Abstract
Avian leukosis virus subgroup J (ALV-J) causes oncogenic disease in chickens in China, resulting in great harm to poultry production, and remains widespread in China. Herein, we employed a cross-priming amplification (CPA) approach and a nucleic acid detection device to establish a visual rapid detection method for ALV-J. The sensitivity of CPA, polymerase chain reaction (PCR) and real-time PCR (RT-PCR) was compared, and the three methods were used to detect ALV-J in the cell cultures which inoculated with clinical plasma. The result showed when the amplification reaction was carried out at 60 °C for just 60 min, the sensitivity of CPA was 10 times higher than conventional PCR, with high specificity, which was comparable with RT-PCR, based on detection of 123 cell cultures which inoculated with clinical plasma, the coincidence rate with real-time PCR was 97.3% (71/73). CPA detection of ALV-J does not require an expensive PCR instrument; a simple water bath or incubator is sufficient for complete DNA amplification, and the closed nucleic acid detection device avoids aerosol pollution, making judgment of results more intuitive and objective. The CPA assay would be a promising simple, rapid and sensitive method for identification of ALV-J.
Highlights
Avian leukosis virus subgroup J (ALV-J) causes oncogenic disease in chickens in China, resulting in great harm to poultry production, and remains widespread in China
The results of Disposable Nucleic Acid Detection Strip showed that the positive control containing the recombinant plasmid as template yield red bands on both the control line (C) and the test line (T), while the negative control group only yielded the C line (Fig. 1b), which suggests that the basic cross-priming amplification (CPA) reaction system could amplify avian leukosis viruses (ALVs)-J effectively
Optimal concentrations for M g2+, betaine, dNTPs and Bst DNA polymerase in the amplification system were 4 mmol L −1 for Mg2+ (Fig. 3), 0.2 mol L−1 for Betaine (Fig. 4), 0.6 mmol L−1 for dNTPs (Fig. 5), and 0.32 units μL−1 for Bst DNA polymerase (Fig. 6)
Summary
Avian leukosis virus subgroup J (ALV-J) causes oncogenic disease in chickens in China, resulting in great harm to poultry production, and remains widespread in China. By labelling one primer with biotin, another primer with FITC, the amplified products were recognized by anti-biotin and anti-FITC monoclonal antibodies on the test line, where gold nanoparticles (AuNPs) were fixed, CPA amplified products could be visualized in nucleic acid test strip[26] It benefits from high specificity and sensitivity, does not require expensive instruments, the operation process is relatively simple, amplification is rapid, and it can be used with disposable nucleic acid detection strip in closed tubes, thereby avoiding aerosol pollution, making results more intuitive and objective[27]. This method has been applied to the diagnosis of infectious diseases and the detection of multiple pathogens[24,28]. We developed a CPA assay for ALV-J, which would be a promising rapid user-friendly detection method
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