Development of an isothermal amplification-based assay for the rapid detection of Cronobacter spp.

Liu, Siying; Geng, Yunyun; Liu, Libing; Sun, Xiaoxia; Shao, Jingyu; Han, Beibei; Wang, Jianchang; Tan, Karsoon

doi:10.3168/jds.2017-13931

Cited by 8 publications

(4 citation statements)

References 22 publications

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“…As a Cronobacter species-specific gene, the ompA gene has been widely used as a target for the detection of Cronobacter spp. in PIF with high specificity and sensitivity (Chen et al, 2017;Liu et al, 2018). These studies indicated that ompA gene exists in all the 7 species of Cronobacter.…”

Section: Specificity and Universality Evaluationmentioning

confidence: 88%

Dual-signal amplification strategy: Universal asymmetric tailing-PCR triggered rolling circle amplification assay for fluorescent detection of Cronobacter spp. in milk

Liu

Zhan

Liang

et al. 2020

Journal of Dairy Science

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Cronobacter spp. are important opportunistic foodborne pathogens in powdered infant formula that cause many serious diseases in neonates and infants. In this study, a novel assay based on dual signal amplification strategy was developed by coupling asymmetric tailing PCR (AT-PCR) with rolling circle amplification (RCA) for the detection of Cronobacter spp. in milk. The tailing single-stranded DNA was generated through AT-PCR and used to initiate RCA, generating tandem repetitive G-quadruplex sequences. In the presence of the fluorescence dye thioflavin T that could intercalate into the G-quadruplex structures, the fluorescence signal was detected with a microplate reader. The AT-PCR coupled with RCA assay was specific for Cronobacter spp. detection because of the highly specific primers chosen for the AT-PCR. The limits of detection were 4.3 × 10 1 cfu/mL in pure culture and 4.5 × 10 2 cfu/mL in spiked milk, respectively. The fixed sequences designed in the hairpin DNA allowed this AT-PCR coupled with RCA assay to serve as a universal platform for the detection of other pathogens by modifying the specificity of the PCR primers.

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Section: Specificity and Universality Evaluationmentioning

confidence: 88%

Dual-signal amplification strategy: Universal asymmetric tailing-PCR triggered rolling circle amplification assay for fluorescent detection of Cronobacter spp. in milk

Liu

Zhan

Liang

et al. 2020

Journal of Dairy Science

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“…Current molecular diagnostic techniques for V. parahaemolyticus mainly rely on PCR-based techniques, such as qPCR and ddPCR. Although these techniques demonstrate strong applicability, they require expensive instruments and professional expertise [ 27 ]. Therefore, strategies for the convenient and rapid detection of V. parahaemolyticus are urgently needed.…”

Section: Discussionmentioning

confidence: 99%

CE–RAA–CRISPR Assay: A Rapid and Sensitive Method for Detecting Vibrio parahaemolyticus in Seafood

Cao²,

Zhang

et al. 2022

Foods

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Vibrio parahaemolyticus is one of the major pathogenic Vibrio species that contaminate seafood. Rapid and accurate detection is crucial for avoiding foodborne diseases caused by pathogens and is important for food safety management and mariculture. In this study, we established a system that combines chemically enhanced clustered regularly interspaced short palindromic repeats (CRISPR) and recombinase-aided amplification (RAA) (CE–RAA–CRISPR) for detecting V. parahaemolyticus in seafood. The method combines RAA with CRISPR-associated protein 12a (Cas12a) for rapid detection in a one-pot reaction, effectively reducing the risk of aerosol contamination during DNA amplifier transfer. We optimized the primers for V. parahaemolyticus, determined the optimal crRNA/Cas12a ratio, and demonstrated that chemical additives (bovine serum albumin and L-proline) could enhance the detection capacity of Cas12a. The limit of detection (at optimal conditions) was as low as 6.7 × 101 CFU/mL in pure cultures and 7.3 × 101 CFU/g in shrimp. Moreover, this method exhibited no cross-reactivity with other microbial pathogens. The CE–RAA–CRISPR assay was compared with the quantitative polymerase chain reaction assay using actual food samples, and it showed 100% diagnostic agreement.

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“…Although many PCR-based methods for detection of Cronobacter spp. have been developed in recent years, and various gene targets such as 16S rRNA (Xu et al, 2014), rpoB (Li et al, 2016), and ompA (Liu et al, 2018b) are widely used, only 2 studies have identified C. turicensis. However, in these studies, the specificity of the target genes rpoB and cgcA could not correctly identify C. turicensis because nonspecific bands appeared often (Stoop et al, 2009;Carter et al, 2013).…”

Section: Short Communicationmentioning

confidence: 99%

Short communication: Bioinformatics-based mining of novel gene targets for identification of Cronobacter turicensis using PCR

Chen

Lü

Qiu

et al. 2019

Journal of Dairy Science

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Cronobacter turicensis is a food-borne pathogen found in dairy products. It has been reported to cause bacteremia and enteritis in immunocompromised people, especially infants. Cronobacter turicensis has been isolated from various food sources, and contaminated powdered infant formula was found to be the most common source of infection among infants. Although some gene targets are used for the identification of C. turicensis, they are not specific at the species level. In this study, we analyzed the genome sequence of C. turicensis by bioinformatics and identified 13 specific gene targets. Primer sets targeting these sequences were designed and selected based on their specificity. Finally, primer set CT11, targeting gene CTU_19580, which codes for a hypothetical protein, was selected for development of the PCR assay because it alone produced positive PCR results for C. turicensis. To our knowledge, this is the first time that this gene target has been used to develop PCR detection assays for C. turicensis. The specific PCR assay had detection limits as low as 760 fg/µL for genomic DNA (approximately 158 copies/µL of DNA) and could detect C. turicensis in powdered infant formula with initial cell concentrations as low as 8.5 cfu per 10 g of powdered infant formula after 10 h of enrichment. Thus, this PCR assay is highly sensitive and can be used for rapid detection of C. turicensis.

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Development of an isothermal amplification-based assay for the rapid detection of Cronobacter spp.

Cited by 8 publications

References 22 publications

Dual-signal amplification strategy: Universal asymmetric tailing-PCR triggered rolling circle amplification assay for fluorescent detection of Cronobacter spp. in milk

Dual-signal amplification strategy: Universal asymmetric tailing-PCR triggered rolling circle amplification assay for fluorescent detection of Cronobacter spp. in milk

CE–RAA–CRISPR Assay: A Rapid and Sensitive Method for Detecting Vibrio parahaemolyticus in Seafood

Short communication: Bioinformatics-based mining of novel gene targets for identification of Cronobacter turicensis using PCR

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