Development Of LAMP-microfluidic High-throughput Diagnostic Chip For Common Dermatophytes Infection

Dermatophytes is a very common superficial fungi,which is a filamentous fungi,often invading keratin-rich matrix growth,lesions involving human skin,hair and nails(toenails)and so on.The common dermatophytes in clinic are divided into three genera:Trichophyton spp.,Microsporum spp.and Epidermophyton spp,which mainly including more than 30 pathogenic bacteria such as Trichophyton rubrum,Trichophyton mentagrophyte,Trichophyton violaceum,Trichophyton tonsurans,Trichophyton verrucosum,Trichophyton schoenleinii,Microsporum canis,Microsporum gypseum,Microsporum ferrugineum and Epidermophyton floccosum and so on.Skin infection caused by dermatophytes is the highest incidence of superficial mycosis.With the gradual aging of the population in China,the wide application of clinical immunosuppressive agents,and the abuse of glucocorticoid preparations for external use,the incidence of superficial mycosis is increasing year by year.At present,the clinical diagnosis of dermatophytes infection is mainly microscopic examination and culture.The presence or absence of spores or hyphae under the microscope is observed by microscope.After culture,it mainly depends on the examination of the total colony morphology and the microscopic characteristics of conidia and other mycelial structures.It is supplemented by mating tests and biochemical tests,such as urease or nutrition tests.However,the positive rate of microscopic examination is low,and the operation requirements of technicians are high,so the fungi can not be identified to the species level.However,the culture took a long time,generally about 2 weeks.Histopathological examination has no practical clinical significance for superficial dermatophytes infection,and it is difficult for patients to accept subjectively.The sample size of clinical dermatophytes infection is huge,so there is an urgent need for an early diagnosis method which can not only diagnose quickly,but also take into account the cost-effectiveness.In order to better meet the needs of early diagnosis of clinical fungal infection,a series of molecular diagnostic techniques for pathogenic fungal nucleic acid and protein have begun experimental research,some of which have been applied in clinical practice.Molecular diagnosis technology achieves the purpose of accurate diagnosis by analyzing and differentiating specific components or sequence fragments such as proteins and nucleic acids of pathogenic microorganisms.Such techniques are specific,sensitive,take a short time from sampling to reporting,and can complete diagnosis in one day or even hours.In this study,ring-mediated isothermal amplification was combined with microfluidic chip carrier to develop a set of diagnostic methods which can realize early and rapid diagnosis,at the same time,it is efficient and cheap.Ring-mediated isothermal amplification(Loop-mediated isothermal amplification,LAMP)is a technique that enables high amplification of nucleic acids at constant temperature,which eliminates the temperature cycle required for polymerase chain reaction(PCR).At the same time,a similar amplified yield was obtained.LAMP technology not only has strong specificity and sensitivity,but also has low requirements for experimental conditions because of its principle of constant temperature amplification,which is convenient for clinical promotion.At present,LAMP technology has been used in the diagnosis of a variety of fungi,viruses,bacteria and mycobacteria and other pathogenic microorganisms.The microfluidic chip technology is the use of microchannels for experimental reaction and sample detection,which has the characteristics of miniaturization and integration.According to the design of the channel,the required microenvironment conditions can be generated on the chip.Using microfluidic chip technology can realize the simultaneous identification of dozens or even hundreds of samples in a short period of time.In this study,three sets of primers for three genera of dermatophytes were designed.Firstly,the rDNA sequences(including 18s,ITS1,5.8s,ITS2 and 28s)of dermatophytes in each genus were downloaded by Pubmed.Through Geneious software analysis and comparison,the target gene of each species in the genus was found to be highly approximated.The target gene fragment should be obviously different from the sequence of other genera or the sequence of adjacent strains on the phylogenetic tree.Then the primers were designed by LAMP primer design software Primer ExplorerV4(two external primers,F3 and B3;two internal primers,FIP and BIP,could increase the ring primers LB and LF to shorten the reaction time).Then the primers were screened,the reaction system was optimized,and the specificity and sensitivity of the system were tested.Then the verified system was loaded into the microfluidic chip for further verification.Finally,the collected clinical specimens were used for clinical verification.A total of 113 strains were studied,there were 20 strains of Trichophyton rubrum,20strains of Trichophyton mentagrophyte,11 strains of Trichophyton violaceum,4 strains of Trichophyton tonsurans,3 strains of Trichophyton verrucosum,8 strains of Trichophyton schoenleinii,16 strains of Microsporum canis,3 strains of Microsporum ferrugineum,6strains of Epidermophyton floccosum,and also there were 5 strains of Candida,5 strains of Malassezia,4 strains of Aspergillus,2 strains of Penicillium,2 strains of Rhodotorula,4 strains of bacteria and 1 human DNA(sigma).The nucleic acid of the strains in each genus was the positive sample of the primers corresponding to the genus,and the nucleic acid of the other strains was the control sample.It was found that the specificity of LAMP for identifying common dermatophytes in Trichophyton spp.,Microsporum spp.and Epidermophyton spp.could reach 100%,and the sensitivity could reach about 1×10~2 copies/?L.After verification,the reaction system has also been well verified in the microfluidic chip,and the specificity and sensitivity are the same as LAMP technology.