Microchemical Journal 197 (2024) 109732

Contents lists available at ScienceDirect

Microchemical Journal
journal homepage: www.elsevier.com/locate/microc

Real-time and visual detection of viable Salmonella in milk from remote

pasture via IMS-LAMP-NALFS
Qianxin Li a, 1, Jingfeng Zhang a, 1, Xiaoxing Chen b, 2, Tingting Jiang a, Li Lin a, Lichao Zhao a, 2, *
a
Guangdong Provincial Key Laboratory of Nutraceuticals and Functional Foods, College of Food Science, South China Agricultural University, Guangzhou 510642, China
b
Yangjiang Jinguo Basket Co., Ltd., Yangjiang 529500, China

A R T I C L E I N F O A B S T R A C T

Keywords: Salmonella is a prevalent foodborne pathogen commonly found in dairy products. Controlling the presence of
Pasture Salmonella during dairy product production is of utmost concern due to the potential for contamination at any
Raw milk stage of the production chain, particularly at the source. Moreover, the unique location of pastures and limited
Salmonella
testing resources further highlight the necessity of establishing a rapid and precise detection method to ensure
On-site detection
Nucleic acid lateral flow strip (NALFS)
milk quality control and enhance overall farm safety. To address these issues, in this study, we constructed a
method as well as a portable toolbox for rapid on-site detection of Salmonella. The method involves immuno­
magnetic separation (IMS) using streptavidin-coupled magnetic nanoparticles and biotinylated antibodies. The
reaction conditions for magnetic capture and enrichment of Salmonella were optimized to efficiently remove
matrix interference from milk and enhance detection sensitivity. Additionally, an improved propidium mono­
azide (PMAxx) treatment was employed to eliminate false positive results caused by dead bacteria. This treat­
ment was combined with loop-mediated isothermal amplification (LAMP) and nucleic acid lateral flow strip
(NALFS) (LAMP-NALFS) for on-site detection of viable bacteria in milk. The resulting IMS-LAMP-NALFA assay
achieved rapid and sensitive detection of viable Salmonella, with a detection sensitivity of 1 × 102 CFU/mL in
milk samples, completed within 90 min under optimal conditions. Based on this achievement, a portable toolbox
was designed for easy transportation and on-site testing in pastures. It is equally applicable for rapid detection of
viable Salmonella contamination in food products in resource-limited areas such as pastures, where the observed
detection rate of Salmonella in unpasteurized raw milk was 7.93 % (28 out of 353 samples tested). Consequently,
the IMS-LAMP-NALFA method and the associated portable toolbox represent highly effective means of enhancing
the detection of viable Salmonella in dairy products.

1. Introduction capable of jeopardizing the quality and safety of dairy products [5,6].
Research has indicated that, the overall prevalence of clinical mastitis in
Salmonella, a Gram-negative short bacillus, is a type of intestinal Chinese dairy cows is 10 %, with the highest prevalence in South China
pathogenic bacteria, that lacks spores, demonstrates aerobic, charac­ reaching 35 %, making mastitis the most common disease in dairy cows
teristics, and can also intermittently function as an anaerobic bacterium [7]. Cows afflicted with mastitis are often susceptible to Salmonella
[1]. In China, this pathogen is responsible for approximately 70 % to 80 infection, leading to the contamination of their milk [8].Since Salmo­
% of bacterial food poisoning cases each year, imposing a substantial nella does not break down proteins, and contaminated dairy products
burden on public health and the economy, especially within the food may not undergo significant changes in appearance or odor, detection
industry [2,3,4]. Being the most prominent foodborne pathogen, Sal­ can be challenging [9,10,11]. In particular, the shortage of detection
monella is frequently found in poultry, eggs, and dairy products. resources in remote farm locations exacerbates this issue. Presently,
Contamination of raw milk, situated at the upstream of the dairy in­ Salmonella detection primarily relies on the complex and time-
dustry chain, represents a crucial source of foodborne pathogens, consuming traditional culture method (GB 4789.4–2016, China) [12].

* Corresponding author.
E-mail address: [email protected] (L. Zhao).
1
These authors contributed equally to this work and should be considered joint first authors.
2
These authors contributed equally to this work and should be considered joint corresponding author.

https://doi.org/10.1016/j.microc.2023.109732
Received 17 September 2023; Received in revised form 21 November 2023; Accepted 27 November 2023
Available online 30 November 2023
0026-265X/© 2023 Elsevier B.V. All rights reserved.
Q. Li et al. Microchemical Journal 197 (2024) 109732

Although nucleic acid amplification-based methods offer higher sensi­ method that utilizes specific antibodies on the surface of magnetic bends
tivity and faster detection speeds, they often entail high instrumentation to capture the target bacteria in the sample, and then with the presence
costs and the need for specialized personnel. Therefore, there is an ur­ of a magnetic field, the bends are separated by adsorption, enabling
gent need for the development of fast and user-friendly field detection enrichment of the target bacteria from the food matrix. IMS and PMAxx
technologies and products suitable for remote areas. combined with real-time polymerase chain reaction (qPCR) (IMS-
In pursuit of on-site detection of foodborne pathogenic bacteria in PMAxx-qPCR) has demonstrated high sensitivity in detecting Vibrio
remote regions, researchers have devised traditional immunochroma­ parahaemolyticus, with a detection limit as low as 1.85 CFU/g, while
tographic methods based on antigenic antibodies. However, these PMAxx-qPCR struggled to quantify the absolute percentage of viable
methods frequently encounter problems such as low sensitivity, leading bacteria when the target was below 0.1 %. This illustrates that IMS can
to missed detections [13]. In recent years, the emergence of Nucleic acid reduce the interference of the food matrix during detection [24].
lateral flow strip (NALFS) has garnered significant attention and has Nevertheless, the application of IMS combined with the PMAxx-LAMP-
found continuous application in the rapid testing and diagnostic in­ NALFS technique for detecting Salmonella in milk samples from farm
dustry due to its high sensitivity [14]. NALFS can provide visualized sites is yet to be explored, and the factors affecting its specificity and
results for the detection of nucleic acid products like polymerase chain sensitivity in this context remain unclear.
reaction (PCR) and loop-mediated isothermal amplification (LAMP) in a Hence, the objective of this study was to establish a rapid and ac­
short time, without the need for any specialized equipment, making it curate method for detecting Salmonella in raw milk by utilizing PMAxx-
highly valuable for field detection of foodborne pathogens [15]. The LAMP-NALFS in conjunction with IMS. We prepared immunomagnetic
process of nucleic acid chromatography involves two stages: nucleic acid beads using streptavidin-coupled magnetic nanoparticles and bio­
amplification and product detection. Primers, such as FAM and biotin, tinylated antibodies, and carefully optimized the reaction conditions for
are labeled to the antigen during nucleic acid amplification, and the magnetic capture and enrichment of Salmonella. Additionally, we
amplified PCR or LAMP product is subsequently captured by NALFS for employed PMAxx treatment and the combination of LAMP-NALFS to
detection [16]. LAMP, being a method that amplifies DNA or RNA create a robust detection method. To facilitate practical use, we
without thermal cycling, allows product detection to be completed designed a convenient equipment kit for carrying and transporting,
within 5 min, faster than the traditional PCR method that requires 30 enabling the application of this portable toolbox for on-site detection of
min of electrophoresis after amplification, making LAMP one of the most viable Salmonella contamination in resource-limited areas using raw
widely used nucleic acid amplification techniques [17]. The LAMP- milk samples from pasture settings.
NALFS method is especially well-suited for the rapid detection of
foodborne pathogens, including Salmonella in resource-limited settings. 2. Materials and methods
Nonetheless, the LAMP-NALFS technique still exhibits some draw­
backs when detecting Salmonella. For instance, it cannot differentiate 2.1. Bacterial strains and culture conditions
between dead and live bacteria [18]. As the DNA of bacteria does not
immediately decompose after death, the DNA of dead bacteria can also The strains utilized in this study are listed in Table 1. All tested
amplify, leading to interference and potential false positives in the strains were preserved in Luria-Bertani (LB) broth (HuanKai Microbial,
actual test [19,20]. Furthermore, in products like milk that are rich in China) supplemented with triglyceride at a temperature of − 20 ◦ C.
carbohydrates, proteins, and lipids, these components typically hinder Subsequently, they were cultured in LB broth on a rotary shaker (180
DNA polymerase activity, inhibiting nucleic acid amplification and rpm) for 24 h at 37 ◦ C. The enumeration of viable Salmonella
reducing the sensitivity and selectivity of molecular detection [21].
Addressing these issues requires a comprehensive and integrated solu­
tion to enhance the sensitivity of detection in real samples and achieve Table 1
rapid on-site Salmonella detection. Bacterial strains used in PMAxx-LAMP-NALFS and IMS-LAMP-NALFA assays.
In our preliminary study, to overcome the interference of dead Serial Species Strain The result of The result of
bacteria in the detection process, we used SYTO-9, a fluorescent dye number PMAxx-LAMP- IMS-LAMP-
capable of distinguishing dead and live bacteria, in combination with PI NALFS NALFA
to develop the Fluorescence immunochromatographic assay (FICA) live 1 Salmonella ATCC14,028 + +
bacteria detection technology for accurate Salmonella detection by typhimurium
immunochromatography. However, this method had a higher detection 2 Salmonella Isolate01 + +
typhimurium
limit, making it less the practical compared to nucleic acid amplification
3 Salmonella Isolate02 + +
techniques. To address this limitation, the literature suggested using typhimurium
propidium monoazide (PMA), a photosensitive dye with a strong bind­ 4 Staphylococcus ATCC − −
ing affinity to nucleic acids. PMA can penetrate the cell membranes of aureus 25,923
damaged or dead cells, and under strong light irradiation, it forms stable 5 Cronobacter ATCC − −
sakazakii 29,544
covalent carbon and nitrogen bonds with DNA, irreversibly modifying it
6 Cronobacter ATCC − −
and preventing the amplification of dead bacteria. Improved propidium sakazakii 12,868
monoazide (PMAxx), an improved version of PMA, functions similarly 7 Escherichia coli CICC 10,662 − −
but with the added advantage of achieving a 3–7 Ct value difference in EIEC
amplification between live and dead bacteria, effectively distinguishing 8 Escherichia coli ATCC − −
O157:H7 35,150
between the two and reducing the detection limit [22]. 9 Escherichia coli ATCC − −
Dealing with the complex food matrix in dairy products, the sepa­ 25,922
ration of bacteria from the food can enhance the sensitivity of detection. 10 Vibrio ATCC − −
Traditional methods like membrane filtration and centrifugation, parahaemolyticus 17,802
11 Pseudomonas ATCC
though simple and cos-effective, lack selectivity in separating and
− −
aeruginosa 15,442
concentrating bacteria from food products. They tend to adsorb to food 12 Bacillus cereus CMCC − −
matrix particles, making complete separation through centrifugation 63,303
challenging, and membrane filters often become clogged with large food 13 Listeria ATCC − −
particles, leading to difficulties in collecting high-purity target bacteria monocytogenes 19,115

[23]. In contrast, Immunomagnetic Separation (IMS) is an effective +: positive result; -: negative result.

2
Q. Li et al. Microchemical Journal 197 (2024) 109732

typhimurium (ATCC 14028) cells was carried out through plate count­ min). Blank controls were prepared using PBS instead of the bacterial
ing. The cultures were subjected to serial dilution with phosphate- solution. Subsequently, samples were centrifuged at 10,000 × g for 5
buffered saline (PBS) and then plated onto tryptone soy agar (TSA, min to eliminate any free PMAxx, followed by washing with PBS two
HuanKai Microbial, China) before being incubated at 37 ◦ C for 24 h. To times.
prepare non-viable cells, the bacterial suspensions were subjected to
heat treatment at 85 ◦ C for 10 min. 2.5. DNA template extraction

2.2. Preparation of artificially contaminated milk samples The DNA extraction was conducted using the Tris-EDTA buffer
(Sangon Biotechnology, Shanghai, China) based boiling method. First, 1
Milk samples were procured from a local supermarket in Foshan, mL of the bacterial culture was centrifuged at 10,000 × g and then boiled
China, and underwent testing using the standard methods outlined in GB in 50 µL of Tris-EDTA buffer for 10 min. Subsequently, the DNA was
4789.4–2016 (China). If Salmonella was detected, the milk samples were placed on ice and cooled for 5 min, followed by centrifugation at 8,000
pasteurized to ensure their subsequent use in experiments was free from × g for 5 min to remove cell debris. The resulting supernatant was
Salmonella contamination. The milk samples (25 mL) were combined wit collected as the DNA template and stored at − 20 ◦ C until further use.
225 mL of PBS in an aseptic homogeneous cup and mixed thoroughly.
Subsequently, the milk samples were contaminated with 1 mL of various 2.6. Primers and probes
Salmonella concentrations, resulting in final concentrations ranging
from 1.0 × 101 to 1.0 × 106 CFU/mL. Salmonella invA virulence gene sequences selected as target genes for
the detection of Salmonella. Table S1 presents the Salmonella primers
employed in this study. For the NALFS detection, primer LF and primer
2.3. Immunomagnetic separation
FIP underwent modifications, with biotin and fluorescein isothiocyanate
(FITC) added at the 5′ ends, respectively. The synthesis of both unlabeled
2.3.1. Preparation of immunomagnetic bends
and labeled primers was carried out by Takara Biomedical Technology
The streptavidin-modified immunomagnetic beads were gently
(Beijing, China).
swirled and mixed for 1 min. Subsequently, 25 µL of streptavidin mag­
netic beads (10 mg/mL) were absorbed into a sterile 1.5 mL centrifuge
2.7. LAMP condition
tube and magnetically separated for 2 min. A sterile PBST solution (0.01
M PBS containing 0.05 % Tween-20, pH 7.4) was used for washing,
During the LAMP amplification process, the reaction mixture was
repeated 2–3 times. The magnetic separation of the centrifuge tube was
composed of the following components 2.5 μL of 10 × Isothermal
performed on a magnetic separation rack, and the supernatant was then
Amplification Buffer, 6 mM Mg2+, 1.4 mM dNTPs, 0.6 mM betaine, 0.2
discarded. Next, the appropriate concentration of biotinylated antibody
μM of each F3 and B3 primer, 1.6 μM of each FIP and BIP primer, 0.8 μM
was added, mixed thoroughly, and left to incubate at room temperature
of each LF and LB primer, 6 U of Bst 2.0 DNA polymerase (8000 U/mL,
for 60 min on a vertical mixing instrument (rotation speed 15 r/min).
HaiGene Biotech, China), 2 μL of target DNA, and ddH2O, resulting in a
After 5 min of magnetic separation, the supernatant was discarded, and
total volume of 25 μL for the reaction. To prevent aerosol pollution, 20
the bends were washed 2–3 times with PBST. The prepared immuno­
μL of paraffin oil was introduced into the reaction mixture before adding
magnetic beads (IMBs) were suspended in PBST solution containing 0.1
the template DNA [25,26]. The reaction was carried out using a block
% BSA and 0.05 % NaN3, and stored at 4 ◦ C for future use.
heater (Shanghai Kunke, China) with incubation at 64 ◦ C for 40 min,
followed by heat inactivation at 85 ◦ C for 5 min.
2.3.2. IMS reaction condition optimization
The optimal capture conditions in the IMS experiment were estab­
2.8. NALFS detection
lished by varying the levels of antibody addition (3, 6, 12, 18, and 24 μg)
and IMBs addition (25, 50, 100, 150, and 200 μL), along with different
As show in Fig. 1a, NALFS comprises four components: the sample
incubation times (15, 30, 45, 60, and 75 min) and magnetic separation
pad, conjugate pad, absorbent pad (Shanghai Kinbio, China), and NC
times (2, 3, 4, 5, and 8 min).
membrane (mdi, India). To detect the LAMP amplicon using NALFS, a
colored latex microspheres (magsphere, Germany) labeled anti-FITC
2.3.3. Capture efficiency values（CEs）
antibody and streptavidin were incorporated into the conjugate pad
The Capture efficiency values（CEs）, which were use to represent
and the test line on the NC membrane, respectively. The control line was
the percentage of total bacteria retained on the IMBs. It was determined
coated with an anti-mouse antibody. For the testing process, 5 μL of the
by dividing the number of isolated Salmonella by the total number of
LAMP product and 75 μL of buffer (PBS containing 2 % S9) were applied
Salmonella present in a sample. The calculation of CE was done using the
to the sample pad, with the flow direction moving from the sample pad
following equation:
to the absorbent pad. Within 6 min, the amplification results became
CE(%) = (1 − B/A) × 100% visible to the naked. A sample yielding two bands at the test line (T line)
and the control line (C line) indicated a positive result. Conversely, a
where A represents the total number of bacteria in the sample (CFU/mL single band at the control line indicated a negative result. If the control
or CFU/g), and B indicates the number of unbound bacteria in the su­ line was not visible, the test strip was deemed invalid.
pernatant (CFU/mL or CFU/g).
2.9. The detection limit and specificity of PMAxx-LAMP-NALFS and IMS-
2.4. PMAxx optimization LAMP-NALFA

Salmonella suspension is diluted to a final concentration of approxi­ Under the meticulously optimized conditions, both PMAxx-LAMP-
mately 106 CFU/mL and subjected to heat inactivation to produce dead NALFS and IMS-LAMP-NALFA techniques were employed for the
cells. PMAxx was dissolved in water and prepared as a 2 mM working detection of 10-fold dilutions raging from 1.0 × 101 to 1.0 × 106 CFU/
solution. Varying volumes of PMAxx were added to 1 mL of dead cells, mL of viable cells. Additionally, 13 strains of bacterial solutions were
resulting in final concentrations of 0, 2, 4, 6, and 8 µM. The PMAxx- diluted to 105 CFU/mL using a sample volume of 1 mL, and to ascertain
treated cells were then incubated in the dark for 5 min at room tem­ specificity, the negative control was replaced with sterile water to
perature and exposed to different light durations (0, 2, 4, 6, 8, and 10 determine the specificity.

3
Q. Li et al. Microchemical Journal 197 (2024) 109732

Fig. 1a. Schematic illustration for the detection of viable Salmonella using the IMS-LAMP-NALFA system.

2.10. Detection limit of viable Salmonella in artificially contaminated 2.11. Practical application in artificially contaminated milk samples
milk samples
To assess the detection capability of various assays under the influ­
Artificially contaminated milk samples were meticulously prepared ence of dead bacteria interference, each assay was subjected to evalu­
with viable Salmonella at concentrations ranging from 10 to 106 CFU/ ation by testing artificially contaminated milk samples containing
mL, following the procedures outlined in section 2.2. The bacterial so­ different ratios of viable and dead Salmonella suspensions. The prepa­
lution was thoroughly mixed with the IMBs and subsequently captured, ration of artificially contaminated milk samples was conducted as
as detailed above. After the capture, PMAxx treatment, DNA extraction, detailed in section 2.8, and 1 mL of the varying proportions of bacteria
and the LAMP-NALFS assay were carried out. Concurrently, samples indicated in Table 3 was incubated with 9 mL of homogenate. The
devoid of Salmonella contamination were utilized as negative controls. standard culture method adhered to the Chinese national standard GB
4789.36–2016.

4
Q. Li et al. Microchemical Journal 197 (2024) 109732

2.12. Design of a portable toolbox suspension (1 × 105 CFU/ mL PBS). Fig. 2b displays the correlation
between the supplemental level of IMBs and the corresponding CE value.
As depicted in Fig. 1b, the toolbox comprised a magnetic separator, AS the IMBs dose increased from 25 μL to 100 μL, the CE gradually
an intelligent temperature-controlled metal bath device, an LED pho­ improved from 75.875 % to 98.025 %, indicating that the added IMBs
toreactor, a bottle of PMAxx dye, a mini high-speed centrifuge, and a set were adequately facilitating bacterial coupling. At the level of 100 μL
of pipettes. This well-equipped toolbox facilitates rapid on-site testing in IMBs, the CE value peaked. Beyond this point, the CE value reached a
resource-limited environments, including pastures. stable state and did not exhibit further growth with additional IMBs.
Therefore, incorporating 100 μL of IMBs to 1 mL of 1 × 105 CFU/mL
2.13. Analysis of actual milk samples bacterial suspension ensures a higher CE.

A comprehensive collection of 353 milk samples was obtained from 3.1.3. Optimization of immune response time
multiple small dairy farms within the Guangdong Province region. Each If the immune response time is too short, the IMBs may not fully bind
case involved the collection of two samples. One sample was subjected with the target bacteria. To determine the optimal immune response
to on-site testing using the portable toolbox, while the other sample time, we conducted experiments using five different periods: 15, 30, 45,
underwent ultra-high-temperature instantaneous sterilization (held at 60, and 75 min. The results, as shown in Fig. 2c, revealed that as the
135 ◦ C for 3 s) before testing. Simultaneously, all the collected samples immune response time increased from 15 min to 45 min, the CE value
were cultured using the standard methods (GB 4789.4–2016, China). also increased and reached its peak at 45 min (CE = 98.349 %). How­
ever, with further extension of the immune response time, the CE value
2.14. Statistical analysis showed minimal change at 75 min. These finding suggest that the
optimal time for immune response is 45 min.
The error bars in the graphs represent the standard deviation (SD).
Graphs were generated using Origin graphing software, ver. 8.5. To 3.1.4. Optimization of immunomagnetic separation time
ensure reproducibility, all experiments were conducted in triplicate. To determine the effect of immunomagnetic separation time on CE,
the immunomagnetic separation time was studied before PMAxx treat­
3. Results and discussion ment. The result is shown in Fig. 2d. When the time of immunomagnetic
separation was 1–4 min, CE increased with the extension of time, and
3.1. Optimization of IMS reaction conditions further extension of immunomagnetic separation time had little effect
on CE value. Considering the overall efficiency, the optimal time of
3.1.1. Optimization of antibody addition amount immunomagnetic separation was 4 min (CE = 97.28 %).
The binding ratio of antibodies to magnetic nanoparticles plays a To wrap up, IMS serves as an effective pretreatment method capable
pivotal role in the CE of Salmonella in IMS. To determine the ideal of selectively isolating target bacteria from food and reducing the in­
coupling rate between biotinylated monoclonal antibodies and magnetic fluence of food substrates on subsequent detection techniques [27,28].
beads, IMBs were prepared with varying antibody concentrations of Several of the above optimizations, all of the resulted in a capture rate of
3–24 µg per 0.25 mg of magnetic beads. Fig. 2a illustrates the rela­ Salmonella exceeding 97 %, signifying excellent capture efficiency. As a
tionship between the quantity of biotinylated polyclonal antibodies and point of comparison, a study by Tsai, HL [29] demonstrated an average
CE. As the amount of antibody added increased from 3 µg to 12 µg, the recovery efficiency 53.5 ± 12.9 % (n = 24) for IMS in different Salmo­
CE also showed an upward trend, peaking at 12 µg (CE = 98.658 %). nella concentrations (ranging from 102 to 105 CFU/mL). The detection
Subsequently, the CE values plateaued when more than 12 µg of anti­ limit of IMS-LAMP-NALFA was ten times lower than PMAxx-LAMP-
body was added. Therefore, 12 µg of antibody addition was identified as NALFS without IMS treatment in artificially contaminated milk sam­
the optimal dose for further studies in the subsequent experiments. ples. Similar observations have been reported in other studied, such as
that of Yuanyi W. et al. [20], where the sensitivity of PMAxx-LAMP-
3.1.2. Optimization of IMBs addition amount NALFS for E. coli detection increased from 8.1 × 102 CFU/mL to 81
To investigate the impact of different doses of IMBs on CE, we CFU/mL after the addition of magnetic bead enrichment, allowing for
introduced 25, 50, 100, 150, and 200 μL of IMBs into 1 mL of bacterial E. coli contamination detection in lettuce within 2 h without the need for

Fig. 1b. The schematic diagram of the portable toolbox.

5
Q. Li et al. Microchemical Journal 197 (2024) 109732

Fig. 2. Optimization of IMS reaction conditions. (a) Optimization of antibody addition amount; (b) Optimization of IMBs addition amount; (c) Optimization of
Incubation time; (d) Optimization of Immunomagnetic separation time.

large instruments. exposure time of 0–6 min, but with an exposure time of 8 min, the
amplification of DNA from dead cells was fully inhibited. The optimized
3.2. Optimization of PMAxx treatment PMAxx concentration was found to be 6 µM, and the ideal light exposure
time was 8 min.
PMAxx, a nucleic acid dye used before nucleic acid amplification, After PMAxx treatment under optimal conditions, the detection re­
acts as an inhibitor of dead bacteria amplification during the process, sults for dead bacterial solutes were negative, affirming the effectiveness
effectively distinguishing between dead and live bacteria [30]. EMA and of the staining treatment inhibiting the amplification of dead bacteria. A
PMA also possesses similar effects, but both dyes, EMA and PMA, have similar study was previously reported in beef samples inoculated with
limitations as they do not sufficiently inhibit the signal in the presence 105 CFU/g heat-killed E. coli O157: H7, where only loop-mediated
of ≥ 105 dead bacteria [31,32]. When LAMP-NALFS is applied to isothermal DNA amplifification (qLAMP) detected 5.15 Log10 CFU/g,
PMAxx-treated bacteria, two factors, namely PMAxx concentration and while PMA-qLAMP and PMAxx-qLAMP yielded negative results [19].
light duration, can impact its ability to differentiate between dead and
live bacteria [33]. If the PMAxx concentration is too high, it may have a 3.3. Verification of the PMAxx treatment effectiveness
toxic effect on living cells; conversely, if it is too low, it might not
adequately inhibit the amplification of dead bacterial DNA. Addition­ Table 2 presents the results of the 10-fold serial dilution of viable,
ally, the duration of illumination significantly affects the results, as dead, and viable containing dead Salmonella cells, with or without
PMAxx permanently modifies dead DNA after strong light irradiation, PMAxx treatment, for comparison. The LAMP-NALFS method exhibited
preventing its amplification during LAMP [34]. To enhance the effi­ the ability to detect pure dead bacteria and pure viable bacteria solu­
ciency of this method, the concentration of PMAxx dye and light dura­ tions at concentrations ranging from 102 to 106 CFU/mL, as well as all
tion were optimized. concentrations of the mixture of dead and viable bacteria. Upon PMAxx
To determine the optimal conditions for PMAxx treatment, a quan­ treatment, the results for the dead bacteria liquid yielded negative sig­
tity of 1.0 × 106 CFU/mL non-viable Salmonella was employed. As nals, indicating the effective inhibition of dead bacteria expansion
illustrated in Fig. S1a, the T line remained red as the PMAxx concen­ through the dyeing treatment. PMAxx-LAMP-NALFS demonstrated the
tration increased from 0 µM to 4 µM. However, at 6 µM PMAxx con­ capacity to detect at least 102 CFU/mL, illustrating the selective inhi­
centration, the T line vanished, indicating complete inhibition of non- bition of dead bacteria DNA interference by PMAxx treatment. Despite a
viable cells. Similarly, as depicted in Fig. S1b, PMAxx treatment failed significant difference between the concentration of viable and dead
to entirely suppress amplification of non-viable cell DNA within a light bacteria, the PMAxx treatment successfully eliminated high-

6
Q. Li et al. Microchemical Journal 197 (2024) 109732

Table 2 effectively enhanced the detection sensitivity by an order of magnitude.

Verification of the PMAxx treatment effectiveness. These findings demonstrate that IMS facilitates selective enrichment of
Concentration of bacterium (CFU/mL) Results target bacteria, mitigating the influence of LAMP inhibitors and milk
(Viable/Dead/Viable containing dead matrices, thereby elevating the overall detection sensitivity in milk
bacteria) samples.
LAMP-NALFS PMAxx-LAMP-NALFS

106 +/+/+ +/− /+ 3.6. Comparison of the application in artificially contaminated milk
105 +/+/+ +/− /+ samples
104 +/+/+ +/− /+
103 /+
+/+/+ +/−
To investigate the practical application of different assays, artificially
102 +/+/+ +/− /+
101 − /− /+ − /− /− contaminated milk samples with varying proportions of viable and dead
NTC − /− /+ − /− /− Salmonella were subjected to detection using the standard culture
method, LAMP-NALFS, PMAxx-LAMP-NALFS, and IMS-LAMP-NALFA
NTC: Negative Control; “+” indicates positive reaction; “− ” indicates negative
reaction.
assays. As indicated in Table 3, both LAMP-NALFS and PMAxx-LAMP-
NALFS demonstrated the capability to detect 103 CFU/mL of Salmo­
nella. However, concerning the same concentrations of viable bacteria,
concentration signals from dead bacteria DNA, ensuring the detection of
different outcomes were observed, with false positive results arising due
low concentrations of viable bacteria. To assess the sensitivity of PMAxx-
to the presence of dead bacteria. Conversely, no false positive results
LAMP-NALFS, the viable Salmonella bacterial solution was serially
were observed in the detection outcomes of PMAxx-LAMP-NALFS and
diluted to achieve final inoculation concentrations ranging from 10 to
IMS-LAMP-NALFA assays. This substantiates the efficacy of PMAxx
1.0 × 106 CFU/mL using sterile PBS. The results revealed that the limit
treatment in eliminating false positives associated with dead bacteria.
of detection (LOD) of PMAxx-LAMP-NALFS for viable Salmonella was
The IMS-LAMP-NALFA method exhibited the capacity to detect 102
1.0 × 102 CFU/mL (Fig. S2).
CFU/mL of Salmonella, displaying a detection sensitivity differing by an
order of magnitude from the standard culture method. The rapid
3.4. Specificity analysis of PMAxx-LAMP-NALFS and IMS-LAMP- detection time of IMS-LAMP-NALFA confers an advantage in the realm
NALFA for Salmonella of swift detection and further demonstrates IMS proficiency in sepa­
rating and collecting bacteria in milk.
To validate the specificity of PMAxx-LAMP-NALFS and IMS-LAMP-
NALFA, 13 strains were subjected to testing. As indicated in Table 1, 3.7. Application of the IMS-LAMP-NALFA in actual raw milk samples
positive results were obtained for all target strains, while negative re­
sults were observed for all non-target strains. This confirms that both To validate the effectiveness of the IMS-LAMP-NALFA method and
IMS-LAMP-NALFA and PMAxx-LAMP-NALFS assays exhibit high speci­ the portable toolbox in practical applications, actual milk samples were
ficity in detecting Salmonella. collected and tested, and the results are presented in Table 4. Out of the
353 milk samples, Salmonella was detected in 28 cases using the IMS-
3.5. Detection limits of viable Salmonella cells in milk
Table 4
To assess the efficacy of PMAxx-LAMP-NALFS and IMS-LAMP- Results of raw milk sample at the pasture.
NALFA in detecting live Salmonella, milk samples with concentrations Sample type IMS-LAMP-NALFA Standard culture method
ranging from 1.0 to 1.0 × 105 CFU/mL were analyzed. As depicted in Positive/ Positive Positive/ Positive
Fig. S3a, the PMAxx-LAMP-NALFS method could detect Salmonella in negative rate negative rate
milk only when the concentration exceeded 1.0 × 103 CFU/mL. On the Unpasteurized raw 28/353 7.93 % 28/353 7.93 %
other hand, as illustrated in Fig. S3b, the IMS-LAMP-NALFA method milk
yielded positive results even at a concentration as low as 1.0 × 102 CFU/ Sterilized raw milk 0/353 0.00 % 0/353 0.00 %
mL. By enriching the sample with magnetic beads, the method

Table 3
Comparison of detection of different proportions of Salmonella in artificially contaminated mlik samples by standard culture method, LAMP-NALFS, PMAxx-LAMP-
NALFS and IMS-LAMP-NALFA assays.
Sample (CFU/mL) Standard culture method LAMP-NALFS PMAxx-LAMP-NALFS IMS-LAMP-NALFA

Viable Dead Result Positive ratio Result Positive ratio Result Positive ratio Result Positive ratio

1 × 104 1 × 106 + 3/3 + 3/3 + 3/3 + 3/3

1 × 104 1 × 104 + 3/3 + 3/3 + 3/3 + 3/3
1 × 104 1 × 102 + 3/3 + 3/3 + 3/3 + 3/3
1 × 103 1 × 106 + 3/3 + 3/3 + 3/3 + 3/3
1 × 103 1 × 104 + 3/3 + 3/3 + 3/3 + 3/3
1 × 103 1 × 102 + 3/3 + 3/3 + 3/3 + 3/3
1 × 102 1 × 106 + 3/3 + 3/3 – 0/3 + 3/3
1 × 102 1 × 104 + 3/3 + 3/3 – 0/3 + 3/3
1 × 102 1 × 102 + 3/3 – 0/3 – 0/3 + 3/3
1 × 101 1 × 106 + 3/3 + 3/3 – 0/3 – 0/3
1 × 101 1 × 104 + 3/3 + 3/3 – 0/3 – 0/3
1 × 101 1 × 102 + 3/3 – 0/3 – 0/3 – 0/3
– 1 × 106 – 0/3 + 3/3 – 0/3 – 0/3
– 1 × 104 – 0/3 + 3/3 – 0/3 – 0/3
– 1 × 102 – 0/3 – 0/3 – 0/3 – 0/3
NTC – 0/3 – 0/3 – 0/3 – 0/3

Positive ratio: the number of positive samples/all analyzed samples; NTC: Negative Control; “+” indicates positive reaction; “− ” indicates negative reaction

7
Q. Li et al. Microchemical Journal 197 (2024) 109732

LAMP-NALFA method, resulting in a detection rate of 7.93 %. This [3] Y. Chen, H. Liu, M. Chen, H.-Y. Sun, Y.-N. Wu, The human health burden of non-
typhoidal Salmonella enterica and Vibrio parahaemolyticus foodborne
detection rate was consistent with that of the standard culture method,
gastroenteritis in Shanghai, east China, Plos One 13, 15 (11) (2020), https://doi.
which also identified Salmonella in the same 28 cases. Notably, Salmo­ org/10.1371/journal.pone.0242156.
nella was not detected in samples that underwent ultra-high temperature [4] Y. He, J. Wang, R. Zhang, L. Chen, H. Zhang, X. Qi, J. Chen, Epidemiology of
transient sterilization. Similarly, Bedassa A et al. reported that the foodborne diseases caused by Salmonella in Zhejiang Province, China, between
2010 and 2021, Frontiers Public Health 11 (2023), 1127925, https://doi.org/
detection rate of Salmonella in raw cow’s milkcollected from the Ethi­ 10.3389/fpubh.2023.1127925.
opian region was approximately 15 %, which was reduced by half after [5] Z. Peng, Y. Li, L. Yan, S. Yang, D. Yang, Correlation analysis of microbial
pasteurization [35]. Such finding indicates that raw milk from pastures contamination and alkaline phosphatase activity in raw milk and dairy products,
Int J Environ Res Public Health. 20 (3) (2023) 1825, https://doi.org/10.3390/
may contain Salmonella contamination, an without timely sterilization, ijerph20031825.
there is a risk of bacterial proliferation, potentially leading to food safety [6] W. Luo, J. Wang, Y. Chen, Y. Wang, R. Li, J. Tang, F. Geng, Quantitative proteomic
incidents. Hence, detecting and controlling raw milk sterilization at the analysis provides insight into the survival mechanism of Salmonella typhimurium
under high-intensity ultrasound treatment, Current Research in Food Science 5
source is crucial. (2022) 1740–1749, https://doi.org/10.1016/j.crfs.2022.09.029.
[7] S. Chen, H. Zhang, J. Zhai, H. Wang, X. Chen, Yanping, Qi, Prevalence of clinical
4. Conclusion mastitis and its associated risk factors among dairy cattle in mainland China during
1982–2022: a systematic review and meta-analysis, Frontiers in Veterinary Science
10 (2023), 1185995, https://doi.org/10.3389/fvets.2023.1185995.
In conclusion, this study established the IMS-LAMP-NALFA method [8] C.L. Holschbach, S.F. Peek, Salmonella in dairy cattle, veterinary clinics of North
for detection of Salmonella in milk, demonstrating that the combination America, Food Animal Practice 34 (1) (2018) 133–154, https://doi.org/10.1016/j.
cvfa.2017.10.005.
of IMS and PMAxx-LAMP-NALFS is more accurate than PMAxx-LAMP- [9] W.u. Xuli, L.u. Yuqin, X.u. Haoxie, M. Lv, H.u. Dongsheng, Z. He, L. Liu, Z. Wang,
NALFS alone. Additionally, the portable toolbox developed based on Y. Feng, Challenges to improve the safety of dairy products in China, Trends Food
this method allows for on-site detection of raw milk samples from pas­ Sci. Technol. 76 (2018) 6–14, https://doi.org/10.1016/j.tifs.2018.03.019.
[10] B.o. Song, P. Zhu, Y. Zhang, J.u. Ning, X. Si, X. Pang, J. Lv, S. Zhang, Preparation
tures. This toolbox can detect various foodborne pathogens such as and quality assessment of processed cream cheese by high hydrostatic pressure
Escherichia coli, Cronobacter sakazakii, and others by replacing strain- combined thermal processing and spore-induced germination, J. Food Eng. 341
specific primers, providing a reference for rapid detection of food­ (2023), 111319, https://doi.org/10.1016/j.jfoodeng.2022.111319.
[11] Y. Zhang, X. Liao, J. Feng, D. Liu, S. Chen, T. Ding, Induction of viable but
borne pathogens.
nonculturable Salmonella spp. in liquid eggs by mild heat and subsequent
resuscitation, Food Microbiology. 109 (2023), 104127, https://doi.org/10.1016/j.
CRediT authorship contribution statement fm.2022.104127.
[12] X. Yang, Y. Wang, Y. Liu, J. Huang, X. Wei, Q. Tan, X. Zeng, X. Ying, S. Li, Rapid,
ultrasensitive, and highly specific identification of Brucella abortus utilizing
Qianxin Li: Conceptualization, Formal analysis, Writing – original multiple cross displacement amplification combined with a gold nanoparticles-
draft. Jingfeng Zhang: Formal analysis, Methodology, Writing – orig­ based lateral flow biosensor, Frontiers Microbiology. 13 (2022), 071928, https://
doi.org/10.3389/fmicb.2022.1071928.
inal draft. Xiaoxing Chen: Investigation. Tingting Jiang: Data cura­
[13] Y. Suda, M. Nagatomo, R. Yokoyama, M. Ohzono, X.u. Kazue Aoyama, K.N. Zhang,
tion, Investigation, Validation. Li Lin: Investigation, Data curation, N. Murakami, T. Shinoda, T. Hirota, S. Yanagihara, J.-I. Nishi, Highly sensitive
Validation. Lichao Zhao: Writing – review & editing, Supervision, detection of influenza virus in saliva by real-time PCR method using sugar chain-
immobilized gold nanoparticles; application to clinical studies, Biotechnology
Project administration.
Report 7 (2015) 64–71, https://doi.org/10.1016/j.btre.2015.05.004.
[14] S. Kim, Jung Ho Kim, Seokjoon Kim, Jung Soo Park, Byung Seok Cha, Eun Sung
Lee, Jinjoo Han, Jiye Shin, Youngjun Jang, Ki Soo Park, Loop-mediated isothermal
Declaration of competing interest amplification-based nucleic acid lateral flow assay for the specific and multiplex
detection of genetic markers, Anal. Chim. Acta 1205 (1) (2022), 339781, https://
The authors declare that they have no known competing financial doi.org/10.1016/j.aca.2022.339781.
[15] Z. Huang, J. Peng, J. Han, G. Zhang, Y. Huang, M. Duan, D. Liu, Y. Xiong, S. Xia,
interests or personal relationships that could have appeared to influence W. Lai, A novel method based on fluorescent magnetic nanobeads for rapid
the work reported in this paper. detection of Escherichia coli O157:H7, Food Chem. 276 (2018) 333–341, https://
doi.org/10.1016/j.foodchem.2018.09.164.
[16] H.-L. Huang, P. Zhu, C.-X. Zhou, S. He, X.-J. Yan, The development of loop-
Data availability
mediated isothermal amplification combined with lateral flow dipstick for
detection of Karlodinium veneficum, Harmful Algae 62 (2017) 20–29, https://doi.
Data will be made available on request. org/10.1016/j.hal.2016.11.022.
[17] J. Li, B. Ma, J. Fang, A. Zhi, E. Chen, X.u. Ying, Y.u. Xiaoping, C. Sun, M. Zhang,
Recombinase polymerase amplification (RPA) combined with lateral flow
Acknowledgements immunoassay for rapid detection of salmonella in food, Foods 9 (2020) 27, https://
doi.org/10.3390/foods9010027.
[18] B. Dolka, Agata Anna Cisek, Piotr Szeleszczuk, The application of the loop-
This work was supported by the Natural Science Foundation of mediated isothermal amplification (LAMP) method for diagnosing Enterococcus
Guangdong Province, China (2023A1515011965); and Special Fund hirae-associated endocarditis outbreaks in chickens, BMC Microbiol. 19 (1) (2019)
Project of Science and Technology in Guangdong Province 48, https://doi.org/10.1186/s12866-019-1420-z.
[19] X. Lv, L.i. Wang, J. Zhang, H. Zeng, X. Chen, L. Shi, H. Cui, X. He, L. Zhao, Rapid
(SDZX2022025); “Jinguo Basket Food Safety Rapid Test Center” labo­
and sensitive detection of VBNC Escherichia coli O157: H7 in beef by PMAxx and
ratory construction planning and technical support (h20230840). real-time LAMP, Food Control 115 (2020), 107292, https://doi.org/10.1016/j.
foodcont.2020.107292.
[20] Y. Wen, Y. Tan, L. Zhao, X. Lv, L.i. Lin, D. Liang, L.i. Wang, Rapid on-site detection
Appendix A. Supplementary data of viable Escherichia coli O157: H7 in lettuce using immunomagnetic separation
combined with PMAxx-LAMP and nucleic acid lateral flow strip, Microchem. J. 178
Supplementary data to this article can be found online at https://doi. (2022), 107348, https://doi.org/10.1016/j.microc.2022.107348.
[21] P. Hari, Dwivedi, Lee-Ann Jaykus, Detection of pathogens in foods: the current
org/10.1016/j.microc.2023.109732.
state-of-the-art and future directions, Crit. Rev. Microbiol. 37 (1) (2011) 40–63,
https://doi.org/10.3109/1040841X.2010.506430.
References [22] T. Huang, Y. Shi, J. Zhang, Q. Han, X.-S. Xia, A.-M. Zhang, Y. Song, Rapid and
simultaneous detection of five, viable, foodborne pathogenic bacteria by
photoinduced PMAxx-coupled multiplex PCR in fresh juice, Foodborne Pathogens
[1] A.B. Alzwghaibi, R. Yahyaraeyat, B. Nayeri Fasaei, A. Ghalyanchi Langeroudi,
and Disease. 18 (9) (2021) 640–646, https://doi.org/10.1089/fpd.2020.2909.
T. Zahraei Salehi, Rapid molecular identification and differentiation of common
[23] M.E. Ersahin, H. Ozgun, R.K. Dereli, I. Ozturk, K. Roest, J.B. van Lier, A review on
Salmonella serovars isolated from poultry, domestic animals and foodstuff using
dynamic membrane filtration: Materials, applications and future perspectives,
multiplex PCR assay, Arch. Microbiol. 200 (2018) 1009–1016, https://doi.org/
Bioresource Technology. 122 (2012) 196–206, https://doi.org/10.1016/j.
10.1007/s00203-018-1501-7.
biortech.2012.03.086.
[2] D.-M. Kim, S.-M. Yoo, Colorimetric systems for the detection of bacterial
[24] L. Zhao, X. Lv, X. Cao, J. Zhang, G.u. Xiaokui, H. Zeng, L.I. Wang, Improved
contamination: strategy and applications, Biosensors-Basel. 12 (7) (2022) 532,
quantitative detection of VBNC Vibrio parahaemolyticus using immunomagnetic
https://doi.org/10.3390/bios12070532.

8
Q. Li et al. Microchemical Journal 197 (2024) 109732

separation and PMAxx-qPCR, Food Control 110 (2020), 106962, https://doi.org/ [30] X. Cao, L. Zhao, J. Zhang, X. Chen, L. Shi, X. Fang, H. Xie, Y. Chang, L.i. Wang,
10.1016/j.foodcont.2019.106962. Detection of viable but nonculturable Vibrio parahaemolyticus in shrimp samples
[25] X. Mei, X. Zhai, C. Lei, X. Ye, Z. Kang, W.u. Xuan, R. Xiang, Y. Wang, H. Wang, using improved real-time PCR and real-time LAMP methods, Food Control 103
Development and application of a visual loop-mediated isothermal amplification (2019) 145–152, https://doi.org/10.1016/j.foodcont.2019.04.003.
combined with lateral flow dipstick (LAMP-LFD) method for rapid detection of [31] W. Randazzo, A. Francisco Lopez-Galvez, R. Allende, G.S. Aznar, Evaluation of
Salmonella strains in food samples, Food Control 104 (2019) 9–19, https://doi. viability PCR performance for assessing norovirus infectivity in fresh-cut
org/10.1016/j.foodcont.2019.04.014. vegetables and irrigation water, Int. J. Food Microbiol. 229 (2016) 1–6, https://
[26] J. Peng, Y. Zhan, F. Zeng, H. Long, Y. Pei, J. Guo, Development of a real-time doi.org/10.1016/j.ijfoodmicro.2016.04.010.
fluorescence loop-mediated isothermal amplification assay for rapid and [32] D. Seinige, C. Krischek, G. Klein, C. Kehrenberg, Comparative analysis and
quantitative detection of Fusarium oxysporum f. sp niveum in soil, FEMS limitations of ethidium monoazide and propidium monoazide treatments for the
Microbiology Letters 349 (2) (2013) 127–134, https://doi.org/10.1111/1574- differentiation of viable and nonviable campylobacter cells, Appl. Environ.
6968.12305. Microbiol. 80 (7) (2014) 2186–2192, https://doi.org/10.1128/AEM.03962-13.
[27] S. Delbeke, S. Ceuppens, K. Holvoet, E. Samuels, I. Sampers, M. Uyttendaele, [33] S.D. Gaoh, O. Kweon, Y.-J. Lee, D. Hussong, B. Marasa, Y. Ahn, A propidium
Multiplex real-time PCR and culture methods for detection of Shiga toxin- monoazide (PMAxx)-droplet digital PCR (ddPCR) for the detection of viable
producing Escherichia coli and Salmonella Thompson in strawberries, a lettuce mix burkholderia cepacia complex in nuclease-free water and antiseptics,
and basil, Int. J. Food Microbiol. 193 (2015) 1–7, https://doi.org/10.1016/j. Microorganisms 10 (5) (2022) 943, https://doi.org/10.3390/
ijfoodmicro.2014.10.009. microorganisms10050943.
[28] R. Najafi, S. Mukherjee, J. Hudson, A. Sharma, P. Banerjee, Development of a rapid [34] J. Zhang, S. Khan, K.K. Chousalkar, Development of PMAxx(TM)-based qPCR for
capture-cum-detection method for Escherichia coli O157 from apple juice the quantification of viable and non-viable load of Salmonella from poultry
comprising nano-immunomagnetic separation in tandem with surface enhanced environment, Front Microbiology 11 (2020), 581201, https://doi.org/10.3389/
Raman scattering, Int. J. Food Microbiol. 189 (2014) 89–97, https://doi.org/ fmicb.2020.581201.
10.1016/j.ijfoodmicro.2014.07.036. [35] A. Bedassa, H. Nahusenay, Z. Asefa, T. Sisay, G. Girmay, J. Kovac, J.L. Vipham,
[29] H.-L. Tsai, B.-M. Hsu, T.-K. Hsu, K.-H. Huang, F.-C. Shih, J.-S. Chen, H.-J. Wang, P.- A. Zewdu, Prevalence and associated risk factors for Salmonella enterica
M. Kao, S.u. Hung-Chang, Evaluation of immunomagnetic separation for the contamination of cow milk and cottage cheese in Ethiopia, Int. J. Food Contam. 10
improvement of Salmonella detection in aquatic environment, Environment Earth (1) (2023) 2, https://doi.org/10.1186/s40550-023-00101-3.
Sciences. 73 (2015) 7909–7914, https://doi.org/10.1007/s12665-014-3948-4.