Journal of Pharmaceutical and Biomedical Analysis 169 (2019) 181–187 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis j o ur na l ho mepage: www.elsevier.com/locate/jpba Sensitive fluorescent detection of Listeria monocytogenes by combining a universal asymmetric polymerase chain reaction with rolling circle amplification Zhongxu Zhan a , Ju Liu a , Leina Yan b , Zoraida P. Aguilar c , Hengyi Xu a,∗ a State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047, PR China b Jiangxi Institute for Drug Control, 330029, PR China c Zystein, LLC., Fayetteville, AR, 72703, USA a r t i c l e i n f o Article history: Received 28 November 2018 Received in revised form 5 March 2019 Accepted 8 March 2019 Available online 9 March 2019 Keywords: Listeria monocytogenes Rolling circle amplification G-quadruplex Asymmetric polymerase chain reaction a b s t r a c t A new, facile, low-cost, and highly sensitive method for detection of Listeria monocytogenes involving a combination of asymmetric polymerase chain reaction (aPCR) and rolling circle amplification (RCA) had been developed. The aPCR-RCA processes were not new but components of the processes made the assay useful. Twenty-one thymine (21-T) tagged forward primer generated universal twenty-one ade- nine (21-A) aPCR amplicons after aPCR amplification. A poly-T sequence dumbbell-like RCA template was produced through the blunt-end ligation activity of T4 DNA ligase. After the mixture of aPCR ampli- cons and dumbbell-like RCA template, the RCA reaction would initiate when the addition of phi29 DNA polymerase, then a large number of G-quadruplex sequences were produced which allowed the interca- lation of Thioflavin T (3,6-dimethyl-2-(4-dimethylaminophenyl) benzo-thiazolium cation, THT) for easy fluorescence detection. Under the optimal conditions, the assay showed a limit of detection (LOD) of 4.8 × 10 1 CFU/mL in pure culture and 4.0 × 10 2 CFU/g in spiked lettuce homogenates. By changing the aPCR primer, the aPCR-RCA method developed in this study had a potential to detect other bacteria without the design an RCA template for each bacterium. © 2019 Elsevier B.V. All rights reserved. 1. Introduction Listeria monocytogenes (L. monocytogenes, LM) is an emergent gram-positive, short rod-shaped, non-spore forming and zoonotic foodborne pathogen which can pose significant threats to public health in both developing and developed countries. [1,2] Newborns, pregnant women, senior citizens and immunodeficient people are susceptible to L. monocytogenes in an invasive way even at low infectious dose. [3] Infection with L. monocytogenes could lead to septicemia, meningitis, gastroenteritis and abortion; plus a high mortality rate ( ˜ 30%). [4,5] L. monocytogenes could survive in soil, water, plant and human or animal feces. [6] Moreover, L. monocy- togenes keeps its viability and growth at refrigeration temperatures, even in some fairly extreme conditions, which makes it a great threat from stored food as well as during cold-chain transportation. [7] Considering that L. monocytogenes poses significant threats to ∗ Corresponding author at: State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang, 330047, PR China. E-mail addresses: kidyxu@163.com, HengyiXu@ncu.edu.cn (H. Xu). public health, a highly specific and accurate method for detection L. monocytogenes in food is urgently needed. Traditional methods for L. monocytogenes detection are based on plate and culture, which are complicated and time-consuming. Polymerase chain reaction (PCR) is a widely used detection strat- egy that has been developed for the rapid and accurate detection of food-borne pathogens. [8,9] However, conventional PCR has been limited by the low resolution of gel electrophoresis when the con- centration of bacteria is low. Even worse, the use of ethidium bromide poses danger to the operator’s health [10]. Therefore, it is important to consider alternative methods to make up for these shortcomings. Recently, many signal amplification methods have been reported by replacing gel electrophoresis detection to improve sen- sitivity such as strand displacement amplification (SDA), nicking enzyme signal amplification (NESA), hybridization chain reaction (HCR) and rolling circle amplification (RCA). [11–14] Among these amplification techniques, RCA seems to be a simple and power- ful isothermal amplification method. RCA amplification needs only a short strand of DNA as a primer and a circular DNA as the tem- plate for amplification and produces long repeating single-stranded https://doi.org/10.1016/j.jpba.2019.03.016 0731-7085/© 2019 Elsevier B.V. All rights reserved.