An Improved Polymerase Cross-Linking Spiral Reaction Assay for Rapid Diagnostic of Canine Parvovirus 2 Infection

Abstract

With increasing complications of canine parvovirus infection cases, disease diagnosis and treatment have become more difficult. In this study, specificity primers for the conserved region of the VP2 gene of canine parvovirus 2 (CPV-2) were synthesized and evaluated. An improved polymerase cross-linking spiral reaction (PCLSR) method for early and rapid diagnosis of CPV-2 was established. The results showed that the amplification reaction was optimal when run at 62 • C for 50 min and could be used to detect CPV-2 without any cross-reactions with other pathogens of canine infectious diseases. Reaction results were directly judged by the naked eyes, with the positive amplification tube shown as luminous yellow and the negative tube as bright purple. Compared with the previously reported polymerase spiral reaction (PSR) method for CPV-2 detection, this reaction was performed using improved primer pairs and a better dye identification method (using an indicator comprising phenol red and cresol red). The detection limit of PCLSR was 3.9 × 10 1 copies using gel electrophoresis or a visible dye. The positive rate of 132 clinical samples was 42.42%, which was identically the same as that of the PSR method and slightly higher than that of the colloidal gold strip method (39.39%). The newly developed CPV-PCLSR assay shows the advantage of rapid visualization of results and offers a convenient and rapid method for early CPV-2 diagnosis with higher sensitivity and specificity than the established methods.

Rapid detection of Actinobacillus pleuropneumoniae targeting the apxIVA gene for diagnosis of contagious porcine pleuropneumonia in pigs by polymerase spiral reaction

“…PSR was not only sensitive but also rapid, already giving results after 30 min of incubation. In most reports, the minimum detection time by PSR was 45–60 min (Ashmi et al ., 2021; Das et al ., 2018; Dutta et al ., 2019; Gupta et al ., 2017; Malla et al ., 2018; Milton et al ., 2021; Xu et al ., 2020).…”

“…The high specificity reported here was probably because of highly specific primers for the A. pleuropneumoniae apxIVA gene (Zhou et al ., 2008). Similar levels of sensitivity of real‐time PCR and PSR have also been reported by other workers (Ashmi et al ., 2021; Das et al ., 2018; Gupta et al ., 2017; Malla et al ., 2018; Milton et al ., 2021; Xu et al ., 2020). And the technology is ten times more sensitive compared to traditional PCR (Milton et al ., 2021).…”

“…The polymerase spiral reaction (PSR) is the most recent inclusion in the list of INAT, which can amplify the target DNA with one pair of primers and a single enzyme (Dong et al ., 2015; Liu et al ., 2015) and it is being adopted for detection of many microbial organisms due to its simplicity, specificity, sensitivity and user‐friendly application (Dutta et al ., 2019). So far, PSR tests have been utilized to identify a variety of animal pathogens all around the world (Ashmi et al ., 2021; Das et al ., 2018; Dutta et al ., 2019; Milton et al ., 2021; Xu et al ., 2020). To date, to the best of our knowledge, no approach has been made to develop a PSR technique for early detection of A. pleuropneumoniae in pigs, which we attempted in the work presented here.…”

“…Recently, various isothermal nucleic acid testing (INAT) assays have been developed to overcome the inherent drawbacks of conventional thermal cycling-based PCR techniques for rapidly amplifying a large amount of DNA under static temperature with high specificity and sensitivity (Yang et al, 2009). A real-time recombinase polymerase amplification (RPA) assay and a loopmediated isothermal amplification (LAMP) assay were developed targeting the apxIVA gene for rapid detection of A. pleuropneumoniae (Li et al, 2019;Yang et al, 2009). Though LAMP is a commonly used isothermal amplification technique, it needs four or more primers and rigorous optimization, which may increase the chance of contamination and the appearance of false-positive results (Yang et al, 2009).…”

“…A real-time recombinase polymerase amplification (RPA) assay and a loopmediated isothermal amplification (LAMP) assay were developed targeting the apxIVA gene for rapid detection of A. pleuropneumoniae (Li et al, 2019;Yang et al, 2009). Though LAMP is a commonly used isothermal amplification technique, it needs four or more primers and rigorous optimization, which may increase the chance of contamination and the appearance of false-positive results (Yang et al, 2009). The polymerase spiral reaction (PSR) is the most recent inclusion in the list of INAT, which can amplify the target DNA with one pair of primers and a single enzyme (Dong et al, 2015;Liu et al, 2015) and it is being adopted for detection of many microbial organisms due to its simplicity, specificity, sensitivity and user-friendly application (Dutta et al, 2019).…”