Abstract
Porcine epidemic diarrhea virus (PEDV) is a highly transmissible coronavirus that causes a severe enteric disease particularly in neonatal piglets. In this study, a rapid method for detecting PEDV was developed based on cross-priming amplification and nucleic acid test strip(CPA-NATS). Five primers specific for the N gene sequence of PEDV were used for the cross-priming amplification. Detection of amplification products based on labeled probe primers was conducted with strip binding antibody of labeled markers. The CPA method was evaluated and compared with a PCR method. The reverse transcription CPA system was further optimized for detecting PEDV RNA in clinical specimens. Results showed that the method was highly specific for the detection of PEDV, and had the same sensitivity as PCR, with detection limit of 10−6 diluted plasmid containing the target gene of PEDV. It was also successfully applied to detecting PEDV in clinical specimens. The reverse transcription CPA-NATS detection system established in this study offers a specific, sensitive, rapid, and simple detection tool for screening PEDV, which can contribute to strategies in the effective control of PEDV in swine.
Highlights
The genome of Porcine epidemic diarrhea virus (PEDV) is approximately 28 kb nucleotides in length
As one-step diagnostic system, RT cross-priming amplification (CPA)-NATS was capable of detecting PEDV in clinical specimens and displayed 100% specificity against several related porcine viruses
It has already been applied to clinical diagnosis of human infectious diseases such as tuberculosis[8], where a double crossing CPA was used to amplify DNA of Mycobacterium tuberculosis
Summary
The genome of PEDV is approximately 28 kb nucleotides in length. At least seven open reading frames (ORFs) have been identified in the PEDV genome, with a characteristic gene order 5′ -replicase (1a/1b)-S-ORF3-E-M-N-3′. Several methods including separation identification, serological method, PCR, and colloidal gold method have been used to detect PEDV. The amplification consisted of three steps: (1) extension and displacement with cross primer, (2) multiple extensions and www.nature.com/scientificreports/. Two red bands indicate a PEDV-positive result, whereas one red band indicates a PEDV-negative result. This method is able to amplify approximately four bacterial cells in less than an hour with a high degree of specificity[7]. It does not require an initial denaturation step or a nicking enzyme and has a high specificity and sensitivity. The system offers a relatively quick and easy method for field diagnostic testing of pathogens
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