Establishment Of The Conventional PCR And Nano-PCR Detection Methods For Eimeria Necatrix

Eimeria necatrix is one of the main pathogenic species causing intestinal coccidiosis in chickens,leading to chicken malabsorption,lower feed conversion rate,blood dysentery,and even mass death.E.necatrix infection often induces Clostridium perfringens infection,causing necrotizing enteritis.The diagnosis of E.necatrix infection is generally based on parasitic site,pathogenicity,intestinal pathology,oocyst morphology.These techniques require professional technicians and are difficult to identify mixed infections.With the development of molecular biology technology,polymerase chain reaction(PCR)has been widely used in detection and identification of pathogens due to its advantages of fast,high efficiency and sensitivity.In recent years,nanoparticle-assisted PCR(nano-PCR)based on the nanotechnology has been proved with higher sensitivity and specificity,but there are no reports about the use of nano-PCRs to detect E.necatrix infection.Based on this,the present study used E.necatrix as the research object,and,through screening specific target genes and optimizing reaction system and conditions,established conventional PCR and nano-PCR methods for specific detection of E.necatrix infection and E.necatrix sporulated oocysts,as well as the duplex PCR and duplex nano-PCR methods for the simultaneous detection of E.necatrix and C.perfringens.The results are as following:1.Established the conventional PCR and nano-PCR methods for the detection of E.necatrix based on the ITS-2 gene: A partial gene fragment of E.necatrix ITS-2 gene was selected,and the conventional PCR and nano-PCR methods for the detection of E.necatrix were established,respectively.Both methods could amplify the target fragment of about 380 bp of E.necatrix.The sensitivity of conventional PCR was 364 pg,while the sensitivity of nano-PCR was 3.64 pg;the conventional PCR had no amplifications for E.tenella,Cryptosporidium baileyi,C.parvum,Giardia lamblia,Blastocystis sp..As for Enterocytozoon bieneusi and Balantidium coli,there were non-specific amplifications but none of them matched the size of the target gene.Nano-PCR had no amplifications of the above pathogens.The conventional PCR and nano-PCR were used to detect E.necatrix infection in 20 clinical chicken faecal samples,and the positive rates were 35.0% for both methods.2.Established the duplex PCR and duplex nano-PCR methods for the detection of E.necatrix and C.perfringens: The partial gene fragment of E.necatrix ITS-2 gene was selected to design specific primers,and the other pair of primers to amplify C.perfringens αtoxin gene was selected from a previous literature.The duplex PCR and duplex nano-PCR detection methods for the simultaneous amplification of E.necatrix and C.perfringens were established,both of which could amplify the target fragments of about 150 bp for E.necatrix and about 400 bp for C.perfringens.Conducting sensitivity test by constructing recombinant plasmids p MD19-T-ITS2 and p MD19-T-cpα,we found that the sensitivities of duplex PCR were 181 copies for E.necatrix and 1050 copies for C.perfringens,while the sensitivities of duplex nano-PCR were 1.81 copies for E.necatrix and 105 copies for C.perfringens.The duplex PCR had no amplifications for E.bieneusi,T.gallinae,C.baileyi,E.tenella.As for G.lamblia,E.bieneusi and Blastocystis sp.,there were non-specific amplifications but none of them matched the size of target genes.The duplex nano-PCR had no amplifications of the above pathogens.The duplex PCR and duplex nano-PCR were used to detect E.necatrix and C.perfringens in 20 samples with g DNA samples artificially added,and the detection rates of E.necatrix were 100% for both methods.The detection rates of C.perfringens were77.8% and 100% for duplex PCR and duplex nano-PCR,respectively.The detection rates of mixed samples of E.necatrix and C.perfringens were 75.0% and 100% for duplex PCR and duplex nano-PCR,respectively.3.Established the conventional PCR and nano-PCR methods for the specific detection of E.necatrix sporulated oocysts: Through RNA-seq analysis of unsporulated oocysts and sporulated oocysts,152 specific genes for sporulated oocysts were found.Six genes were selected for PCR verification,and two sporulated oocyst-specific genes(ENH＿00008300and ENH＿00076120)were identified.The conventional PCR and nano-PCR for specific detection of E.necatrix sporulated oocysts were established based on these two genes.Both conventional PCR and nano-PCR based on ENH＿00008300 gene could specifically amplify about 450 bp target fragment of E.necatrix sporulated oocysts,while there were no amplifications for unsporulated oocysts;the sensitivities of conventional PCR and nano-PCR were 25 ng and 3 ng;the conventional PCR and nano-PCR based on ENH＿00076120 gene could specifically amplify about 280 bp target fragment of E.necatrix sporulated oocysts,but there were no amplifications for unsporulated oocysts.The sensitivities of conventional PCR and nano-PCR for this gene were 400 pg and 50 pg.In conclusion,the conventional PCR and nano-PCR methods for the detection of E.necatrix,as well as the duplex PCR and duplex nano-PCR method for the simultaneously detection of E.necatrix and C.perfringens were established in our study.The results provide technical support for the rapid diagnosis of E.necatrix infection and the differential diagnosis of secondary C.perfringens infection.In addition,the conventional PCR and nano-PCR methods for the specific detection of E.necatrix sporulated oocysts were established,which realized the specific identification of E.necatrix sporulated oocysts from unsporulated oocysts at the molecular level with high sensitivity,making up for the deficiency of traditional morphological observation.