Detection Of Cryptococcus Neoformans By Recombinant Polymerase Technique And Retrospective Analysis Of Clinical Data

Objective 1.The diagnosis and treatment of Cryptococcus neoformans were discussed by analyzing the medical history of patients infected with Cryptococcus neoformans.2.Preliminary establishment of a point-of-care,POC method for immediate detection of Cryptococcus neoformans.Methods: 1.The medical history of 38 patients with Cryptococcus neoformans infection treated in Tianjin first Central Hospital from February 2015 to August 2018 were analyzed retrospectively,and their basic diseases,clinical manifestations,laboratory examination,treatment and prognosis were analyzed.2.Using recombinant enzyme polymerase amplification technique(recombinase polymera-se amplification,RPA),specific primers were designed for the conservative sequence of Cryptococcus neoformans genome,and a simple,rapid,efficient and sensitive method for the detection of Cryptococcus neoformans was established..Results:1.38 patients were diagnosed by etiology,including 31 cases of blood,5cases of cerebrospinal fluid and 2 cases of pathological tissue.Treatment:amphotericin B,5-flucytosine,fluconazole,voriconazole or combination.Among the38 patients,1 case was transferred to hospital,3 cases died,2 cases gave up treatment,and 32 cases were discharged.2.The results of gel RPA detection of Cryptococcus neoformans: the primers were designed to amplify the genomic DNA extracted by ATCC32609 and detected by gel electrophoresis,and the genomic DNA of the extracted Cryptococcus neoformans was diluted at 1:5,1:10,1:50,1:100,1:500 and1:1000,respectively,and the sensitivity of the primer and reagent was semi-quantitatively detected.The positive bands of Cryptococcus neoformans could still be seen by 1:1000 diluted template DNA gel electrophoresis.The sequencing results of the amplified products were compared by blast on the pubmed website,which belonged to the DNA fragment encoding ribosomal RNA of Cryptococcus neoformans,and the coincidence rate was 100%.The RPA of Cryptococcus neoformans was consistent in qualitative detection of clinical samples compared with real-time fluorescence PCR,RPA and oasig real-time fluorescence PCR.Conclusion: 1.Cryptococcus neoformans can not only cause central nervous systeminfection,but also cause multi-site infection of the whole body,so we should be on guard against non-central nervous system occult infection.Early etiological detection is very important for the diagnosis,treatment and prognosis of the disease.2.It is proved by experiments that the gel RPA technique can qualitatively detect Cryptococcus neoformans and the designed primers are highly specific.