New rapid detection by using a constant temperature method for avian leukosis viruses

Xiuhong Wu; Fengsheng Chu; Luxuan Zhang; Sheng Chen; Liguo Gao; Hao Zhang; Haohua Huang; Jin Wang; Mengjun Chen; Zi Xie; Feng Chen; Xinheng Zhang; Qingmei Xie

doi:10.3389/fmicb.2022.968559

New rapid detection by using a constant temperature method for avian leukosis viruses

Front Microbiol. 2022 Aug 18:13:968559. doi: 10.3389/fmicb.2022.968559. eCollection 2022.

Authors

Xiuhong Wu^{1

2

3}, Fengsheng Chu^{1

2

3}, Luxuan Zhang⁴, Sheng Chen^{1

2

3}, Liguo Gao^{1

2

3}, Hao Zhang^{1

2}, Haohua Huang^{1

2}, Jin Wang^{1

2}, Mengjun Chen^{1

2}, Zi Xie^{1

2

3}, Feng Chen^{1

2

3}, Xinheng Zhang^{1

2

3}, Qingmei Xie^{1

2

3}

Affiliations

¹ Heyuan Branch, Guangdong Provincial Laboratory of Lingnan Modern Agricultural Science and Technology and Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China.
² Guangdong Engineering Research Center for Vector Vaccine of Animal Virus, Guangzhou, China.
³ South China Collaborative Innovation Center for Poultry Disease Control and Product Safety, Guangzhou, China.
⁴ School of Pharmaceutical Science, Sun Yat-sen University, Guangzhou, China.

Abstract

The avian leukemia virus causes avian leukemia (AL), a severe immunosuppressive disease in chickens (ALV). Since the 1990s, the diversity of ALV subpopulations caused by ALV genome variation and recombination, and the complexity of the infection and transmission, with currently no effective commercial vaccine and therapeutic for ALV, has resulted in severe economic losses to the chicken business in various parts of the world. Therefore, as a key means of prevention and control, an effective, rapid, and accurate detection method is imperative. A new real-time reverse transcription recombinase-aided amplification (RT-RAA) assay for ALV with rapid, highly specific, low-cost, and simple operational characteristics have been developed in this study. Based on the amplification of 114 base pairs from the ALV P12 gene, real-time RT-RAA primers and a probe were designed for this study. The lowest detection line was 10 copies of ALV RNA molecules per response, which could be carried out at 39°C in as fastest as 5 min and completed in 30 min, with no cross-reactivity with Marek's disease virus, avian reticuloendothelial virus, Newcastle disease virus, infectious bronchitis virus, infectious bursal disease virus, infectious laryngotracheitis virus, and avian influenza virus. Furthermore, the kappa value of 0.91 (>0.81) was compared with reverse transcription-polymerase chain reaction (RT-PCR) for 44 clinical samples, and the coefficients of variation were within 5.18% of the repeated assays with three low-level concentration gradients. These results indicate that using a real-time RT-RAA assay to detect ALV could be a valuable method.

Keywords: avian leukosis viruses; clinical diagnosis; detection by constant temperature; p12; real-time reverse-transcription recombinase-aided amplification assay.