key: cord-0985920-g2lod9nh
authors: Zhou, Ling; Chen, Yonghui; Fang, Xueen; Liu, Yanhong; Du, Mengkan; Lu, Xiandong; Li, Qianniu; Sun, Yuan; Ma, Jingyun; Lan, Tian
title: Microfluidic-RT-LAMP chip for the point-of-care detection of emerging and re-emerging enteric coronaviruses in swine
date: 2020-05-19
journal: Anal Chim Acta
DOI: 10.1016/j.aca.2020.05.034
sha: 64570c31c33d1f472894e24bab32f683ff3301c1
doc_id: 985920
cord_uid: g2lod9nh

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome-coronavirus (SADS-CoV) are three emerging and re-emerging coronaviruses (CoVs). Symptoms caused by these three viruses are extremely similar, including acute diarrhea, vomiting and even death in piglets. To date, strict biosecurity is still the most effective disease prevention and control measures, and the early detection of pathogens is the most important link. Here, we developed a microfluidic-RT-LAMP chip detection system for the first time, which could detected PEDV, PDCoV and SADS-CoV simultaneously, and had advantages of rapid, simple, sensitive, high-throughput, and accurate at point-of-care settings. The lowest detection limits of the microfluidic-RT-LAMP chip method are 10(1) copies/μL, 10(2) copies/μL and 10(2) copies/μL for PEDV, PDCoV and SADS-CoV, respectively. The whole detection procedure can be finished rapidly in 40 min without any cross-reaction with other common swine viruses. A total of 173 clinical swine fecal samples characterized with diarrheal symptoms were used to evaluate the performance of the newly developed system, which presented good stabilities (C.V.s<5%) and specificities (100%), and possessed sensitivities of 92.24%, 92.19% and 91.23% for PEDV, PDCoV and SADS-CoV respectively. In summary, the established microfluidic-RT-LAMP chip detection system could satisfy the demanding in field diagnoses, which was suitable for promotion in remote areas due to its fast, portable and cost-effective characters.

Diarrhea disease is one of major threats that afflict the global pork industry in 55 recent years, which has caused an estimated loss of multi-million dollar annually. 56 Various pathogens that can cause diarrhea in swine have been identified, and swine 57 enteric coronaviruses are the most destructive pathogens that are able to result 58 vomiting, dehydration and lethal watery diarrhea in piglets [1] . To date, four swine 59 enteric coronaviruses have been defined, including three Alphacoronaviruses widely applied for detecting and identifying pathogens [27] [28] [29] [30] . 89 Up to now, the predominant control measures of swine diarrhea disease are the 90 rapid and accurate diagnoses of pathogens at the early stage to prevent further 91 circulations in swine. Currently, the most commonly detection methods for swine 92 enteric coronaviruses such as ELISA (enzyme-linked immunosorbent assay), PCR and 93 IHC (immunohistochemistry), all depend on laboratory diagnosis and possessed the 94 advantages of high sensitivity and accuracy, and have been widely used [31] [32] [33] [34] . 95 However， most of these diagnostic assays are complex, time-consuming, and require a 96 large of reagents and well-equipped laboratories, which limit their application in 97 remote areas. Therefore, it is urgent to develop portable POC devices with characters 98 of simple, timely, multi-target and strong pertinence, so as to detect pathogens early, showed that the developed microfluidic-RT-LAMP chip system was accurate, 111 sensitive, specific and reproducible for point-of-care diagnosis, which was advantaged 112 for the early diagnosis of diarrhea in swine farms, especially in remote areas, and 113 adopting effective measures to control the spread of the diseases. The microfluidic chip made of PMMA that was used in this study was a CD-shape 132 with a diameter of 80 mm and a thickness of 2.5 mm, which was fabricated by the 133 micro-injection molding technology. To form an integrated microfluidic chip, a kind 134 of pressure-sensitive film was applied to seal the micro-channel of chip via suitable 135 pressure. The microfluidic chip comprised four independent units, each of which was 136 consisted of a sample well, a vent and eight micro reaction wells ( Figure 1A ). The 137 ball valve played a significant role throughout the process ensuring that the reaction 138 mixture diffused into the adjacent pipe and the reaction wells were filled with liquid.

The volume of each micro reaction well was 5 μL. The supporting microfluidic To ensure the accuracy of the three LAMP systems, the amplification products were 172 further determined by standard agarose gel electrophoresis. Table 1 .

To determine the accuracy of the LAMP reaction, the target templates were 227 successfully amplified from the target-contained sample and presented ladder-like 228 bands with agarose gel electrophoresis ( Figure 2D ). 287 The specificity, sensitivity and reproducibility test results have illustrated that the 288 microfluidic-RT-LAMP chip detection system is stable, rapid, convenient and portable.

To further verify the actual performance of the microfluidic-RT-LAMP chip detection 

Real-time reverse 466 transcription recombinase polymerase amplification assay for rapid detection of 467 porcine epidemic diarrhea virus

Point-of-care diagnostic assay for rapid detection of porcine 470 deltacoronavirus using the recombinase polymerase amplification method

Microfluidic 473 tectonics: a comprehensive construction platform for microfluidic systems

Point-of-Care Detection of African Swine Fever Virus with a Median Time to 477 Threshold in about 10 min

In this research, we firstly integrated microfluidic chip with the RT-LAMP technology for the high-throughput and multi-target detection of three emerging and re-emerging swine coronaviruses

The microfluidic-RT-LAMP chip system established greatly facilitated the rapid, specific and sensitive diagnosis of the PEDV

The application of cost-effective microfluidic chip device is characterized with excellent prospects in swine enteropathogenic coronaviruses monitoring and control for remote livestock farms

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: