Development And Preliminary Application Of Duplex Nest-PCR Detection Method For Three Intestinal Protozoa In Swine

Cystoisospora suis,Cryptosporidium,Enterocytozoon bieneusi are severely harmful,and often transmitted widely via the fecal-oral route,they not only has caused great economic losses to China’s animal husbandry industry,for the late two are zoonotic protozoa,so they also threaten public health security.There is only one species known as Cystoisospora suis,which is considered to be the main cause of swine coccidiosis and it can infected pigs of different breeds at different growth stages and led clincal symptoms of water-like non-hemorrhagic diarrhea and significant weight loss,especially inlactating and weaning piglets.Delayed treatment can lead to piglet death or stiffness.Cryptosporidium infection in humans and animals gastrointestinal tract usually manifests as acute and chronic diarrhea,especially in patients with immunodeficiency syndrome,can cause self-limited diarrhea,even death.There is no obvious clinical symptom after E.bieneusi infection.HIV patients and children with low immune function often develop persistent diarrhea with fever,decreased appetite,and weight loss.Piglets infected with E.bieneusi can cause malabsorption and thus affect its growth and development.At present,the detection of the three pathogens is mainly a conventional PCR method,but the method can detect only one pathogen at a time,which is time-consuming and laborious in clinical application.So far,there is no duplex nest-PCR method for simultaneous detection of Cystoisospora suis and Cryptosporidium/E.bieneusi,,this study aims to establish a duplex-nest-PCR detection method with strong specificity,high sensitivity,and good reproducibility,in order to be able to detect Cystoisospora suis and Ctyptosporidium/E.bieneusi simultaneously,and provide efficient and rapid detection means of these three pathogens for pig farms.1.Establishment and preliminary application of duplex nest-PCR detection method for Cystocystis suis and E.bieneusi enteritidisWith Cystoisospora suis SSU rRNA and E.bieneusi ITS primers were synthesised based on references,the duplex nest-PCR reaction system and reaction conditions for the detection of the two pathogens were optimized,and specificity,sensitivity,repeatability tests,and then the clinical sample tests were performed.The results showed that the two-step annealing temperature of this method was determined to be 57℃ and 55℃,respectively,and when Cystoisospora suis/E.bieneusi primer ratio is 0.25/1.0 μL,Cystoisospora suis(530 bp)and E.bieneusi(392 bp)can be specifically amplified.,while C.andersoni,G.duodenalis,Blastocystis,E.coli have no specific amplification,the result showed that the method specificity is good;The minimum DNA dilution of Cystoisospora suis and E.bieneusi is 1×10-5 and 1×10-7,that is,the minimum DNA detection amounts are 209×10-5 ng/μL and 527×10-7 ng/μL,respectively;and the method repeatability is good.Cystoisospora suis and E.bieneusi were used to amplify 48 swine stool DNA samples using single nest-PCR and duplex-nest-PCR.The results showed that there was no significant difference in the detection rate of Cystoisospora suis and E.bieneusi between single-nest and duplex-nest-PCR(P>0.05).The rapid,accurate,and simple duplex-PCR method for the simultaneous diagnosis of Cystoisospora suis and E.bieneusi established in this study provides a basis for the diagnosis of intestinal protozoosis in pig farms,and it is useful for the prevention and control of two intestinal protozoa disease.2.Establishment and preliminary application of duplex nest-PCR detection method for Cystocystis suis and CryptosporidiumA duplex-nest-PCR method was developed based on the SSU rRNA loci of Cystoisospora suis and Cryptosporidium to detect the two pathogens(the primers were synthesised according to references).First,the main parameters of the reaction system were optimized,and the established method was tested for specificity,sensitivity,repeatability,and then the clinical samples were detected too.The Cystoisospora Suis and Cryptosporidium duplex nest-PCR method was successfully established.The results showed that the annealing temperature was finally determined to be 55 0C,and the optimal primer ratio of is 0.25/1.0 μL.The nest-PCR established could amplify targeted fragments of 530 bp(Cystoisospora suis)and 830 bp(Cryptosporidium),respectively,and no cross-infection with E.bieneusi,G.duodenalis,E.coli,the result showed that the method specificity is good;the minimum DNA dilutions which can be detected of Cystoisospora suis and Cryptosporidium are 1×10-4 and 1×10-3;that is,the minimum detection amounts are 80.6×10-4 ng/μL and 9.8×10-3 ng/μL,respectively;and the repeatability test is good too.Cystoisospora suis and Cryptosporidium were amplified from 62 pig stool DNA samples using the duplex-nest-PCR method established in this experiment.The results showed that there was no significant difference in the detection results of Cystoisospora suis and Cryptosporidium by single nest-PCR or duplex nest-PCR(P>0.05).It shows that the duplex nest-PCR of Cystoisospora suis and Cryptosporidium can be used for clinical sample detection.