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Article

Polymerase Chain Reaction-Lateral Flow Strip for Detecting Escherichia coli and Salmonella enterica Harboring blaCTX-M

by
Rujirat Hatrongjit
1,2,*,
Sumontha Chaisaeng
2,
Kulsatree Sitthichotthumrong
3,
Parichart Boueroy
2,3,
Peechanika Chopjitt
2,3,
Ratchadaporn Ungcharoen
3 and
Anusak Kerdsin
2,3,*
1
Department of General Science, Faculty of Science and Engineering, Kasetsart University Chalermphrakiat, Sakon Nakhon Campus, Sakon Nakhon 47000, Thailand
2
Special Research Unit of Emerging Foodborne Pathogens Surveillance, Kasetsart University, Bangkok 10900, Thailand
3
Faculty of Public Health, Kasetsart University Chalermphrakiat, Sakon Nakhon Campus, Sakon Nakhon 47000, Thailand
*
Authors to whom correspondence should be addressed.
Antibiotics 2025, 14(8), 745; https://doi.org/10.3390/antibiotics14080745
Submission received: 4 May 2025 / Revised: 4 July 2025 / Accepted: 22 July 2025 / Published: 24 July 2025
(This article belongs to the Special Issue Foodborne Pathogens: Detection, Resistance Mechanisms, and Control Strategies)
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Abstract

Background: Salmonella enterica and Escherichia coli are common foodborne pathogens of global concern, particularly due to their antimicrobial resistance, notably to cephalosporins. Objective: This study aimed to evaluate a polymerase chain reaction-based lateral flow strip (PCR-LFS) assay for the detection of Salmonella spp. and E. coli harboring the blaCTX-M gene, which confers resistance to third-generation cephalosporins. Methods: Two duplex PCRs (dPCR) were established to detect E. coli-harboring blaCTX-M (set 1) and Salmonella-harboring blaCTX-M (set 2). 600 bacterial isolates and raw pork mince spiked with blaCTX-M-harboring E. coli and Salmonella were used to evaluated. Results: Both dPCR assays successfully detected blaCTX-M-positive E. coli or Salmonella strains, while strains lacking the gene showed no amplification. Non-E. coli and non-Salmonella strains were PCR-negative unless they carried blaCTX-M. The dPCR-LFS showed 100% validity including accuracy, sensitivity, specificity, positive predictive value, and negative predictive value for both E. coli or Salmonella spp. harboring or lacking blaCTX-M. The assay accurately detected target strains without cross-reactivity with other bacteria or antimicrobial resistance genes. Cohen’s Kappa coefficient indicated perfect agreement (κ = 1), reflecting the high reliability of the dPCR-LFS. The assay could detect as low as 25 CFU/mL for blaCTX-M-positive E. coli and 40 CFU/mL for blaCTX-M-positive Salmonella in spiked raw pork mince. Conclusions: This assay is rapid, easy to interpret, and suitable for large-scale screening in surveillance programs.

1. Introduction

Foodborne pathogens in contaminated food are a significant concern in food safety and require close monitoring. These pathogens include viruses, bacteria, fungi, and protozoa [1]. Generally, infection occurs through the consumption of food or water contaminated with foodborne pathogens or their toxins. Detection and control of foodborne pathogens are prerequisites for protecting human and animal health and maintaining international trade. Rapid detection of foodborne pathogens is key to effective surveillance systems, ensuring a safe food supply, and preventing foodborne diseases [2,3,4].
Among these foodborne pathogens, Salmonella enterica and Escherichia coli have been implicated in numerous foodborne diseases associated with the consumption of contaminated food and water [1]. These bacterial pathogens are critical targets for the monitoring and surveillance of both phenotypic and genotypic antimicrobial resistance (AMR) in food-producing animals, fresh and processed meat, vegetables, and drinking water. Additionally, Salmonella, E. coli, Campylobacter spp., and Enterococcus spp. are recommended as indicators for AMR surveillance in human and non-human sectors (i.e., food supply chains, animals, and the environment) [5,6,7].
The emergence of extended-spectrum β-lactamase (ESBL)-producing bacteria has conferred resistance to penicillin and third- and fourth-generation cephalosporins, which are critically important antibiotics in both human and veterinary medicine [8]. These pathogens are recognized by the World Health Organization (WHO) as among the most challenging to treat [9]. Infections caused by ESBL-producing bacteria in humans and animals have been widely reported, with documented transmission routes including food, water, and contact with contaminated environments [10]. Thus, monitoring ESBL-producing Salmonella spp. and E. coli is essential for public health.
ESBL has been classified into three main groups, including Ambler class A ESBL, miscellaneous ESBL, and ESBL that degrade carbapenems [11]. Most ESBLs belong to Ambler class A, which includes sulfhydryl reagent variable (SHV), Temoniera (TEM), and cefotaxime-M (CTX-M) types [11]. Among these, CTX-M-type ESBLs are the most widely distributed globally [12,13,14]. CTX-M type-ESBLs have increased in prevalence since 2000, and this is an important mechanism for developing resistance to cephalosporins, posing a major difficulty in clinical treatment, with restricted options to treat infections caused by CTX-M-producing bacteria [12,14]. This trend has led to increased reliance on carbapenems [12]. CTX-M-producing bacteria are now frequently detected in Europe, Asia, and the Americas [14]. Over 80 CTX-M variants have been reported across hospitals, communities, food animals, fresh produce, water, and the environment [15].
Routine screening and rapid detection of ESBL-producing bacteria in clinical laboratory settings are critical for infection control and therapeutic decision-making. The Clinical and Laboratory Standards Institute (CLSI) recommends a two-step approach for ESBL detection [16]. Initial screening methods include the Kirby–Bauer disk diffusion test and automated systems such as Vitek. Confirmatory tests involve the double-disk synergy test (DDST), combination disk methods, or E-test ESBL strips. In addition to phenotypic confirmatory methods, genotypic assays such as polymerase chain reaction (PCR) and nucleotide sequencing are employed to detect specific ESBL-encoding genes and their variants [11]. Other molecular methods include isoelectric point determination, DNA probes, oligotyping, PCR-restriction fragment length polymorphism (PCR-RFLP), PCR-single-strand conformational polymorphism (PCR-SSCP), real-time PCR, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF), and the NG-Test CTX-M MULTI, a rapid immunochromatographic lateral flow strip assay (LFS) [11].
Among these techniques, nucleic acid amplification followed by LFS, such as PCR-LFS and recombinase polymerase amplification (RPA)-LFS, is simple and widely applied for detecting various pathogens and antimicrobial resistance genes [17,18,19,20,21,22]. LFS is a paper-based device that uses colloidal gold nanoparticles as signal labels [23]. Combining PCR or RPA with LFS improves laboratory efficiency and enables rapid visualization of target genes, eliminating the need for gel electrophoresis and staining, thereby reducing detection time and processing complexity. The principles of PCR-LFS or RPA-LFS involve labeling the 5′ end of the forward primer with fluorescein isothiocyanate (FITC) and the 5′ end of the reverse primer with biotin or digoxigenin, followed by amplification using PCR or RPA [21,24]. The labeled amplicons bind to specific antibodies on the test line of the colloidal gold strip, producing a visual colorimetric signal interpretable by the naked eye.
Salmonella spp. and E. coli are important indicator organisms for pathogen and AMR surveillance, particularly in relation to cephalosporin nonsusceptibility due to blaCTX-M. Although many PCR assays have been developed to detect Salmonella spp. and E. coli harboring antimicrobial resistance genes such as blaCTX-M [25,26], neither PCR-LFS nor RPA-LFS currently allow simultaneous detection of both bacterial species and resistance genes in a single assay [18,27,28,29]. In this study, we evaluated a polymerase chain reaction-based lateral flow strip (PCR-LFS) assay for detecting Salmonella spp. and E. coli harboring blaCTX-M, aimed at infection control and surveillance in environmental, livestock, food, or feed contamination, particularly in resource-limited laboratories. This assay is rapid, easy to perform and interpret, and suitable for high-throughput screening in AMR surveillance programs.

2. Results

Two duplex PCR (dPCR) assays were established to detect E. coli harboring blaCTX-M (set 1) and Salmonella harboring blaCTX-M (set 2). Both assays successfully detected blaCTX-M-positive E. coli and Salmonella strains, while E. coli and Salmonella strains lacking blaCTX-M showed no blaCTX-M amplification, and the negative control (Klebsiella pneumoniae ATCC13883) did not show any bands (Figure 1 and Figure 2). Non-E. coli and non-Salmonella strains tested negative in the dPCR assays unless they carried blaCTX-M, in which case only the blaCTX-M band was detected. For bacterial species other than E. coli, Salmonella, or Enterobacterales that do not harbor blaCTX-M, the PCR-LFS produced negative results for both target bands, while the internal strip-control band remained positive. These results confirm the high specificity of the duplex PCR-LFS for E. coli and Salmonella.
The PCR-LFS assay was evaluated using 200 E. coli isolates and 30 Salmonella isolates harboring blaCTX-M (Table 1). All isolates showed positive results for both the species-specific and blaCTX-M bands. E. coli and Salmonella strains carrying other antimicrobial resistance genes (but not blaCTX-M) showed amplification of only the species-specific band. In contrast, blaCTX-M-harboring strains of other species (e.g., Klebsiella pneumoniae, Enterobacter cloacae, Citrobacter freundii) showed positive results only for the blaCTX-M band. Sanger sequencing of representative amplicons confirmed the identities of E. coli, Salmonella, and blaCTX-M. Non-Enterobacterales species tested negative for all targets, indicating no cross-reactivity.
As shown in Table 2, Table 3 and Table 4, the dPCR-LFS assay demonstrated 100% validity, including accuracy, sensitivity, specificity, positive predictive value, and negative predictive value, for both E. coli and Salmonella strains harboring or lacking blaCTX-M. The assay accurately detected the target species without cross-reactivity with non-target bacteria or other antimicrobial resistance genes. Cohen’s kappa coefficient indicated perfect agreement (κ = 1), confirming the high reliability of the dPCR-LFS. Statistical analysis revealed a significant association (p < 0.01) for true positives. The coefficient of determination (R2) was 0.92952 for simultaneous detection of E. coli and Salmonella with blaCTX-M, and 0.96621 and 0.96642 for detecting either target species or blaCTX-M alone, respectively. A receiver operating characteristic (ROC) curve confirmed that the assay’s sensitivity and specificity were consistent with theoretical expectations (Figure 3).
The limit of detection of the duplex PCR-LFS assay was 72 fg and 105 fg of genomic DNA for E. coli and Salmonella, respectively. In the case of blaCTX-M detection, the assay identified as little as 7.2 pg for E. coli carrying blaCTX-M and 105 fg for Salmonella carrying blaCTX-M. When tested in artificially spiked raw pork mince, the assay could detect as low as 25 CFU/mL for blaCTX-M-positive E. coli and 40 CFU/mL for blaCTX-M-positive Salmonella (Figure 4). However, the signal at these lowest concentrations was noticeably weaker compared to 102 CFU/mL. In contrast, dPCR–gel electrophoresis had a detection limit of 103 CFU/mL for both targets. The duplex PCR-LFS assay was more time-efficient than PCR followed by gel electrophoresis. Excluding DNA extraction, the cost per sample for dPCR-LFS was approximately USD 2.25–2.45 per set. The total running time for dPCR-LFS was approximately 60–70 min.

3. Discussion

In 2017, the WHO released guidance on the integrated surveillance of antimicrobial resistance (AMR) in foodborne bacteria, recommending a One Health approach and suggesting the monitoring of Salmonella, Campylobacter spp., E. coli, and Enterococcus spp. as priority organisms [6]. Similarly, in 2019, the Food and Agriculture Organization (FAO) designated the same bacterial species as targets for AMR surveillance [5]. In 2021, the WHO introduced the Tricycle Surveillance Program, which emphasizes the monitoring of ESBL-producing E. coli across human, food supply chain, and environmental sectors [7]. These organisms serve as key indicators in global AMR surveillance systems.
Rapid diagnostic methods are critical to reducing the spread of antimicrobial-resistant bacteria, enabling timely isolation and appropriate treatment. A variety of rapid detection technologies have been developed, including nucleic acid amplification (NAA) techniques, immunochromatographic tests, electrochemical assays, microarrays, nanoparticle-based systems, and mass spectrometry [30,31,32,33]. Among these, NAA-based methods such as PCR, real-time PCR, loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA) are particularly favored. These have been applied for the detection of Salmonella spp., E. coli, Klebsiella pneumoniae, and other Enterobacterales harboring blaCTX-M or other antimicrobial resistance genes [18,34,35,36,37].
In this study, two sets of duplex PCR-based lateral flow strip (dPCR-LFS) assays were evaluated to detect E. coli, Salmonella, and blaCTX-M for use in surveillance of foods, the environment, livestock, and hospital infection control purposes. The method is rapid and user-friendly, with results easily interpreted without the need for gel electrophoresis or specialized visualization instruments. These characteristics make it suitable for application in both human and non-human sectors, especially in the food safety program part of surveillance.
NAA-based lateral flow assays (NAA-LFS) have been developed for detecting various pathogens, including Vibrio parahaemolyticus, E. coli, Salmonella, Bacillus anthracis, Yersinia pestis, Listeria monocytogenes, Staphylococcus aureus, and coliforms [20,38,39,40,41,42,43,44]. For example, mPCR-LFS assays have enabled the simultaneous detection of E. coli O157:H7 and S. Typhimurium [45]. PCR-LFS has also been applied for detecting E. coli, coliform bacteria, and total bacterial load [42]. A lateral flow biosensor based on LAMP-CRISPR/Cas12a has been developed for Salmonella detection in food samples [46]. However, few NAA-LFS assays enable simultaneous detection of both bacterial species and resistance genes, as demonstrated in this study. For example, RPA-LFS assays have detected antimicrobial resistance genes such as blaCTX-M, blaSHV, blaOXA, blaKPC, blaNDM, mcr-1, and tet(X4), but without identifying the bacterial species carrying them [18,37,47,48]. One prior study used mPCR-LFS to detect blaKPC and blaNDM in Enterobacterales [49].
A comparison of detection limits between dPCR-LFS and dPCR–gel electrophoresis, using raw pork mince spiked with E. coli and S. enterica harboring blaCTX-M, showed that dPCR-LFS had a lower detection limit. Our dPCR-LFS assay detected bacterial concentrations as low as 25–40 CFU/mL, consistent with previous findings demonstrating low detection limits for NAA-LFS in food matrices [45,50,51,52]. For instance, a previous study reported that mPCR-LFS detected E. coli O157:H7 and S. Typhimurium in spiked cabbage at limits of 104 CFU/25 g and 103 CFU/25 g, respectively [45]. Zeng et al. (2020) reported a detection limit of 50 CFU/mL for Vibrio parahaemolyticus using PCR-LFS [52]. Another study detected methicillin-resistant Staphylococcus aureus at 200 CFU/100 g of pork products using PCR-LFS [53]. Wang et al. (2020) showed that RPA-LFS detected as low as 1 CFU of Listeria monocytogenes per reaction without DNA purification [51]. LAMP-LFS has also been used to detect Salmonella in milk, pork, beef, and chicken samples at 144 CFU/g or mL, without an enrichment step [50]. These findings collectively demonstrate that NAA-LFS assays, including PCR-LFS, offer highly sensitive detection of target organisms. However, a unique advantage of our PCR-LFS is its ability to detect both the bacterial species (E. coli or Salmonella spp.) and the antimicrobial resistance gene blaCTX-M in a single reaction.
Our current study demonstrated high validity of the dPCR-LFS assay in terms of accuracy, sensitivity, specificity, positive predictive value, and negative predictive value when tested with pure bacterial cultures. Although the assay shows promise for direct application to clinical or environmental samples, further validation using reference methods is necessary. One limitation of the current dPCR-LFS is its ability to detect only two targets per reaction, due to constraints in the availability of label molecules such as biotin and digoxigenin. A triplex format that includes E. coli-specific, Salmonella-specific, and blaCTX-M targets with distinct labels would improve efficiency and reduce cost. Therefore, future development is warranted. Moreover, while the assay was validated using pure isolates, its application to direct specimens remains a key challenge for further research.

4. Materials and Methods

4.1. Bacteria

E. coli and Salmonella strains harboring blaCTX-M, as well as other Enterobacterales species, are listed in Table 1. The 287 E. coli strains were isolated from humans (n = 162), ready-to-eat foods (n = 50), meats (n = 40), and environmental water (n = 35). A total of 95 Salmonella strains were isolated from humans (n = 50), ready-to-eat foods (n = 15), and meats (n = 30). Other Enterobacterales species (Table 1) were isolated from humans (n = 118) and environmental sources (n = 100). In addition, various non-Enterobacterales species were included to evaluate potential nonspecific reactions. These strains included: Streptococcus pneumoniae ATCC33400, S. anginosus ATCC33397, S. pyogenes SF370, S. agalactiae ATCC 13813, Enterococcus faecalis ATCC29212, E. faecium ATCC10541, Lactiplantibacillus plantarum ATCC 43199, Leuconostoc lactis ATCC19256, Micrococcus luteus ATCC10240, Bacillus subtilis ATCC6633, Staphylococcus aureus ATCC700698, Haemophilus influenzae ATCC10211, Achromobacter xylosoxidans ATCC27061, Pseudomonas aeruginosa ATCC9027, Acinetobacter baumannii ATCC 19606, Burkholderia cepacia LMG0122, Vibrio parahaemolyticus ATCC17802, Aeromonas hydrophila ATCC7966, and Neisseria flavescens ATCC13119. All bacterial strains were kept at −80 °C before used in this study.

4.2. DNA Extraction

DNA was extracted from cultured bacterial colonies using a simple alkaline lysis method [54]. Briefly, one or two colonies (incubated for 18–24 h) were suspended in 20 µL of lysis buffer containing 0.25% (v/v) sodium dodecyl sulfate and 0.05 M NaOH, then heated at 95 °C for 15 min. After lysis, samples were briefly centrifuged, and 180 µL of sterile deionized water was added. The crude DNA supernatant was stored at −20 °C and used as the template for PCR.

4.3. PCR Reaction

The PCR reaction mixture consisted of 1× PCRBIO HS Taq Mix Red (PCR Biosystems, London, UK) and 0.4 µM of each primer (Table 5). The thermal cycling conditions were as follows: initial denaturation at 95 °C for 2 min; 35 cycles of denaturation at 95 °C for 30 s; annealing at 58 °C for 30 s; and extension at 72 °C for 45 s. E. coli ATCC BAA-3303 (carrying blaCTX-M-15) and S. enterica strain SW060-1 (carrying blaCTX-M) were used as positive controls. Klebsiella pneumoniae ATCC 13883 was used as the negative control. PCR products were analyzed by 2% agarose gel electrophoresis (Mupid-ExU, Takara, Japan) in 0.5× TBE buffer for 30 min. Gels were stained with ethidium bromide and visualized under UV light (GeneGenius Bioimaging System; SynGene, Cambridge, UK). PCR product sizes were determined using a 100 bp Plus DNA ladder (GeneRuler™, Thermo Fisher Scientific, Waltham, MA, USA).

4.4. Lateral-Flow Strip (LFS)

LFS devices and reagents (K-AmpDetect 2T) were purchased from K.Bio Sciences (Bangkok, Thailand). Each strip had three lines (C, T1, and T2), with T1 and T2 lines immobilized with anti-biotin and anti-digoxigenin, respectively. The LFS reaction was done according to the manufacturer’s protocol. Briefly, strips were immersed in the PCR product for 10–20 s, after which 100 µL (or three drops) of DNA running buffer was added to the application pad. Results were observed within 2–10 min. The presence of both test lines (T1, T2, or both) and the control line (C) indicated a positive result. The appearance of only the control line indicated a negative result.

4.5. Confirmation of PCR Products by DNA Sequencing

To confirm the dPCR-LFS results, PCR products from strains testing positive for E. coli, Salmonella, or blaCTX-M were subjected to Sanger DNA sequencing using the respective forward and reverse primers without labelling molecules (Table 5). Sequences were analyzed using BLASTN (https://blast.ncbi.nlm.nih.gov/Blast.cgi; accessed on 5 June 2025).

4.6. Detection Limit

The detection limit of the duplex PCR was evaluated using blaCTX-M-harboring E. coli and Salmonella strains. Serial 10-fold dilutions were prepared from an initial DNA concentration of 72 ng/µL for E. coli and 105 ng/µL for Salmonella, based on OD260 readings. The detection limit was defined as the highest dilution at which a positive result was observed. Each test was performed in triplicate.

4.7. Artificially Spiked Raw Pork Mince Sample

Raw pork mince confirmed to be free of blaCTX-M-harboring E. coli and Salmonella by conventional culture and PCR methods was used for spiking experiments. Pure colonies of E. coli ATCC BAA-3303 and S. enterica strain SW060-1 were suspended in 1 mL of sterile saline, vortexed for 30 s, and adjusted to a turbidity of 1.00 using a turbidimeter. Serial 10-fold dilutions were prepared. Then, 10 g of pork mince was mixed with 99 mL of buffered peptone water (BPW; Oxoid, Basingstoke, UK) and inoculated with 1 mL of the appropriate dilution. One uninoculated sample served as a negative control. After inoculation, samples were stomached for 1 min. One milliliter of the homogenate was subjected to DNA extraction using ZymoBIOMICS DNA Kits (Zymo Research, Tustin, CA, USA) following the manufacturer’s instructions. Each dilution was tested in duplicate. Colony-forming units (CFU) were determined by plating on MacConkey agar for E. coli and XLD agar for Salmonella (Oxoid, Basingstoke, UK).

4.8. Statistical Analysis

Diagnostic performance measures—including sensitivity, specificity, and accuracy—were calculated [58]. The kappa statistic was used to evaluate interrater agreement [59]. Fisher’s exact test (p < 0.01 considered significant) was performed using GraphPad tools (https://www.graphpad.com/quickcalcs/, accessed on 5 June 2025) [60,61]. McFadden’s R2 was used to estimate model fit [62]. Receiver operating characteristic (ROC) curve analysis was performed using a 2 × 2 contingency table comparing PCR results with genotypic/phenotypic data [63]. Sensitivity and 1 − specificity were plotted to generate the ROC curve. All measurements were done in triplicate. A fixed SD of 0.02 was applied to illustrate variability, similar to approaches used in simulations or illustrative ROC analyses. A dotted reference line indicating ideal performance (100% sensitivity and specificity) was added as a benchmark. All analyses and visualizations were performed using Stata version 17.0.

4.9. Data Availability

Nucleotide sequence of blaCTX-M is available in the GenBank databases under the accession numbers PV809620-PV809640.

5. Conclusions

The dPCR-LFS assay in this study demonstrated high diagnostic performance for detecting E. coli and Salmonella strains harboring blaCTX-M from pure cultures. However, its application to direct specimens requires further validation. This method is rapid and user-friendly, offering simple interpretation without the need for gel electrophoresis. It holds significant potential for application in AMR surveillance across both human and non-human sectors.

Author Contributions

Conceptualization, R.H., P.C., P.B. and A.K.; methodology, R.H., R.U., K.S., P.C., P.B. and S.C.; validation, R.H., P.C., P.B. and A.K.; formal analysis, R.H., R.U., P.B., P.C. and K.S.; investigation, R.H., S.C., P.C. and P.B.; resources, A.K.; writing—original draft preparation, R.H. and A.K.; writing—review and editing, R.H. and A.K.; visualization, R.H.; supervision, A.K.; funding acquisition, A.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by Kasetsart University Research and Development Institute (KURDI) under Grant No. FF(KU-SRIU)16.67.

Institutional Review Board Statement

Not applicable because this study did not involve humans or animals.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Duplex PCR-LFS (A) and duplex PCR–gel electrophoresis (B) detection of E. coli with and without blaCTX-M, and non-E. coli with and without blaCTX-M. (A) Top line = control line; middle line = E. coli; bottom line = blaCTX-M. E. coli carrying blaCTX-M shows two bands (species-specific and blaCTX-M) (first strip from left). E. coli lacking blaCTX-M shows only the E. coli line (second strip). Non-E. coli strains carrying blaCTX-M show only the blaCTX-M line (third strip). Non-E. coli strains without blaCTX-M show no target lines (strips 4–8). Negative control = Klebsiella pneumoniae ATCC 13883. (B) Lane M = 100 bp DNA ladder; Lane 1 = E. coli with blaCTX-M; Lane 2 = E. coli without blaCTX-M; Lane 3 = non-E. coli with blaCTX-M; Lanes 4–8 = non-E. coli without blaCTX-M; Lane 9 = negative control (Klebsiella pneumoniae ATCC 13883).
Figure 1. Duplex PCR-LFS (A) and duplex PCR–gel electrophoresis (B) detection of E. coli with and without blaCTX-M, and non-E. coli with and without blaCTX-M. (A) Top line = control line; middle line = E. coli; bottom line = blaCTX-M. E. coli carrying blaCTX-M shows two bands (species-specific and blaCTX-M) (first strip from left). E. coli lacking blaCTX-M shows only the E. coli line (second strip). Non-E. coli strains carrying blaCTX-M show only the blaCTX-M line (third strip). Non-E. coli strains without blaCTX-M show no target lines (strips 4–8). Negative control = Klebsiella pneumoniae ATCC 13883. (B) Lane M = 100 bp DNA ladder; Lane 1 = E. coli with blaCTX-M; Lane 2 = E. coli without blaCTX-M; Lane 3 = non-E. coli with blaCTX-M; Lanes 4–8 = non-E. coli without blaCTX-M; Lane 9 = negative control (Klebsiella pneumoniae ATCC 13883).
Antibiotics 14 00745 g001
Antibiotics 14 00745 g002
Figure 2. Duplex PCR-LFS (A) and duplex PCR-gel electrophoresis; (B) detection of Salmonella spp. with and without blaCTX-M, and non-Salmonella spp. with and without blaCTX-M. (A) Top line = control line; middle line = blaCTX-M; bottom line = Salmonella. Salmonella carrying blaCTX-M shows two lines (first strip). Salmonella without blaCTX-M shows only the species-specific line (second strip). Non-Salmonella carrying blaCTX-M shows the blaCTX-M line (third strip). Non-Salmonella without blaCTX-M shows no target lines (strips 4–8). Negative control = Klebsiella pneumoniae ATCC 13883. (B) Lane M = 100 bp DNA ladder; Lane 1 = Salmonella with blaCTX-M; Lane 2 = Salmonella without blaCTX-M; Lane 3 = non-Salmonella with blaCTX-M; Lanes 4–8 = non-Salmonella without blaCTX-M; Lane 9 = negative control (Klebsiella pneumoniae ATCC 13883).
Figure 2. Duplex PCR-LFS (A) and duplex PCR-gel electrophoresis; (B) detection of Salmonella spp. with and without blaCTX-M, and non-Salmonella spp. with and without blaCTX-M. (A) Top line = control line; middle line = blaCTX-M; bottom line = Salmonella. Salmonella carrying blaCTX-M shows two lines (first strip). Salmonella without blaCTX-M shows only the species-specific line (second strip). Non-Salmonella carrying blaCTX-M shows the blaCTX-M line (third strip). Non-Salmonella without blaCTX-M shows no target lines (strips 4–8). Negative control = Klebsiella pneumoniae ATCC 13883. (B) Lane M = 100 bp DNA ladder; Lane 1 = Salmonella with blaCTX-M; Lane 2 = Salmonella without blaCTX-M; Lane 3 = non-Salmonella with blaCTX-M; Lanes 4–8 = non-Salmonella without blaCTX-M; Lane 9 = negative control (Klebsiella pneumoniae ATCC 13883).
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Antibiotics 14 00745 g003
Figure 3. ROC curve showing triplicate standard deviation and theoretical sensitivity of duplex PCR-LFS detecting E. coli and Salmonella carrying blaCTX-M.
Figure 3. ROC curve showing triplicate standard deviation and theoretical sensitivity of duplex PCR-LFS detecting E. coli and Salmonella carrying blaCTX-M.
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Antibiotics 14 00745 g004
Figure 4. Detection limit of duplex PCR–gel electrophoresis (A,C) and duplex PCR-LFS (B,D) detecting E. coli (A,B) and Salmonella spp. (C,D) harboring blaCTX-M, spiked in raw pork mince. Dilutions: 2.5 × 105 to 2.5 CFU/mL for E. coli, and 4 × 105 to 4 CFU/mL for Salmonella. M = 100 bp DNA ladder; B = blank control (uninoculated).
Figure 4. Detection limit of duplex PCR–gel electrophoresis (A,C) and duplex PCR-LFS (B,D) detecting E. coli (A,B) and Salmonella spp. (C,D) harboring blaCTX-M, spiked in raw pork mince. Dilutions: 2.5 × 105 to 2.5 CFU/mL for E. coli, and 4 × 105 to 4 CFU/mL for Salmonella. M = 100 bp DNA ladder; B = blank control (uninoculated).
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Table 1. Evaluation of dPCR-LFS for detection of blaCTX-M in E. coli, Salmonella, and other Enterobacterales.
Table 1. Evaluation of dPCR-LFS for detection of blaCTX-M in E. coli, Salmonella, and other Enterobacterales.
SpeciesNAntimicrobial-Resistant Genotypes/PhenotypesCiprofloxacin Disk DiffusionESBL-ProducingPCR-LFS
SpeciesblaCTX-M
E. coli
(N = 287)		100blaCTX-M/blaTEMR+++
100blaCTX-M/blaNDM/blaOXA-48-like (CRE)R+++
7blaNDM (CRE)R−+−
5blaOXA-48-like (CRE)R−+−
25blaTEM/blaSHVND−+−
10blaTEM/blaSHVND++−
30Non-cephalosporins resistanceND−+−
10blaTEMND−+−
Salmonella sp.
(N = 95)		30blaCTX-M, Ciprofloxacin-susceptibleS+++
50Ciprofloxacin-resistanceR−+−
15Cephalosporins and Ciprofloxacin-susceptibleS−+−
Klebsiella pneumoniae
(N = 95)		45blaCTX-M/blaSHVND+−+
25blaCTX-M/blaNDM/blaOXA-48-like (CRE)R+−+
5blaNDM/blaOXA-48-like (CRE)R−−−
12blaTEM/blaSHVND+−−
8blaSHVND+−−
Klebsiella oxytoca10blaCTX-M/blaSHVND+−+
Klebsiella aerogenes5blaCTX-M/blaSHVND+−+
Enterobacter asburiae10blaCTX-M/blaNDM/blaOXA-48-like (CRE)R+−+
Enterobacter cloacae complex (N = 25)10blaCTX-MND+−+
10blaCTX-M/blaNDM/blaOXA-48-like (CRE)R+−+
5blaNDM/blaOXA-48-like (CRE)R−−−
Citrobacter freundii
(N = 28)		6blaCTX-MND+−+
14blaCTX-M/blaNDM/blaOXA-48-like (CRE)ND+−+
8Cephalosporins susceptibleND−−−
Proteus mirabilis12blaCTX-MND+−+
Proteus vulgaris3noneND−−−
Cronobacter sakazakii5noneND−−−
Shigella sonnei2noneND−−−
Edwardsiella tarda2noneND−−−
Morganella morganii6noneND−−−
Serratia marcescens15noneND−−−
Total600
Table 2. Diagnostic performance of dPCR-LFS in detecting E. coli and Salmonella harboring blaCTX-M.
Table 2. Diagnostic performance of dPCR-LFS in detecting E. coli and Salmonella harboring blaCTX-M.
PCR-LFSGenotypic/Phenotypic Resultsp-Value
PositiveNegative
Positive200 (E. coli)/30 (Salmonella)0<0.001
Negative071
Total23071
Accuracy = 100% (95% CI = 98.78% to 100.00%); Sensitivity = 100% (95% CI = 98.41% to 100.00%); Specificity = 100% (95% CI = 94.94% to 100.00%); PPV = 100% (95% CI = 98.41% to 100.00%); NPV = 100% (95% CI = 94.94% to 100.00%); κ = 1; R2 = 0.92952.
Table 3. Diagnostic performance of dPCR-LFS in detecting E. coli and Salmonella lacking blaCTX-M.
Table 3. Diagnostic performance of dPCR-LFS in detecting E. coli and Salmonella lacking blaCTX-M.
PCR-LFSGenotypic/Phenotypic Resultsp-Value
PositiveNegative
Positive287 (E. coli)/95 (Salmonella)0<0.001
Negative0218
Total382218
Accuracy = 100% (95% CI = 99.39% to 100.00%); Sensitivity = 100% (95% CI = 99.04% to 100.00%); Specificity = 100% (95% CI = 98.32% to 100.00%); PPV = 100% (95% CI = 99.04% to 100.00%); NPV = 100% (95% CI = 98.32% to 100.00%); κ = 1; R2 = 0.96621.
Table 4. Diagnostic performance of dPCR-LFS for detection of blaCTX-M among Enterobacterales.
Table 4. Diagnostic performance of dPCR-LFS for detection of blaCTX-M among Enterobacterales.
PCR-LFSGenotypic/Phenotypic Resultsp-Value
PositiveNegative
Positive3770<0.001
Negative0223
Total377223
Accuracy = 100% (95% CI = 99.39% to 100.00%); Sensitivity = 100% (95% CI = 99.03% to 100.00%); Specificity = 100% (95% CI = 98.36% to 100.00%); PPV = 100% (95% CI = 99.03% to 100.00%); NPV = 100% (95% CI = 98.36% to 100.00%); κ = 1; R2 = 0.96642.
Table 5. Primer used in this study.
Table 5. Primer used in this study.
Primer NameSequence (5′–3′)Product Size (bp)TargetPCR SetReference
uidA-F5′-FITC-AAAACGGCAAGCAAAAGCAG147E. coli1[55]
uidA-R5′-Digoxigenin-ACGCGTGGTTAACAGTCTTGCG
CTX-M-U15′-FITC-ATGTGCAGYACCAGTAARGTKATGGC594blaCTX-M[35]
CTX-M-U25′-Biotin-TGGGTRAARTARGTSACCAGAAYCAGCGG
invA-F5′-FITC-GAGGAAAAAGAAGGGTCG780Salmonella spp.2[56]
invA-R5′-Biotin-CTCAACTTCAGCAGATACCA
CTX-M-F5′-FITC-TTTGCGATGTGCAGTACCAGTAA544blaCTX-M[57]
CTX-M-R5′-Digoxigenin-CGATATCGTTGGTGGTGCCATA
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Hatrongjit, R.; Chaisaeng, S.; Sitthichotthumrong, K.; Boueroy, P.; Chopjitt, P.; Ungcharoen, R.; Kerdsin, A. Polymerase Chain Reaction-Lateral Flow Strip for Detecting Escherichia coli and Salmonella enterica Harboring blaCTX-M. Antibiotics 2025, 14, 745. https://doi.org/10.3390/antibiotics14080745

AMA Style

Hatrongjit R, Chaisaeng S, Sitthichotthumrong K, Boueroy P, Chopjitt P, Ungcharoen R, Kerdsin A. Polymerase Chain Reaction-Lateral Flow Strip for Detecting Escherichia coli and Salmonella enterica Harboring blaCTX-M. Antibiotics. 2025; 14(8):745. https://doi.org/10.3390/antibiotics14080745

Chicago/Turabian Style

Hatrongjit, Rujirat, Sumontha Chaisaeng, Kulsatree Sitthichotthumrong, Parichart Boueroy, Peechanika Chopjitt, Ratchadaporn Ungcharoen, and Anusak Kerdsin. 2025. "Polymerase Chain Reaction-Lateral Flow Strip for Detecting Escherichia coli and Salmonella enterica Harboring blaCTX-M" Antibiotics 14, no. 8: 745. https://doi.org/10.3390/antibiotics14080745

APA Style

Hatrongjit, R., Chaisaeng, S., Sitthichotthumrong, K., Boueroy, P., Chopjitt, P., Ungcharoen, R., & Kerdsin, A. (2025). Polymerase Chain Reaction-Lateral Flow Strip for Detecting Escherichia coli and Salmonella enterica Harboring blaCTX-M. Antibiotics, 14(8), 745. https://doi.org/10.3390/antibiotics14080745

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