微小隐孢子虫RPA-CRISPR/Cas12a可视化检测方法的建立及应用

doi:10.16431/j.cnki.1671-7236.2023.12.002

Abstract

Abstract: 【Objective】 This study was aimed to developed a rapid,accurate,and sensitive visualization detection method for Cryptosporidium parvum (C. parvum). 【Method】 In this study,the recombinase polymerase amplification (RPA) was combined with the CRISPR/Cas12a system,and the results were read by the fluorescence reporter system and the lateral flow strip biosensor. The crRNA and RPA primers were designed and screened against the small subunit ribosome RNA (SSU rRNA) gene of C. parvum,and two different single-stranded DNA (ssDNA) reporters were used to present the detection results by fluorescence values and the LFS biosensor.The specificity of the RPA-CRISPR/Cas12a assay was verified by detecting C. parvum and 7 other pathogens.The sensitivity of the RPA-CRISPR/Cas12a detection method was evaluated by serially diluted recombinant plasmids and C. parvum genome,and the method was applied to clinical sample detection.【Result】 The RPA-CRISPR/Cas12a assay established in this study could detect C. parvum in samples within 90 min,and had no cross-reactivity with Escherichia coli,Giardia duodenalis,Clonorchis sinensis,Toxoplasma gondii,Fasciola hepatica,Pentatrimonas hominis,and Salmonella,the method could specifically detect C. parvum.The sensitivity test results showed that the minimum detection limit was 10 oocysts/mL.Using this method to detect 70 cattle feces samples and 50 human feces samples,the positive rate of C. parvum was 15.7% (11/70) and 6.0% (3/50) after reading the results by the fluorescent reporter system and lateral flow strips biosensor,which was completely consistent with the results of the nested PCR.【Conclusion】 The RPA-CRISPR/Cas12a assay for C. parvum had high specificity and sensitivity with intuitive results,without relying on expensive instrumentation,and had broad application value as a new strategy for more rapid and portable identification of C. parvum on-site and risk monitoring.

Key words: CRISPR/Cas12a; Cryptosporidium parvum; recombinase polymerase amplification; visual diagnostic method

CLC Number:

S852.72

WANG Li, LI Shan, LI Lu, ZHANG Xichen, WANG Xiaocen, LI Xin, ZHANG Nan, GONG Pengtao. Establishment and Application of the RPA-CRISPR/Cas12a Visual Diagnostic Method for Cryptosporidium parvum[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(12): 4793-4804.

References

[1] HASSAN E M,ÖRMECI B,DEROSA M C,et al.A review of Cryptosporidium spp.and their detection in water[J].Water Science and Technology,2021,83(1):1-25.
[2] KING P,TYLER K M,HUNTER P R.Anthroponotic transmission of Cryptosporidium parvum predominates in countries with poorer sanitation:A systematic review and meta-analysis[J].Parasites&Vectors,2019,12(1):16.
[3] BOULTER-BITZER J I,LEE H,TREVORS J T.Molecular targets for detection and immunotherapy in Cryptosporidium parvum[J].Biotechnology Advances,2007,25(1):13-44.
[4] RYAN U,ZAHEDI A,PAPARINI A.Cryptosporidium in humans and animals-A one health approach to prophylaxis[J].Parasite Immunology,2016,38(9):535-547.
[5] CHEN Y,HUANG J,QIN H,et al.Cryptosporidium parvum and gp60 genotype prevalence in dairy calves worldwide:A systematic review and meta-analysis[J].Acta Tropica,2023,240:106843.
[6] HIJJAWI N,ZAHEDI A,AL-FALAH M,et al.A review of the molecular epidemiology of Cryptosporidium spp.and Giardia duodenalis in the Middle East and North Africa (MENA) region[J].Infection Genetics and Evolution,2022,98:105212.
[7] WANG R J,LI J Q,CHEN Y C,et al.Widespread occurrence of Cryptosporidium infections in patients with HIV/AIDS:Epidemiology,clinical feature,diagnosis,and therapy[J].Acta Tropica,2018,187:257-263.
[8] 满达,兰丽,王艳霞,等.牛微小隐孢子虫表面抗原CP15基因原核表达及其表达产物的鉴定[J].中国畜牧兽医,2012,39(6):50-53. MAN D,LAN L,WANG Y X,et al.Prokaryotic expression and identification of Cryptosporidium parvum surface antigen CPl5 gene in Escherichia coli BL21[J].China Animal Husbandry&Veterinary Medicine,2012,39(6):50-53.(in Chinese)
[9] 李家诚,张龙现,何国声.微小隐孢子虫研究进展[J].国际医学寄生虫病杂志,2008,35(2):100-105. LI J C,ZHANG L X,HE G S.Research advance on Cryptosporidium parvum[J].International Journal of Medical Parasitic Diseases,2008,35(2):100-105.(in Chinese)
[10] 杨子吟,曹建平,沈玉娟,等.几种新现人体隐孢子虫种和基因型流行现状[J].中国人兽共患病学报,2017,33(2):148-155. YANG Z Y,CAO J P,SHEN Y J,et al.Epidemiology status of some new emerging Cryptosporidium species and genotypes in humans[J].Chinese Journal of Zoonoses,2017,33(2):148-155.(in Chinese)
[11] 黎巧,肖冉,陶婧,等.奶牛隐孢子虫虫种和基因型的国内研究进展[J].中国奶牛,2020,6:26-30. LI Q,XIAO R,TAO J,et al.Advances in the species and genotypes of Cryptosporidium among dairy cows in China[J].China Dairy Cattle,2020,6:26-30.(in Chinese)
[12] HELMY Y A,HAFEZ H M.Cryptosporidiosis:From prevention to treatment,a narrative review[J].Microorganisms,2022,10(12):2456.
[13] FOX L M,SARAVOLATZ L D.Nitazoxanide:A new thiazolide antiparasitic agent[J].Clinical Infectious Diseases,2005,40(8):1173-1180.
[14] RYAN U,HIJJAWI N.New developments in Cryptosporidium research[J].International Journal for Parasitology,2015,45(6):367-373.
[15] CHALMERS R M,CAMPBELL B M,CROUCH N,et al.Comparison of diagnostic sensitivity and specificity of seven Cryptosporidium assays used in the UK[J].Journal of Medical Microbiology,2011,60(Pt 11):1598-1604.
[16] JOHNSTON S P,BALLARD M M,BEACH M J,et al.Evaluation of three commercial assays for detection of Giardia and Cryptosporidium organisms in fecal specimens[J].Journal of Clinical Microbiology,2003,41(2):623-626.
[17] ALALI F,ABBAS I,JAWAD M,et al.Cryptosporidium infection in humans and animals from Iraq:A review[J].Acta Tropica,2021,220:105946.
[18] WANG J,WANG X,GONG P,et al.The protective role of TLR2 mediates impaired autophagic Flux by activating the mTOR pathway during Neospora caninum infection in mice[J].Frontiers in Cellular and Infection Microbiology,2021,11:788340.
[19] MOHANRAJU P,MOUGIAKO I,ALBERS J,et al.Development of a Cas12a-based genome editing tool for moderate thermophiles[J].Crispr Journal,2021,4(1):82-91.
[20] CHEN J S,MA E,HARRINGTON L B,et al.CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity[J].Science,2018,360(6387):436-439.
[21] KIM S,JI S,KOH H R.CRISPR as a diagnostic tool[J].Biomolecules,2021,11(8):1162.
[22] DAI Y,WU Y,LIU G,et al.CRISPR mediated biosensing toward understanding cellular biology and point-of-care diagnosis[J].Angewandte Chemie-international Edition,2020,59(47):20754-20766.
[23] WANG M,ZHANG R,LI J.CRISPR/cas systems redefine nucleic acid detection:Principles and methods[J].Biosensors&Bioelectronics,2020,165:112430.
[24] YIN L,MAN S,YE S,et al.CRISPR-Cas based virus detection:Recent advances and perspectives[J].Biosensors&Bioelectronics,2021,193:113541.
[25] LI J,MACDONALD J,VON STETTEN F.Review:A comprehensive summary of a decade development of the recombinase polymerase amplification[J].Analyst,2018,144(1):31-67.
[26] TSOU J H,LENG Q,JIANG F.A CRISPR test for detection of circulating nuclei acids[J].Translational Oncology,2019,12(12):1566-1573.
[27] HUANG W,WEI H,WANG C,et al.Establishment and preliminary evaluation of a fluorescent recombinase-aided amplification/CRISPR-Cas12a system for rapid detection of Plasmodium falciparum[J].Chinese Journal of Schistosomiasis Control,2023,35(1):38-43.
[28] CHENG P,WU Y,GUO S,et al.RPA assay coupled with CRISPR/Cas12a system for the detection of seven Eimeria species in chicken fecal samples[J].Veterinary Parasitology,2022,311:109810.
[29] LI S,WANG X,YU Y,et al.Establishment and application of a CRISPR-Cas12a-based RPA-LFS and fluorescence for the detection of Trichomonas vaginalis[J].Parasites&Vectors,2022,15(1):350.
[30] DUEÑAS E,NAKAMOTO J A,CABRERA-SOSA L,et al.Novel CRISPR-based detection of Leishmania species[J]. Frontiers in Microbiology,2022,13:958693.
[31] MA Q N,WANG M,ZHENG L B,et al.RAA-Cas12a-Tg:A nucleic acid detection system for Toxoplasma gondii based on CRISPR-Cas12a combined with recombinase-aided amplification (RAA)[J].Microorganisms,2021,9(8):1644.
[32] KANITCHINDA S,SRISALA J,SUEBSING R,et al.CRISPR-Cas fluorescent cleavage assay coupled with recombinase polymerase amplification for sensitive and specific detection of Enterocytozoon hepatopenaei[J].Biotechnology Reports(Amsterdam, Netherlands),2020,27:e00485.
[33] ALVES M,XIAO L,SULAIMAN I,et al.Subgenotype analysis of Cryptosporidium isolates from humans,cattle,and zoo ruminants in Portugal[J].Journal of Clinical Microbiology,2003,41(6):2744-2747.
[34] HUANG W,GUO Y,LYSEN C,et al.Multiple introductions and recombination events underlie the emergence of a hyper-transmissible Cryptosporidium hominis subtype in the USA[J].Cell Host&Microbe,2023,31(1):112-123.e4.
[35] MTHETHWA N P,AMOAH I D,REDDY P,et al.Fluorescence and colorimetric LAMP-based Real-time detection of human pathogenic Cryptosporidium spp.from environmental samples[J].Acta Tropica,2022,235:106606.
[36] WU Z,NAGANO I,MATSUO A,et al.Specific PCR primers for Cryptosporidium parvum with extra high sensitivity[J].Molecular and Cellular Probes,2000,14(1):33-39.
[37] OCHIAI Y,TAKADA C,HOSAKA M.Detection and discrimination of Cryptosporidium parvum and C.hominis in water samples by immunomagnetic separation-PCR[J].Applied and Environmental Microbiolog,2005,71(2):898-903.
[38] LOBATO I M,O'SULLIVAN C K.Recombinase polymerase amplification:Basics,applications and recent advances[J].Trac-trends in Analytical Chemistry,2018,98:19-35.
[39] LIU P F,ZHAO K R,LIU Z J,et al.Cas12a-based electrochemiluminescence biosensor for target amplification-free DNA detection[J].Biosensors&Bioelectronics,2021,176:112954.
[40] JIANG Y,HU M,LIU A A,et al.Detection of SARS-CoV-2 by CRISPR/Cas12a-enhanced colorimetry[J].Acs Sensors,2021,6(3):1086-1093.
[41] MYHRVOLD C,FREIJE C A,GOOTENBERG J S,et al.Field-deployable viral diagnostics using CRISPR-Cas13[J]. Science,2018,360(6387):444-448.
[42] DING X,YIN K,LI Z,et al.Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay[J].Nature Communications,2020,11(1):4711.
[43] ZHANG A,SUN B,ZHANG J,et al.CRISPR/Cas12a coupled with recombinase polymerase amplification for sensitive and specific detection of Aphelenchoides besseyi[J].Frontiers in Bioengineering and Biotechnology,2022,10:912959.
[44] GONG C,CAO X F,DENG L,et al.Epidemiology of Cryptosporidium infection in cattle in China:A review[J].Parasite,2017,24:1.
[45] ZHANG Z,HU S,ZHAO W,et al.Population structure and geographical segregation of Cryptosporidium parvum Ⅱd subtypes in cattle in China[J].Parasites&Vectors,2020,13(1):425.
[46] LI N,WANG R,CAI M,et al.Outbreak of cryptosporidiosis due to Cryptosporidium parvum subtypeⅡdA19G1 in neonatal calves on a dairy farm in China[J].International Journal for Parasitology,2019,49(7):569-577.
[47] FENG Y,XIAO L.Molecular epidemiology of cryptosporidiosis in China[J].Frontiers in Microbiology,2017,8:1701.
[48] FOZOUNI P,SON S,DÍAZ DE LEÓN DERBY M,et al.Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy[J].Cell,2021,184(2):323-333.e9.
[49] NGUYEN L T,SMITH B M,JAIN P K.Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection[J].Nature Communications,2020,11(1):4906.
[50] GOOTENBERG J S,ABUDAYYEH O O,KELLNER M J,et al.Multiplexed and portable nucleic acid detection platform with Cas13,Cas12a,and Csm6[J].Science,2018,360(6387):439-444.

Establishment and Application of the RPA-CRISPR/Cas12a Visual Diagnostic Method for Cryptosporidium parvum

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 4

Recommended Articles

Metrics

Comments

[1]	SHI Meiyi, YANG Falong. RPA and Its Application in Detection of Animal Pathogens [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(2): 467-476.
[2]	LV Ji-zhou, FAN Yan-ru, FENG Chun-yan, YUAN Xiang-fen, WU Shao-qiang. Establishment of Real-time Recombinase Polymerase Amplification for Detection of Porcine Epidemic Diarrhoea Virus [J]. , 2017, 44(12): 3434-3439.
[3]	HADENG Chu-riya, FAN Xiao-xu, ZHAO Yong-gang, WANG Shu-juan, ZHANG Zhi-cheng, GE Sheng-qiang, LI Lin, WU Xiao-dong, WANG Zhi-liang. Establishment of a Real-time Fluorescent Recombinase Polymerase Amplification (RPA) for the Detection of African Swine Fever Virus [J]. , 2017, 44(11): 3270-3277.
[4]	LIU Li-bing, WANG Jian-chang, JING Mei, WANG Jin-feng, YUAN Wan-zhe. Development of the Recombinase Polymerase Amplification Assay for Rapid Detection of Porcine Parvovirus [J]. , 2017, 44(11): 3320-3326.