scholarly journals Development and Clinical Validation of a Potential Penside Colorimetric Loop-Mediated Isothermal Amplification Assay of Porcine Circovirus Type 3

2022 ◽  
Vol 12 ◽  
Author(s):  
Jie Zhang ◽  
Miaomiao Li ◽  
Yunwen Ou ◽  
Danian Chen ◽  
Yaozhong Ding ◽  
...  
Keyword(s):  
Epidemiological Study ◽  
Mainland China ◽  
Porcine Circovirus Type ◽  
Isothermal Amplification ◽  
Porcine Circovirus ◽  
Lower Sensitivity ◽  
Loop Mediated Isothermal Amplification ◽  
One Step ◽  
Type 3 ◽  
Taqman Qpcr

Porcine circovirus type 3 (PCV3), a novel circovirus, imposes great burdens on the global pig industry. The penside tests for detecting PCV3 are critical for assessing the epidemiological status and working out disease prevention and control programs due to the unavailability of a commercial vaccine. A one-step molecular assay based on visual loop-mediated isothermal amplification (vLAMP) was developed for simple and rapid detection of PCV3. We compared its sensitivity and specificity with TaqMan quantitative real-time polymerase chain reaction (qPCR) and applied the developed assay in the epidemiological study of (n = 407) pooled swine sera collected from almost the entire mainland China during the years 2017–2018. We also explored the feasibility of the vLAMP assay for detecting raw samples without a prior DNA isolation step to expand its application capability. Results showed that the vLAMP assay could reliably detect the PCV3 cap gene with a detection limit of 10 DNA copies equal to that of the Taqman qPCR assay. In the epidemiological study, the PCV3 positive detection rate for 407 swine pooled sera detected by the vLAMP assay was 37.35% (152/407), whereas it was 39.01% (159/407) for Taqman qPCR. For the detection method without genome extraction, the results kept satisfactory specificity (100%) but displayed lower sensitivity (100% for CT < 32), indicating the direct detection is not sensitive enough to discriminate the samples with low viral loads. The one-step vLAMP is a convenient, rapid, and cost-effective diagnostic for penside detection and will enable the epidemiological surveillance of PCV3, which has widely spread in mainland China.

scholarly journals Detection of porcine circovirus type 1 in commercial porcine vaccines by loop-mediated isothermal amplification

2011 ◽  
Vol 56 (6) ◽  
pp. 483-489 ◽  
Author(s):  
Chun Wang ◽  
Victor Fei Pang ◽  
Chian-Ren Jeng ◽  
Fan Lee ◽  
Yu-Wen Huang ◽  
...  
Keyword(s):  
Porcine Circovirus Type ◽  
Isothermal Amplification ◽  
Porcine Circovirus ◽  
Loop Mediated Isothermal Amplification

scholarly journals Development of a reverse transcription loop mediated isothermal amplification assay for the detection of Mouse reovirus type 3 in laboratory mice

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Taofeng Lu ◽  
Lingyun Tao ◽  
Haibo Yu ◽  
Hui Zhang ◽  
Yanjun Wu ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Laboratory Mice ◽  
Blood Samples ◽  
S1 Gene ◽  
Loop Mediated Isothermal Amplification ◽  
Reovirus Type ◽  
One Step ◽  
Type 3

AbstractMouse reovirus type 3 (Reo-3) infection is a viral disease that is harmful for laboratory mice. No rapid and accurate detection methods are currently available for this infection. In this study, we describe a rapid, simple, closed-tube, one step, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for Reo-3 and compare our assay with indirect enzyme-linked immunosorbent assay (ELISA). Three sets of RT-LAMP primers were designed by sequence analysis of a specific conserved sequence of the Reo-3 S1 gene. Using RS2 primer set, the RT-LAMP assay required 60 min at 65 °C to amplify the S1 gene in one step by using Reo-3 RNA template and had no cross-reactivity with the other related pathogens, such as Sendai virus (SV), pneumonia virus of mice (PVM), mouse hepatitis virus (MHV), Ectromelia virus (Ect), minute virus of mice (MVM), P. pneumotropica, B. bronchiseptica, K. pneumonia and P. aeruginosa. in our LAMP reaction system. The limit of detection (LOD) of our RT-LAMP assay is 4 fg/μL. The established RT-LAMP assay enabled visual detection when fluorescence detection reagents were added, and was demonstrated to be effective and efficient. We tested 30 clinical blood samples and five artificial positive samples from SPF mice, the concordance between the two methods for blood samples was 100% compared with indirect ELISA and RT-PCR. Considering its performance, specificity, sensitivity, and repeatability, the developed RT-LAMP could be a valuable tool to supply a more effective Reo-3 detection method in laboratory animal quality monitoring.

scholarly journals Loop-mediated isothermal amplification for detection of porcine circovirus type 2

2011 ◽  
Vol 8 (1) ◽  
pp. 497 ◽  
Author(s):  
Shun Zhou ◽  
Si Han ◽  
Jianli Shi ◽  
Jiaqiang Wu ◽  
Xiaoyuan Yuan ◽  
...  
Keyword(s):  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Isothermal Amplification ◽  
Porcine Circovirus ◽  
Loop Mediated Isothermal Amplification

scholarly journals Development and validation of a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of ZIKV in patient samples from Brazil

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Severino Jefferson Ribeiro da Silva ◽  
Keith Pardee ◽  
Udeni B. R. Balasuriya ◽  
Lindomar Pena
Keyword(s):  
Reverse Transcription ◽  
Rapid Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Serum Samples ◽  
Low Resource ◽  
Loop Mediated Isothermal Amplification ◽  
One Step

AbstractWe have previously developed and validated a one-step assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of the Zika virus (ZIKV) from mosquito samples. Patient diagnosis of ZIKV is currently carried out in centralized laboratories using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR), which, while the gold standard molecular method, has several drawbacks for use in remote and low-resource settings, such as high cost and the need of specialized equipment. Point-of-care (POC) diagnostic platforms have the potential to overcome these limitations, especially in low-resource countries where ZIKV is endemic. With this in mind, here we optimized and validated our RT-LAMP assay for rapid detection of ZIKV from patient samples. We found that the assay detected ZIKV from diverse sample types (serum, urine, saliva, and semen) in as little as 20 min, without RNA extraction. The RT-LAMP assay was highly specific and up to 100 times more sensitive than RT-qPCR. We then validated the assay using 100 patient serum samples collected from suspected cases of arbovirus infection in the state of Pernambuco, which was at the epicenter of the last Zika epidemic. Analysis of the results, in comparison to RT-qPCR, found that the ZIKV RT-LAMP assay provided sensitivity of 100%, specificity of 93.75%, and an overall accuracy of 95.00%. Taken together, the RT-LAMP assay provides a straightforward and inexpensive alternative for the diagnosis of ZIKV from patients and has the potential to increase diagnostic capacity in ZIKV-affected areas, particularly in low and middle-income countries.

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