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Listeria monocytogenes is an important foodborne pathogen worldwide, with 20-30% fatality rate in vulnerable persons. The hypervirulent L. monocytogenes clonal complex (CC) 87 strains have emerging both in food production environments and clinic cases. The objective of this study was to develop a multiplex PCR to simultaneously detect L. monocytogenes CC87 and CC88 strains based on pan-genome analysis. A novel multiplex PCR comprised of genes A6K41_13255 (specific for CC87 and 88), BCW_4260_01987 group_8135 (specific for CC88) and 02-1103_01073 group_5869 (specific for L. monocytogenes) were designed. The specificity of this multiplex PCR was robust verified with other CCs of L. monocytogenes and other species strains. The detection limit of this multiplex PCR for CC87 and CC88 were 1.7 × 10 4  cfu/mL and 2.1 × 10 4  cfu/mL, respectively. This multiplex PCR could accurately detect CC87 and CC88 strains with the interference of different ratios of L. monocytogenes CC8, CC9, CC121, CC155, and L. innocua strains. Furthermore, this multiplex PCR method could successfully detect 1.9 × 10 4  cfu/mL of L. monocytogenes CC87 and 1.7 × 10 4  cfu/mL CC88 strains in artificially contaminated milk after 9 h enrichment, respectively. In addition, this multiplex PCR could accurately detect CC87 isolates in food samples within 48 h, which was faster than the routine MLST analysis. In conclusion, this novel multiplex PCR offers a promising approach for accurate, inexpensive, and rapid detection of L. monocytogenes CC87 and CC88 strains simultaneously, which could apply to surveillance the prevalence of CC87 and CC88 strains in both food and food production environments and to evaluate the effect of disinfection measures for controlling the persistent L. monocytogenes contamination., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Listeria monocytogenes is an important foodborne pathogen that can cause listeriosis with high patient mortality. Accordingly, it is necessary to develop a L. monocytogenes detection platform with high specificity, sensitivity, and exploitability. CRISPR/Cas systems have shown great potential in the development of next-generation biosensors for nucleic acid detection, owing to the trans-cleavage capabilities of the Cas effector proteins. Herein, we introduce the trans-cleavage activity of CRISPR/Cas12a into an electrochemical biosensor (E-CRISPR), combined with recombinase-assisted amplification (RAA), to establish a cost-effective, specific and ultrasensitive method; namely RAA-based E-CRISPR. The concept behind this approach is that the target will induce the number change of the surface signaling probe (containing an electrochemical tag), which leads to a variation in the electron transfer of the electrochemical tag. The introduction of an RAA-based Cas12a system into the E-CRISPR sensor achieves a more prominent signal change between the presence and absence of the target. Under optimized conditions, RAA-based E-CRISPR can detect as low as 0.68 aM of genomic DNA and 26 cfu/mL of L. monocytogenes in pure cultures. More importantly, the RAA-based E-CRISPR enables rapid and ultrasensitive detection of L. monocytogenes in spiked and natural Flammulina velutipes samples. Moreover, no cross-reactivity with other non-target bacteria was observed. This system thus demonstrates to be a simple, high-sensitivity, and high-accuracy platform for L. monocytogenes detection., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The CRISPR/Cas12a system has displayed remarkable potential in the development of new methods for nucleic acid detection owing to the trans-cleavage activity of Cas12a. Despite the tremendous development in recent years, existing CRISPR/Cas12a-based methods have several limitations such as the time-consuming process, which takes up to 2 h, and the risk of aerosol contamination during DNA amplicon transfer. Herein, we propose a CRISPR/Cas12a-based fluorescence detection platform named "Cas12aFDet" for rapid nucleic acid detection that overcomes these limitations. By integrating PCR or recombinase-aided amplification (RAA) methods with Cas12a-mediated cleavage in a sealed reaction tube, Cas12aFDet-based detection of amplified products could be accomplished within 15 min, while avoiding amplicon contamination. The detection limits of PCR-based Cas12aFDet and RAA-based Cas12aFDet were determined to be 3.37 × 10 1  cfu/mL and 1.35 × 10 2  cfu/mL of Listeria monocytogenes serotype 4c in pure culture, respectively. Most importantly, RAA-based Cas12aFDet exhibited 0.64 aM sensitivity for DNA detection, and showed high specificity for detection of other serotypes of Listeria and non-Listeria strains. Furthermore, the feasibility of the RAA-based Cas12aFDet method was evaluated in spiked and natural samples, enabling the quantitative detection of 1.35 × 10 8 -1.35 × 10 3  cfu/g fresh grass carp of the target L. monocytogenes serotype 4c, and the results obtained for 22 natural aquatic samples were highly consistent with those of the culture-based serotyping method. The established Cas12aFDet platform is expected to provide a new paradigm for the sensitive and specific detection of pathogens in food safety and clinical diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The abundant information provided by the pan-genome analysis approach reveals the diversity among Listeria monocytogenes serotypes. The objective of this study was to mine novel target genes using pan-genome analysis for multiplex PCR detection and differentiation of the major L. monocytogenes serotypes present in food. Pan-genome analysis and PCR validation revealed a total of 10 specific targets: one for lineage I, two for serogroup I.1, one for serogroup I.2, two for lineage II, one for serogroup II.1, three for lineage III. Primers for the novel targets were highly specific in individual reactions. The detection limits were 10 3 -10 4 colony-forming units (CFU)/mL in pure bacterial cultures, meeting the requirements of molecular detection. Based on these novel targets, two new "lineage" multiplex PCR assays were developed to simultaneously distinguish between three lineages (I, II, and III) and five major serotypes (1/2a, 1/2b, 1/2c, 4b, and 4c) of L. monocytogenes. The detection limits of lineage I and lineage II&III mPCRs were 0.771 pg/μL and 1.76 pg/μL genomic DNA, respectively. The specificity of the mPCRs was robustly verified using other L. monocytogenes and non-L. monocytogenes serotypes. These results suggest that the two "lineage" multiplex PCRs based on novel targets offer a promising approach for accurate, sensitive, and rapid identification of L. monocytogenes serotypes., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Ready-to-eat (RTE) vegetables have become increasingly popular along with the trend of moving towards a healthy lifestyle. However, RTE vegetables are at a higher risk of containing pathogens, maybe owing to lack of rigorous sanitization procedures. To understand the prevalence and potential risk of Listeria monocytogenes in RTE vegetables, we investigated the contamination level and characteristics of L. monocytogenes isolated from fresh vegetables., Results: Twenty-three (5.49%) of the 419 vegetables samples were positive for L. monocytogenes. Phylogenetic group I.1 (1/2a-3a) and II.2 (1/2b-3b-7) strains were predominant in 30 isolates, which accounted for 33.3 and 50.0%, respectively. Multilocus sequence typing of the 30 isolates grouped them into nine sequence types (STs). The most common STs were ST87 (36.7%) and ST8 (26.7%). Virulence analysis showed that all 30 isolates harbored eight classical virulence genes, 10.0% isolates harbored the llsX gene (ST3 and ST1 strains), and 36.7% carried the ptsA gene and belonged to ST87. Approximately 83.3% isolates carried full-length inlA, whereas five isolates had premature stop codons in inlA, three of which belonged to ST9 and two to ST8. Antibiotic susceptibility showed the isolates were varyingly resistant to 13 antibiotics, 26.7% of the isolates were multi-drug resistant., Conclusions: The fresh vegetables contain some potential hypervirulent L. monocytogenes (ST1 and ST87) in the Chinese markets. In addition, the high rate of L. monocytogenes isolates was multi-drug resistant. Fresh raw vegetables may be a possible transmission route for L. monocytogenes infection in consumers. Therefore, sanitization of raw fresh vegetables should be strengthened to ensure their microbiological safety when used as RTE vegetables.

Listeria monocytogenes is a foodborne pathogen with a high mortality rate in humans. This study aimed to identify the pathogenic potential of L. monocytogenes isolated from ready-to-eat (RTE) foods and pasteurized milk in China on the basis of its phenotypic and genotypic characteristics. Approximately 7.7% (44/570) samples tested positive for L. monocytogenes among 10.8% (39/360) RTE and 2.4% (5/210) pasteurized milk samples, of which 77.3% (34/44) had < 10 MPN/g, 18.2% (8/44) had 10–110 MPN/g, and 4.5% (2/44) had > 110 MPN/g. A total of 48 strains (43 from RTE foods and five from milk samples) of L. monocytogenes were isolated from 44 positive samples. PCR-serogroup analysis revealed that the most prevalent serogroup was II.2 (1/2b-3b-7), accounting for 52.1% (25/48) of the total, followed by serogroup I.1 (1/2a-3a) accounting for 33.3% (16/48), serogroup I.2 (1/2c-3c) accounting for 12.5% (6/48), and serogroup II.1 (4b-4d-4e) accounting for 2.1%. All isolates were grouped into 11 sequence types (STs) belonging to 10 clonal complexes (CCs) and one singleton (ST619) via multi-locus sequence typing. The most prevalent ST was ST87 (29.2%), followed by ST8 (22.9%), and ST9 (12.5%). Virulence genes determination showed that all isolates harbored eight virulence genes belonging to Listeria pathogenicity islands 1 (LIPI-1) (prfA , actA , hly , mpl , plcA , plcB , and iap) and inlB. Approximately 85.4% isolates carried full-length inlA , whereas seven isolates had premature stop codons in inlA , six of which belonged to ST9 and one to ST5. Furthermore, LLS (encoded by llsX gene, representing LIPI-3) displays bactericidal activity and modifies the host microbiota during infection. LIPI-4 enhances neural and placental tropisms of L. monocytogenes. Results showed that six (12.5%) isolates harbored the llsX gene, and they belonged to ST1/CC1, ST3/CC3, and ST619. Approximately 31.3% (15/48) isolates (belonging to ST87/CC87 and ST619) harbored ptsA (representing LIPI-4), indicating the potential risk of this pathogen. Antimicrobial susceptibility tests revealed that > 95% isolates were susceptible to 16 antimicrobials; however, 60.4 and 22.9% isolates were intermediately resistant to streptomycin and ciprofloxacin, respectively. The results show that several isolates harbor LIPI-3 and LIPI-4 genes, which may be a possible transmission route for Listeria infections in consumers. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a globally notorious foodborne pathogen. This study aimed to qualitatively and quantitatively detect L. monocytogenes from meat and meat products in China and to establish their virulence profiles and population diversity. From 1212 meat and meat product samples, 362 (29.9%) were positive for L. monocytogenes. Of these positive samples, 90.6% (328/362) had less than 10 MPN/g, 5.5% (20/364) samples had 10–110 MPN/g, and 3.9% (14/362) of the positive samples had over 110 MPN/g. Serogroup analysis showed that the most prevalent serogroup of L. monocytogenes was I.1 (1/2a-3a), which accounted for 45.0% (123/458) of the total, followed by serogroup I.2 (1/2c-3c) that comprised 26.9%, serogroup II.1 (4b-4d-4e) that comprised 4.8%, and serogroup II.2 (1/2b-3b-7) that comprised 23.3%. A total of 458 isolates were grouped into 35 sequence types (STs) that belonged to 25 clonal complexes (CCs) and one singleton (ST619) by multi-locus sequence typing. The most prevalent ST was ST9 (26.9%), followed by ST8 (17.9%), ST87 (15.3%), ST155 (9.4%), and ST121 (7.6%). Thirty-seven isolates harbored the llsX gene (representing LIPI-3), and they belonged to ST1/CC1, ST3/CC3, ST288/CC288, ST323/CC288, ST330/CC288, ST515/CC1, and ST619, among which ST323/CC288, ST330/CC288, and ST515/CC1 were newly reported to carry LIPI-3. Seventy-five isolates carried ptsA , and they belonged to ST87/CC87, ST88/CC88, and ST619, indicating that consumers may be exposed to potential hypervirulent L. monocytogenes. Antibiotics susceptibility tests revealed that over 90% of the isolates were susceptible to 11 antibiotics; however, 40.0% of the isolates exhibited resistance against ampicillin and 11.8% against tetracycline; further, 45.0 and 4.6% were intermediate resistant and resistant to ciprofloxacin, respectively. The rise of antibiotic resistance in L. monocytogenes suggests that stricter regulations should be formulated to restrict the use of antibiotic agents in human listeriosis treatment and livestock breeding. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a hazardous foodborne causative agent, commonly found in foods and food production environments. The persistent contamination of L. monocytogenes sequence type (ST) 121 in food production environments is one of the challenging issues. The objective of this study was to develop a multiplex PCR for identification of L. monocytogenes ST121 strains. A total of 50 specific candidate genes of ST121 were screened by the comparative genomics approach. A novel multiplex PCR for ST121 identification, which comprised of ST121 strain-specific primers (LM6179_0609 and LM6179_0171) and L. monocytogenes -specific primers (prfA), was designed. The specificity of this multiplex PCR was robustly verified by other STs of L. monocytogenes and non- L. monocytogenes strains. The detection limit of the multiplex PCR was 253 fg/μL genomic DNA. This multiplex PCR could avoid the interference of L. monocytogenes ST9, ST8, ST87, and ST155 in different ratios, which are common contaminants in food items. In addition, this multiplex PCR method could successfully detect 2.5 × 104–2.5 × 100 cfu/10 mL of L. monocytogenes ST121 in artificially contaminated milk after 4–12 h enrichment. These results suggested that the newly developed multiplex PCR in this study offers a promising approach for specific, rapid, and accurate detection of L. monocytogenes ST121 strains. • Comparative genome analysis was performed for 656 L. monocytogenes strains. • Multiplex PCR was developed to detect L. monocytogenes sequence type 121 strains. • This novel method is specific, sensitive, economical, and had a run-time of 7–15 h. • Rapid screening of L. monocytogenes sequence type 121 in food items is possible. [ABSTRACT FROM AUTHOR]

Detection of the foodborne pathogen Listeria monocytogenes serotype 4c, one of the predominant serotypes in aquatic products, is difficult and time-consuming. Our objective was to identify novel target genes for real-time PCR detection of L. monocytogenes serotype 4c using comparative genomics to facilitate its specific identification in food products. A total of 160 Listeria genome sequences, including those of 12 L. monocytogenes serotypes and 14 other Listeria species, were employed to identify serotype 4c-specific targets. Two serotype 4c-specific genes (SLCC2376_00062 and SLCC2376_00822) were obtained. Using primers for SLCC2376_00822 , a real-time PCR assay for the detection of serotype 4c was developed. The specificty of this assay was robustly verified by other serotypes of L. monocytogenes and non- L. monocytogenes strains. In addition, this real-time PCR method could accurately detect serotype 4c even when mixed with different concentrations of serotypes 1/2a and 1/2b. Furthermore, the assay could successfully detect 1.4 × 109–1.4 × 103 CFU/g of L. monocytogenes serotype 4c in artificially contaminated grass carp without enrichment. These results demonstrate that the assay, which has high accuracy and sensitivity, comprises a promising approach for the detection and identification of L. monocytogenes serotype 4c strains in foods. • Listeria monocytogenes serotype 4c is increasingly being found in aquatic products. • Current serotyping methods are expensive and time-consuming. • We used comparative genomics to identify serotype 4c-specific genes for a PCR assay. • Novel assay targeting SLCC2376_ 00822 has high serotype specificity and sensitivity. • Novel PCR assay is rapid and costs only one-tenth of that of traditional serotyping. [ABSTRACT FROM AUTHOR]