Development of A Super-Sensitive Diagnostic Method for African Swine Fever Using CRISPR Techniques

Ren, Meishen; Hong, Ming; Zhou, Ming; Fu, Zhen F.; Han, Hao; Bi, Dingren; Peng, Fuhu; Zhao, Ling

doi:10.1007/s12250-020-00323-1

Cited by 16 publications

(10 citation statements)

References 40 publications

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“…RPA assays for the detection of ASFV based on separate steps of amplification and visualization using lateral flow were developed [ 33 , 46 ]. The clinical sensitivities of these assays were ranging between 70 and 100% with a turnaround time of 30 min.…”

Section: Discussionmentioning

confidence: 99%

“…This approach is subjective to high cross-contamination risk since the post-amplification pipetting step is needed to transfer the amplicon to the lateral flow cartridge. RPA assay relying on CRISPR as a reporter is highly sensitive but has a runtime similar to the real-time PCR and the reagents must be stored at −20 °C [ 46 ]. Other ASFV-RPA amplification monitoring based on SYBRE Green dye is not field applicable because of the need to open the post-amplification tube to add the dye [ 47 ].…”

Section: Discussionmentioning

confidence: 99%

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Rapid Extraction and Detection of African Swine Fever Virus DNA Based on Isothermal Recombinase Polymerase Amplification Assay

Ceruti

Kobialka

Ssekitoleko

et al. 2021

Viruses

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African swine fever virus (ASFV) is the causative agent of a deadly disease in pigs and is spread rapidly across borders. Samples collected from suspected cases must be sent to the reference laboratory for diagnosis using polymerase chain reaction (PCR). In this study, we aimed to develop a simple DNA isolation step and real-time recombinase polymerase amplification (RPA) assay for rapid detection of ASFV. RPA assay based on the p72 encoding B646L gene of ASFV was established. The assays limit of detection and cross-reactivity were investigated. Diagnostic performance was examined using 73 blood and serum samples. Two extraction approaches were tested: silica-column-based extraction method and simple non-purification DNA isolation (lysis buffer and heating, 70 °C for 20 min). All results were compared with well-established real-time PCR. In a field deployment during a disease outbreak event in Uganda, 20 whole blood samples were tested. The assay’s analytical sensitivity was 3.5 DNA copies of molecular standard per µL as determined by probit analysis on eight independent assay runs. The ASFV RPA assay only detected ASFV genotypes. Compared to real-time PCR, RPA diagnostic sensitivity and specificity were 100%. Using the heating/lysis buffer extraction procedure, ASFV-RPA revealed better tolerance to inhibitors than real-time PCR (97% and 38% positivity rate, respectively). In Uganda, infected animals were identified before the appearance of fever. The ASFV-RPA assay is shown to be as sensitive and specific as real-time PCR. Moreover, the combination of the simple extraction protocol allows its use at the point of need to improve control measures.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Rapid Extraction and Detection of African Swine Fever Virus DNA Based on Isothermal Recombinase Polymerase Amplification Assay

Ceruti

Kobialka

Ssekitoleko

et al. 2021

Viruses

View full text Add to dashboard Cite

show abstract

“…Chen et al have combined the RPA and Cas12a-crRNA complex to establish a novel method for the rapid identification of HPV16 and 18, and named it the DETECTR (DNA endonuclease-targeted CRISPR trans reporter) detection system [ 23 ]. Ren et al have developed an RPA-CRISPR-lwCas13a-based nucleic acid detection method to diagnose ASF, which can detect as little as a single copy of ASFV plasmid and genomic DNA with sensitivity comparable to or even higher than those of qPCR methods [ 26 ]. Broughton et al have reported a CRISPR-Cas12a-based diagnostic tool for rapid detection of SARS-CoV-2.…”

Section: Discussionmentioning

confidence: 99%

Reverse transcription–enzymatic recombinase amplification coupled with CRISPR-Cas12a for rapid detection and differentiation of PEDV wild-type strains and attenuated vaccine strains

Yang

Liang

et al. 2021

Anal Bioanal Chem

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Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes acute watery diarrhea and vomiting in unweaned piglets, and is associated with high mortality, thus causing severe economic losses in the pig industry. Currently, although attenuated vaccines are commonly used in commercial pig farms in China, they do not completely protect against all mutated wild-type strains. Existing nucleic acid assays have high sensitivity and specificity, but the complexity of the assay process and expensive instrumentation hinder disease detection. Here, reverse transcription–enzymatic recombinase amplification (RT-ERA) was combined with the CRISPR-Cas12a system to develop a rapid diagnostic method to distinguish PEDV wild-type strains from attenuated vaccine strains. The protocol used crRNA and RT-ERA amplification primers against open reading frame 3 (ORF3), followed by Cas12a/crRNA complex detection of predefined target sequences at 37 °C for 30 min, thus producing results visible to the naked eye under LED blue light. The assay is highly sensitive and specific, detecting as few as two copies of the target gene per test and showing no cross-reactivity with other porcine pathogens. Overall, this integrated RT-ERA pre-amplification and Cas12a/crRNA cleavage assay is a practical tool for reliable and rapid detection of PEDV for diagnostic differentiation. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03716-7.

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“…The assays based on immunology include colloidal gold test strip assay [ 4 ], time-resolved fluorescence immunoassay [ 5 ], fluorescent immunochromatography test strip [ 6 ], amplified luminescent proximity homogeneous assay (AlphaLISA) [ 7 ], triplex bead-based assay for the simultaneous detection of antibodies [ 8 ], Eu (III) chelate microparticle-based lateral flow test strip and blocking enzyme-linked immunosorbent assay [ 2 9 ]. Many nucleic acid amplification methods have been improved or modified for the detection of ASFV, such as multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay [ 10 ], triplex real-time PCR assay [ 11 ], real-time loop-mediated isothermal amplification (LAMP) assay [ 12 13 ], LAMP combined with lateral flow dipstick [ 14 ], semi-quantitative colorimetric LAMP [ 15 ], recombinase polymerase amplification (RPA) combined with lateral flow [ 16 17 18 ], PCR with a lateral flow strip [ 19 ], improved real-time PCR system with probe modification [ 20 ], CRISPR-Cas12a system coupled with PCR or LAMP [ 21 ], recombinase aided amplification combined with CRISPR/Cas12a [ 22 ], RPA-CRISPR-based nucleic acid detection method [ 23 ] and Crispr/cas12a technology combined with immunochromatographic strips [ 24 ]. The microfluidic-circular fluorescent probe-mediated isothermal nucleic acid amplification (CFPA) system has been newly developed for the detection of ASFV [ 25 ], and advanced nanopore sequencing has also been used for the diagnosis of ASFV [ 26 ].…”

Section: Introductionmentioning

confidence: 99%

Development of a ladder-shape melting temperature isothermal amplification (LMTIA) assay for detection of African swine fever virus (ASFV)

Wang

et al. 2022

J Vet Sci

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Background Due to the unavailability of an effective vaccine or antiviral drug against the African swine fever virus (ASFV), rapid diagnosis methods are needed to prevent highly contagious African swine fever. Objectives The objective of this study was to establish the ladder-shape melting temperature isothermal amplification (LMTIA) assay for the detection of ASFV. Methods LMTIA primers were designed with the p72 gene of ASFV as the target, and plasmid pUC57 was used to clone the gene. The LMTIA reaction system was optimized with the plasmid as the positive control, and the performance of the LMTIA assay was compared with that of the commercial real-time polymerase chain reaction (PCR) kit in terms of sensitivity and detection rate using 200 serum samples. Results Our results showed that the LMTIA assay could detect the 10 4 dilution of DNA extracted from the positive reference serum sample, which was the same as that of the commercial real-time PCR kit. The coincidence rate between the two assays was 100%. Conclusions The LMTIA assay had high sensitivity, good detection, and simple operation. Thus, it is suitable for facilitating preliminary and cost-effective surveillance for the prevention and control of ASFV.

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Development of A Super-Sensitive Diagnostic Method for African Swine Fever Using CRISPR Techniques

Cited by 16 publications

References 40 publications

Rapid Extraction and Detection of African Swine Fever Virus DNA Based on Isothermal Recombinase Polymerase Amplification Assay

Rapid Extraction and Detection of African Swine Fever Virus DNA Based on Isothermal Recombinase Polymerase Amplification Assay

Reverse transcription–enzymatic recombinase amplification coupled with CRISPR-Cas12a for rapid detection and differentiation of PEDV wild-type strains and attenuated vaccine strains

Development of a ladder-shape melting temperature isothermal amplification (LMTIA) assay for detection of African swine fever virus (ASFV)

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