种,如裂褶菌、红色单囊菌等。共鉴定菌株158株,其中113株可鉴定到种,31株鉴定到属,无法鉴定14株。结论新建立的丝状真菌样本快速前处理方法,大大缩短了样本前处理实验流程,具有快速、简便、高效、极低实验成本、鉴定正确率较高的特点,可快速为临床提供丝状真菌鉴定结果,使患者得到及时有效的抗真菌治疗。

Objective To solve the disadvantages of the Ccmplicated and time-consuming operation of sample processing tech- nology for the identification of Filamentous fungi in the clinical microbiology laboratory by the bruker microflex LT MAL- DI-TOF mass spectrometer and establish a fast and simple method for pre-processing samples in the identification of Fila- mentous fungi by mass spectrometry. Methods Various Filamentous fungi isolated from clinical specimens of outpatients and inpatients in the Shaanxi Provincial People＇s Hospital from January 2017 to March 2018 were selected as research ob- jects. Using ＂double formate sandwich＂ method for pre-processing,that was, 1 μ1 of formic acid was added dropwise to a 96- well stainless steel target plate,and a mycelium was picked by a sterile toothpick with formic acid solution. The mycelium was mixed with a formic acid solution,and once it was naturally dried, 1μ1 of formicacid was again dropped on the surface. The solution was dried and then added with 1 μ1 of the matrix solution. After drying,the lysate was ready for use. The test results were confirmed by morphological and gene sequencing methods. Results Filaments,Penicillium, Paecilomyces,Fu- sarium, Dermatophytes, Zgomycetes, Dark-colored fungi, Bidirectional fungi, and some rare genus fungi could be correctly identified in newly estabished methods. 158 strains were identified, among which 113 strains could be identified as species, 31 strains were identified as genus,and 14 strains could hot be identified. Conehtsion The newly established rapid pretreatment method for Filamentous fungal samples had greatly shortened the sample pretreatment process and it has the characteristics of rapidity, simplicity, high efficiency, extremely low experimental cost, and high accuracy. It can provide clinically rapid iden- tification of Filamentous fungi. As a result,the patient is given timely and effective antifungal treatment.