scholarly journals LAMP assay coupled with CRISPR/Cas12a system for portable detection of African swine fever virus

2021 ◽  
Author(s):  
Bo YANG ◽  
zhengwang shi ◽  
Yuan Ma ◽  
Lijuan Wang ◽  
Liyan Cao ◽  
...  
Keyword(s):  
Real Time ◽  
Detection System ◽  
African Swine Fever Virus ◽  
Low Cost ◽  
African Swine Fever ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Portable Detection ◽  
Real Time Qpcr

African swine fever (ASF) is one of the most severe infectious diseases of pigs. In this study, a LAMP assay coupled with the CRISPR Cas12a system was established in one tube for the detection of the ASFV p72 gene. The single-strand DNA-fluorophore-quencher (ssDNA-FQ) reporters and CRISPR-derived RNA (crRNAs) were screened and selected for the CRISPR detection system. In combination with LAMP amplification assay, the detection limit for the LAMP-CRISPR assay can reach 7 copies/μl of p72 gene per reaction. Furthermore, this method displays no cross-reactivity with other porcine DNA or RNA viruses. The performance of the LAMP-CRISPR assay was compared with real-time qPCR tests for clinical samples, a good consistency between the LAMP-CRISPR assay and real-time qPCR was observed. In the current study, a LAMP coupled with the CRISPR detection method was developed. The method shed a light on the convenient, portable, low cost, highly sensitive and specific detection of ASFV, demonstrating a great application potential for monitoring on-site ASFV in the field.

scholarly journals LAMP assay coupled with CRISPR/Cas12a system for portable detection of African swine fever virus

2021 ◽  
Author(s):  
Bo YANG ◽  
zhengwang shi ◽  
Yuan Ma ◽  
Lijuan Wang ◽  
Liyan Cao ◽  
...  
Keyword(s):  
Real Time ◽  
Detection System ◽  
African Swine Fever Virus ◽  
Low Cost ◽  
African Swine Fever ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Portable Detection ◽  
Real Time Qpcr

African swine fever (ASF) is one of the most severe infectious diseases of pigs. In this study, a LAMP assay coupled with the CRISPR Cas12a system was established in one tube for the detection of the ASFV p72 gene. The single-strand DNA-fluorophore-quencher (ssDNA-FQ) reporter and CRISPR-derived RNA (crRNAs) were screened and selected for the CRISPR detection system. In combination with LAMP amplification assay, the detection limit for the LAMP-CRISPR assay can reach 7 copies/μl of p72 gene per reaction. Furthermore, this method displays no cross-reactivity with other porcine DNA or RNA viruses. The performance of the LAMP-CRISPR assay was compared with real-time qPCR tests for clinical samples, a good consistency between the LAMP-CRISPR assay and real-time qPCR was observed. The method shed a light on the convenient, portable, low cost, highly sensitive and specific detection of ASFV, demonstrating a great application potential for monitoring on-site ASFV in the field.

scholarly journals Field Verification of an African Swine Fever Virus Loop-Mediated Isothermal Amplification (LAMP) Assay during an Outbreak in Timor-Leste

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1444
Author(s):  
Peter T. Mee ◽  
Shani Wong ◽  
Kim J. O’Riley ◽  
Felisiano da Conceição ◽  
Joanita Bendita da Costa Jong ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Lamp Assay ◽  
Fever Virus ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification ◽  
Field Verification ◽  
Extraction Step ◽  
Timor Leste

Recent outbreaks of African swine fever virus (ASFV) have seen the movement of this virus into multiple new regions with devastating impact. Many of these outbreaks are occurring in remote, or resource-limited areas, that do not have access to molecular laboratories. Loop-mediated isothermal amplification (LAMP) is a rapid point of care test that can overcome a range of inhibitors. We outline further development of a real-time ASFV LAMP, including field verification during an outbreak in Timor-Leste. To increase field applicability, the extraction step was removed and an internal amplification control (IAC) was implemented. Assay performance was assessed in six different sample matrices and verified for a range of clinical samples. A LAMP detection limit of 400 copies/rxn was determined based on synthetic positive control spikes. A colourmetric LAMP assay was also assessed on serum samples. Comparison of the LAMP assay to a quantitative polymerase chain reaction (qPCR) was performed on clinical ASFV samples, using both serum and oral/rectal swabs, with a substantial level of agreement observed. The further verification of the ASFV LAMP assay, removal of extraction step, implementation of an IAC and the assessment of a range of sample matrix, further support the use of this assay for rapid in-field detection of ASFV.

scholarly journals Development of Real-Time PCR Based on A137R Gene for the Detection of African Swine Fever Virus

2021 ◽  
Vol 8 ◽  
Author(s):  
Dan Yin ◽  
Renhao Geng ◽  
Hui Lv ◽  
Chunhui Bao ◽  
Hongxia Shao ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Economic Losses ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Taqman Pcr ◽  
Control And Eradication

African swine fever virus (ASFV) can infect domestic pigs and wild boars and causes huge economic losses in global swine industry. Therefore, early diagnosis of ASFV is important for the control and eradication of African swine fever (ASF). In this study, a SYBR Green-based real-time polymerase chain reaction (PCR) assay targeting the viral encoded A137R gene was established for the detection of ASFV infection. For the evaluation of the established real-time PCR, 34 clinical samples were assessed by both the A137R gene-based real-time PCR and OIE-recommended TaqMan PCR. The results showed that 85.29% (29/34) were detected by A137R gene-based real-time PCR, but only 79.41% (27/34) positive using OIE-recommended TaqMan PCR. Moreover, no cross-reaction with other common swine pathogens was found in the A137R gene-based real-time PCR. These results demonstrated that the established real-time PCR assay in this study showed better performance than the OIE-recommended method in detecting ASFV from clinical samples, which could be applied for control and eradication programs of ASF.

scholarly journals Direct colorimetric LAMP assay for in-field detection of African swine fever virus: a validation study during an outbreak in Vietnam

2020 ◽  
Author(s):  
Diem Hong Tran ◽  
Hau Thi Tran ◽  
Uyen Phuong Le ◽  
Xuan Dang Vu ◽  
Thi Bich Ngoc Trinh ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
African Swine Fever Virus ◽  
Animal Health ◽  
Virus Detection ◽  
African Swine Fever ◽  
Lamp Assay ◽  
Serum Samples ◽  
Field Detection

ABSTRACTAfrican Swine Fever (ASF) is a highly infectious viral disease with high mortality. The most recent ASF outbreak in Vietnam occurred in 2019, posing a threat to spread to the neighboring Asian countries. Without a commercial vaccine or efficient chemotherapeutics successfully developed, rapid diagnosis and necessary biosecurity procedures are required to control the disease. While the diagnosis method of ASF recommended by the World Organization of Animal Health is real-time PCR, it is not suitable for in-field detection of the disease. In this study, a colorimetric Loop-Mediated Isothermal Amplification (LAMP) assay was developed and evaluated for ASF virus detection using crude serum samples collected from domestic pigs in Vietnam during the 2019 outbreak. The LAMP results can be readily visualized to naked eyes within 30 minutes without the requirement of DNA extraction and sophisticated equipment. The sensitivity, specificity, and limit of detection of colorimetric LAMP assay were comparable to a commercial diagnostic real-time PCR kit. Results strongly indicate that the developed colorimetric LAMP assay is highly recommended for the in-field diagnosis of ASF.

scholarly journals An improvement of real-time polymerase chain reaction system based on probe modification is required for accurate detection of African swine fever virus in clinical samples in Vietnam

2020 ◽  
Vol 33 (10) ◽  
pp. 1683-1690
Author(s):  
Ha Thi Thanh Tran ◽  
Anh Kieu Dang ◽  
Duc Viet Ly ◽  
Hao Thi Vu ◽  
Tuan Van Hoang ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Binding Sites ◽  
African Swine Fever Virus ◽  
False Negative ◽  
African Swine Fever ◽  
Clinical Samples ◽  
Probe Sequence ◽  
Field Samples ◽  
Polymerase Chain

Objective: The rapid and reliable detection of the African swine fever virus (ASFV) plays an important role in emergency control and preventive measures of ASF. Some methods have been recommended by FAO/OIE to detect ASFV in clinical samples, including realtime polymerase chain reaction (PCR). However, mismatches in primer and probe binding regions may cause a false-negative result. Here, a slight modification in probe sequence has been conducted to improve the qualification of real-time PCR based on World Organization for Animal Health (OIE) protocol for accurate detection of ASFV in field samples in Vietnam.Methods: Seven positive confirmed samples (four samples have no mismatch, and three samples contained one mutation in probe binding sites) were used to establish novel real-time PCR with slightly modified probe (Y = C or T) in comparison with original probe recommended by OIE.Results: Both real-time PCRs using the OIE-recommended probe and novel modified probe can detect ASFV in clinical samples without mismatch in probe binding site. A high correlation of cycle quantification (Cq) values was observed in which Cq values obtained from both probes arranged from 22 to 25, suggesting that modified probe sequence does not impede the qualification of real-time PCR to detect ASFV in clinical samples. However, the samples with one mutation in probe binding sites were ASFV negative with OIE recommended probe but positive with our modified probe (Cq value ranked between 33.12-35.78).Conclusion: We demonstrated for the first time that a mismatch in probe binding regions caused a false negative result by OIE recommended real-time PCR, and a slightly modified probe is required to enhance the sensitivity and obtain an ASF accurate diagnosis in field samples in Vietnam.

scholarly journals Evaluation of mobile real-time polymerase chain reaction tests for the detection of severe acute respiratory syndrome coronavirus 2

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chukwunonso Onyilagha ◽  
Henna Mistry ◽  
Peter Marszal ◽  
Mathieu Pinette ◽  
Darwyn Kobasa ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Point Of Care ◽  
Middle Eastern ◽  
Turnaround Time ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Diarrhea Virus ◽  
Highly Sensitive ◽  
Transmissible Gastroenteritis

AbstractThe coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), calls for prompt and accurate diagnosis and rapid turnaround time for test results to limit transmission. Here, we evaluated two independent molecular assays, the Biomeme SARS-CoV-2 test, and the Precision Biomonitoring TripleLock SARS-CoV-2 test on a field-deployable point-of-care real-time PCR instrument, Franklin three9, in combination with Biomeme M1 Sample Prep Cartridge Kit for RNA 2.0 (M1) manual extraction system for rapid, specific, and sensitive detection of SARS-COV-2 in cell culture, human, and animal clinical samples. The Biomeme SARS-CoV-2 assay, which simultaneously detects two viral targets, the orf1ab and S genes, and the Precision Biomonitoring TripleLock SARS-CoV-2 assay that targets the 5′ untranslated region (5′ UTR) and the envelope (E) gene of SARS-CoV-2 were highly sensitive and detected as low as 15 SARS-CoV-2 genome copies per reaction. In addition, the two assays were specific and showed no cross-reactivity with Middle Eastern respiratory syndrome coronavirus (MERS-CoV), infectious bronchitis virus (IBV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis (TGE) virus, and other common human respiratory viruses and bacterial pathogens. Also, both assays were highly reproducible across different operators and instruments. When used to test animal samples, both assays equally detected SARS-CoV-2 genetic materials in the swabs from SARS-CoV-2-infected hamsters. The M1 lysis buffer completely inactivated SARS-CoV-2 within 10 min at room temperature enabling safe handling of clinical samples. Collectively, these results show that the Biomeme and Precision Biomonitoring TripleLock SARS-CoV-2 mobile testing platforms could reliably and promptly detect SARS-CoV-2 in both human and animal clinical samples in approximately an hour and can be used in remote areas or health care settings not traditionally serviced by a microbiology laboratory.

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