AUTHOR=Shen Haiyan , Wen Junping , Liao Xinmeng , Lin Qijie , Zhang Jianfeng , Chen Kaifeng , Wang Shaojun , Zhang Jianmin 

TITLE=A Sensitive, Highly Specific Novel Isothermal Amplification Method Based on Single-Nucleotide Polymorphism for the Rapid Detection of Salmonella Pullorum

JOURNAL=Frontiers in Microbiology

VOLUME=Volume 11 - 2020

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.560791

DOI=10.3389/fmicb.2020.560791

ISSN=1664-302X

ABSTRACT=S. Pullorum (Salmonella enterica serovar Gallinarum biovars Pullorum) is an infectious pathogen that causes the acute systemic disease called Pullorum disease in poultry. This disease causes huge losses to the poultry industry and seriously affect the yield and quality of chicken product. It is not easy to distinguish with fowl typhoid caused by S. Gallinarum (Salmonella enterica serovar Gallinarum biovars Gallinarum), hence the development of a specific and rapid detection method for this pathogen is highly desired. In this study, we propose a novel single-nucleotide polymorphism (SNP) detection strategy termed loop-primer probe introduced loop-mediated isothermal amplification (LP-LAMP) for S. Pullorum detection. Based on the original primer sets, we targeted the nucleotide position 237 of the rfbS gene sequence to designed a new modified loop-primer probe with a ribonucleotide insertion, where activity of the enzyme ribonuclease H2 (RNase H2) is only activated when the probe is perfectly complementary, leading to hydrolytic release of a quencher moiety and thus an amplified signal. The method exhibits robust specificity and a lowest detection limit of copy number and genomic DNA is 21 copies/μL and 4.92pg/μL respectively. This method shows great performance in real sample testing of 130 samples of embryos, livers and anal swabs from chickens in poultry farms. The experimental results are mainly consistent with traditional identification methods and a PCR methods reported in the past. However, the other two methods are still exist some false negative results, while our method without miss detection. The entire closed-tube reaction process can be accomplished within 40 min at a constant temperature (61 ℃) without the need for expensive instruments or complicated operation. The LP-LAMP strategy established in this study not only overcomes the existing difficulties of S. Pullorum rapid detection, it also provides a novel, sensitive, and highly specific detection platform for SNPs that is suitable for clinical use.