Detection of Listeria monocytogenes in Food Using the Proofman-LMTIA Assay.

Microbial factors, including bacteria, viruses, and other pathogens, are significant contributors to foodborne illnesses, posing serious food safety risks due to their potential for rapid growth and contamination. Listeria monocytogenes is one of the most common types of foodborne bacteria that can cause serious foodborne diseases or even fatalities. In this study, a novel nucleic acid amplification method called Proofman-LMTIA was employed to detect Listeria monocytogenes contamination in food. This method combines proofreading enzyme-mediated probe cleavage with ladder-shape melting temperature isothermal amplification. A positive recombinant plasmid was used as a control to ensure the accuracy of the detection results, and primers and Proofman probes were specifically designed for the LMTIA. Genomic DNA was extracted, the reaction temperature was optimized, and the primers' specificity was verified using foodborne pathogens like Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella. The sensitivity was assessed by testing serial dilutions of genomic DNA, and the method's applicability was confirmed by detecting artificially contaminated fresh pork. The established LMTIA method exhibited both high specificity and sensitivity. At the optimal reaction temperature of 63 °C, the primers specifically identified Listeria monocytogenes contamination in pork at a concentration of 8.0 ± 0.7 colony-forming units (CFUs) per 25 g. Furthermore, the Proofman-LMTIA method was applied to test Listeria monocytogenes DNA in 30 food samples purchased from a Chinese retail market, and reassuringly, all results indicated no contamination. Proofman-LMTIA can serve as a reliable and rapid method for detecting Listeria monocytogenes in food, contributing to public health by safeguarding consumers from foodborne illnesses, and strengthening food safety regulations.

Micro-Executor of Natural Products in Metabolic Diseases.

Obesity, diabetes, and cardiovascular diseases are the major chronic metabolic diseases that threaten human health. In order to combat these epidemics, there remains a desperate need for effective, safe, and easily available therapeutic strategies. Recently, the development of natural product research has provided new methods and options for these diseases. Numerous studies have demonstrated that microRNAs (miRNAs) are key regulators of metabolic diseases, and natural products can improve lipid and glucose metabolism disorders and cardiovascular diseases by regulating the expression of miRNAs. In this review, we present the recent advances involving the associations between miRNAs and natural products and the current evidence showing the positive effects of miRNAs for natural product treatment in metabolic diseases. We also encourage further research to address the relationship between miRNAs and natural products under physiological and pathological conditions, thus leading to stronger support for drug development from natural products in the future.

Silencing Protease-Activated Receptor-2 alleviates ox-LDL-induced lipid accumulation, inflammation and apoptosis via activation of Wnt/ß-catenin signaling.

Macrophages conversion to foam cells strongly promoted atherosclerosis progression by plaque formation and plaque rupture. Macrophages swallow oxidized-low density lipoprotein (ox-LDL) to promote foam cell formation. Protease-activated receptor 2 (PAR2) has been reported to take part in atherosclerotic development. However, the effects of PAR2 in macrophages were rarely investigated. In this study, human monocyte, THP-1 was induced to macrophages by using phorbol 12-myristate 13-acetate (PMA). Subsequently, an in vitro model was arranged by using ox-LDL to treat the macrophages. The data showed that inhibition of PAR2 reduced ox-LDL-induced foam cell formation, inflammation, and apoptosis. Additionally, ox-LDL increased PAR2 and inhibited Dickkopf-related protein 1 (DKK1) expression, which is a Wnt signaling inhibitor. PAR2 knocked-down decreased DKK1 and enhanced expression of Wnt3a, ß-catenin. Meanwhile, DKK1 overexpression reversed the effects of PAR2 on foam cell formation, inflammation, and apoptosis. In summary, PAR2 is essential for the formation of foam cells, inflammation, and apoptosis in macrophages which plays a critical role during atherosclerosis. PAR2 plays roles in macrophages treated with ox-LDL via DKK1/Wnt/ß-catenin signaling.

Self-paired monoclonal antibody lateral flow immunoassay strip for rapid detection of Acidovorax avenae subsp. citrulli.

We screened a highly specific monoclonal antibody (McAb), named 6D, against Acidovorax avenae subsp. citrulli (Aac). Single McAb 6D was used as both nanogold-labeled antibody and test antibody to develop a single self-paired colloidal gold immunochromatographic test strip (Sa-GICS). The detection limit achieved using the Sa-GICS approach was 10(5) CFU/mL, with a result that can be observed by the naked eye within 10 min. Moreover, Sa-GICS can detect eight strains of Aac and display no cross-reactions with other pathogenic plant microorganisms. Artificial contamination experiments demonstrated that Sa-GICS would not be affected by impurities in the leaves or stems of the plants and were consistent with the PCR results. This is the first report on the development of a colloidal gold immunoassay strip with self-paired single McAb for the rapid detection of Aac. Graphical Abstract Schematic representation of the test strip.

Simultaneous quantification of Escherichia coli O157:H7 and Shigella boydii using a visual-antibody-macroarray.

Being high throughput, rapid, automated, economical, convenient to operate and highly sensitive, protein arrays have been widely used in the analysis of tumor markers and veterinary drug residues. Pathogenic microbes also can be detected qualitatively by DNA array or protein array; however, their high throughput detection and quantification remains a big obstacle. To evaluate the potentiality of protein arrays for multiple quantitative detection of microorganisms with naked eye examination without the help of any equipment, here we developed a visual-antibody-macroarray (VAMA) aiming at rapid and simultaneous quantification of Escherichia coli O157:H7 and Shigella boydii. The results show that this VAMA is highly specific and is able to distinguish mixed Escherichia coli O157:H7 and Shigella boydii synchronously. The detection limits are equivalent to 3.4 × 10(5) CFU mL(-1) and 3.2 × 10(5) CFU mL(-1), respectively, which conform to the results of plate counting and ELISA. Importantly, the examination can be solely performed with the naked eye. Therefore, we provide an easy, reliable and rapid method for quantitative analysis of microorganisms.

[Research of Hippophae rhamnoides fruits on serum lipids and liver protection effects in high-fat-diet rats].

OBJECTIVE: To study the effects o Hippophae rhamnoides fruits on serum lipids and liver protection in high-fat-diet rats. METHODS: Wistar rats were divided into 5 groups,including control group, high lipid model group and Hippophae rhamnoides low-, medium- and high- dose groups,every group wastaken high-fat diet except control group. The rats in control group and high lipid group were lavaged with physiological saline. The doses of Hippophae rhamnoides in low, middle and high groups were determined based on the 1x, 5x, and 10x standard human doses (50 g/60 kg BW), respectively. The rat were orally given test sample respectively for 28 days, once a day. Observed the changes of serum lipids and hepatic tissues pathology. RESULTS: Compared with the control group, the serum levels of total cholesterol (TC), triglyceride (TG) and low density lipoprotein cholesterol (LDL-C) were increased significantly (P < 0.05) in high-fat-diet rats. Compared with the high lipid group, Hippophae rhamnoides of different doses could significantly reduce the content of TG (P < 0.01). Hippophae rhamnoides reduced the tendency of TC and LDL-C in serum and reduced the fatty degeneration of liver cells. CONCLUSION: The Hippophae rhamnoides fruits can reduce the level of serum lipids and prevent the occurrence of fatty liver, can be used for the prevention of hyperlipidemia.

Platelet-Derived Microvesicles Mediate Cardiomyocyte Ferroptosis by Transferring ACSL1 During Acute Myocardial Infarction.

Acute myocardial infarction (AMI) is one of the critical health conditions often caused by the rupture of unstable coronary artery plaque, triggering a series of events, such as platelet activation, thrombus formation, coronary artery blockage, lasted severe ischemia, and hypoxia in cardiomyocytes, and culminating in cell death. Platelet-derived microvesicles (PMVs) act as intermediates for cellular communication. Nevertheless, the role of PMVs in myocardial infarction remains unclear. Initially, AMI-related messenger ribose nucleic acid (mRNA) and micro RNA (miRNA) datasets from the Gene Expression Omnibus (GEO) database were analyzed, specifically focusing on the expressed genes associated with Ferroptosis. Further, a miRNA-mRNA regulatory network specific to AMI was constructed. Then, the effect of PMVs on cardiomyocyte survival was further confirmed through in vitro experiments. High ACSL1 expression was observed in the platelets of AMI patients. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that ACSL1, located in the mitochondria, played a key role in the PPAR signaling pathway. The elevated ACSL1 expression in a co-culture model of PMVs and AC16 cardiomyocytes significantly increased the AC16 cell Ferroptosis. Further, we validated that the platelet ACSL1 expression could be regulated by hsa-miR-449a. Together, these findings suggested that platelet ACSL1 could trigger myocardial cell death via PMV transport. In addition, this research provided a theoretical framework for attenuating myocardial cell Ferroptosis in patients with acute myocardial infarction.

[Research of grape seed powder on serum lipids and hepatic protection effects in high-fat diet rats].

OBJECTIVE: To research of grape seed powder on serum lipids and hepatic protection effects in high-fat diet rats. METHODS: The rats were divided into five groups, every group wastaken high-fat diet except control group, the physiological saline was administered to the control group and the high-fat diet groups. The doses of grape seed power in low, middle and high groups were determined based on the 1x , 5x , and 10x standard human doses (20 mg/60 kg BW), respectively. The rats were orally given test sample respectively for 28 days, once a day. Observed the changes of erum lipids and live pathology. RESULTS: Compared to the control group, the serum levels of total cholesterol (TC), triglyceride (TG) and low density lipoprotein cholesterol (LDL-C) were increased significantly (P < 0.05) and HDL-C markedly decreased (P < 0.05) in high-fat diet group. The histological data with light microscopy demonstrated that the fatty degeneration in liver was significantly attenuated in grape seed powder rats as compared to high-fat diet group. CONCLUSION: The grape seed powder can reduce the level of serum lipids and prevent the occurrence of fatty liver.

A novel regulatory mechanism of geniposide for improving glucose homeostasis mediated by circulating RBP4.

BACKGROUND: Systemic insulin signal transduction is influenced by the inter-tissue crosstalk, which might be the potential therapeutic strategy for T2DM. Although anti-diabetic function of geniposide has been previously reported, the underlying mechanism was not completely clear in light of the complex pathogenesis of T2DM. PURPOSE: The present experiment is devoted to investigate the potential effects of geniposide on systemic insulin sensitivity mediated by hepatokine-RBP4 in high fat diet (HFD)-fed mice. METHODS: The HFD-fed wild type mice were administered with geniposide (25 or 50 mg/kg/d) by intraperitoneal injection, and the normal saline and Metformin were used as negative control group and positive control group, respectively. After administration for 4 weeks, the food intake, body weight, glucose tolerance tests, insulin tolerance tests and serum biochemical indices were examined, along with insulin signaling pathway-associated proteins and hepatic histomorphological analysis. The liver, gastrocnemius and mouse primary hepatocytes were also harvested for molecular mechanism study. RESULTS: After geniposide treatment for 4 weeks, the blood glucose level was reduced in HFD-fed mice. Furthermore, geniposide treatment improved insulin sensitivity both in the liver and gastrocnemius (GAS). In terms of mechanism, geniposide disturbed circulating RBP4 level including its synthesis, secretion and homeostasis. Moreover, geniposide modified fuel selection and promoted glucose uptake in skeletal muscle and reduced glycogen storage, which were closely related to impaired circulating RBP4 homeostasis, leading to ameliorative systemic insulin sensitivity. CONCLUSION: Our current study proposes a novel regulatory mechanism of geniposide for improving glucose homeostasis through regulating circulating RBP4 level, which also provides new strategies for the prevention and treatment of T2DM.

Heparin-Binding Protein Enhances NF-κB Pathway-Mediated Inflammatory Gene Transcription in M1 Macrophages via Lactate.

In early-stage sepsis, glucose metabolism is increased primarily through glycolysis in the inflammatory response of M1 macrophages. Heparin-binding protein (HBP) has been linked to sepsis, which can promote macrophage activation and inflammatory factor release. However, the mechanism by which glucose metabolism regulates the inflammatory response is unclear. We show that HBP contributes to sepsis by modulating the inflammatory response via lactate-dependent glycolysis in macrophages. Peritoneal macrophages from BALB/c mouse were treated with lipopolysaccharide (LPS). The expression of M1-related proinflammatory genes was investigated by PCR array. IL-1ß, iNOS, TNF-α, and IL-6 mRNA expression was determined by qRT-PCR. Intracellular lactate levels were measured using lactate assays. Nuclear factor-kappaB (NF-κB) activity was determined by electrophoretic mobility shift assays (EMSAs). TNF-α levels were measured by qRT-PCR. HBP enhanced inflammatory gene expression in mouse peritoneal macrophages and intracellular lactate accumulation and significantly increased LPS-stimulated NF-κB transcriptional activity and TNF-α expression through lactate. Lactate was essential for the HBP-induced increase in LPS-stimulated TNF-α expression. The critical role of lactate in HBP-induced NF-κB signaling was confirmed, as α-CHCA-mediated (MCT) suppression significantly inhibited NF-κB activity and TNF-α expression. HBP plays an important role in the initial inflammatory reaction, presumably by activating M1 macrophages, increasing lactate levels, and regulating proinflammatory factor release via NF-κB pathway activation.

Hydroxysafflor Yellow A Alters Fuel Selection From Glucose to Fat by Activating the PPARδ Pathway in Myocytes.

Modulation of fuel selection is critical in skeletal muscle function. Hydroxysafflor yellow A (HSYA) is the major bioactive component in safflower (Carthamus tinctorius L.) and, in our previous study, has been demonstrated to promote a shift from fast to slow myofiber. However, the effects of HSYA on fuel selection in skeletal muscle and its underlying mechanisms remain unclear. In this study, the in vitro experiments found that water extracts of safflower, rich in HSYA, significantly suppressed the expressions of the genes related to glucose utilization and activated the expressions of the lipolysis genes. Furthermore, HSYA resulted in a shift in substrate utilization toward fat relative to carbohydrates in C2C12 myotubes. Animal tests showed HSYA could significantly reduce the respiratory exchange ratio and prolonge endurance performance in mice and also trigger a switch in intramuscular fuel selection preference from carbohydrates to fat at rest and during exercise. Mechanistic studies revealed that HSYA converted this fuel selection by activating peroxisome proliferator activated receptor δ (PPARδ), and these effects of HSYA could be reversed by specific suppression of PPARδ by PPARδ siRNA. Collectively, our study demonstrated that HSYA can switch substrate utilization from glucose to fat in myocytes by activating PPARδ signaling, resulting in prolonged endurance performance. These findings provided direct evidence for the endurance performance enhancement effect of HSYA and explored new perspectives for the innovation and application of HSYA in the health care industry.

Ladder-shape melting temperature isothermal amplification of nucleic acids.

A novel method, termed ladder-shape melting temperature isothermal amplification (LMTIA), was developed in this study. As a proof of concept, one pair of primers or two pairs of nested primers and a thermostable DNA polymerase were employed to amplify the internal transcribed spacer of Oryza sativa with the ladder-shape melting temperature curve. Our results demonstrated that the LMTIA assay with nested primers was 50-fold more sensitive than the LAMP assay with the same level of specificity. The LMTIA method has the potential to be used for the prevention and control of emerging epidemics caused by different types of pathogens.

Liquid phase exfoliated boron nanosheets for all-optical modulation and logic gates.

Boron nanosheets possess unique photoelectric properties, including photosensitivity, photoresponse, and optical nonlinearity. In this article, we show the interaction between light and boron nanosheets in which concentric rings formed in the far field, which attributed to the strong Kerr nonlinearity of boron nanosheets. Furthermore, the distortion, regulation and relationship between the Kerr nonlinearity and effective mass or carrier mobility of the diffraction rings of boron nanosheets have been investigated. Our work shows that the spatial self-phase modulation effect of boron nanosheets is indeed caused by nonlocal electronic coherence. In addition, we have implemented all-light modulation and all-light logic gates based on the prepared boron nanosheets. We believe that our results will provide a powerful demonstration of nonlinear photonic devices based on boron nanosheets and a reference for photonic devices based on two-dimensional materials.

Hapten Synthesis and the Development of an Ultrasensitive Indirect Competitive ELISA for the Determination of Diethylstilbestrol in Food Samples.

An ultrasensitive indirect competitive enzyme-linked immunosorbent assay (ic ELISA) using monoclonal antibodies (mAbs) was developed for the specific detection of diethylstilbestrol (DES) residues. To establish an ELISA based on mAbs, hapten diethylstilbestrol mono-carboxypropyl-ether (DES-MCPE) was chemically synthetized and then conjugated to bovine serum albumin (BSA) for immunization in mice. This ic ELISA was further optimized for DES determination. The sensitivity of the ic ELISA was found to be 0.49 µg/kg and the limit of detection was 0.075 µg/kg. DES residues in salmon meat and pork were tested with the recovery range from 74.0 to 85.2% and the coefficient of variation (CV) was less than 10%. Parallel analysis of DES samples from salmon meat showed comparable results from the ic ELISA with high-performance liquid chromatography. The ic ELISA provides a useful screening method for the quantitative detection of DES residues in animal-derived food.

MHC-Based Mate Choice in Wild Golden Snub-Nosed Monkeys.

The genes of the major histocompatibility complex (MHC) are an important component of the vertebrate immune system and play a significant role in mate choice in many species. However, it remains unclear whether female mate choice in non-human primates is based on specific functional genes and/or genome-wide genes. The golden snub-nosed monkey (Rhinopithecus roxellana) lives in a multilevel society, which consists of several polygynous one-male-several-female units. Although adult females tend to mainly socialize with one adult male, females often initiate extra-pair copulations with other males resulting in a high proportion of offspring being fathered by extra-pair males. We investigated the effects of adaptive MHC genes and neutral microsatellites on female mate choice in a wild R. roxellana population. We sequenced 54 parent-offspring triads using two MHC class II loci (Rhro-DQA1 and Rhro-DQB1) and 20 microsatellites from 3 years of data. We found that the paternities of offspring were non-randomly associated with male MHC compositions not microsatellite genotypes. Our study showed that the fathers of all infants had significantly less variance for several estimates of genetic similarity to the mothers compared with random males at both MHC loci. Additionally, the MHC diversity of these fathers was significantly higher than random males. We also found support for choice based on specific alleles; compared with random males, Rhro-DQA1∗ 05 and Rhro-DQB1∗ 08 were more common in both the OMU (one-male unit) males and the genetic fathers of offspring. This study provides new evidence for female mate choice for MHC-intermediate dissimilarity (rather than maximal MHC dissimilarity) and highlights the importance of incorporating multiple MHC loci and social structure into studies of MHC-based mate choice in non-human primates.

Neuroprotective effects of chlorogenic acid against apoptosis of PC12 cells induced by methylmercury.

Chlorogenic acid (CGA) widely exists in edible and medicinal plants. We aimed to evaluate the effect of CGA on the protection from apoptosis by methylmercury (MeHg) in PC12 cells. Cell viability was evaluated by MTT assay. Apoptosis was assayed by flow cytometry detection. Caspase-3 activity was measured by confocal microscopy. Intracellular GSH levels were determined by bicinchoninic acid protein assay. Intracellular reactive oxygen species (ROS) was assessed by means of chloromethyl-dihydrodichlorofluorescein diacetate. Glutathione peroxidase (GPx) activity was determined by UV. In order to elucidate the action of CGA, the protective effects of CGA were compared to Vit.E. CGA was effective at protecting PC12 cells against MeHg-induced damage in dose-dependent manner. CGA not only suppressed the generation of ROS, the decrease of activity in GPx and the decrease of GSH, but also attenuated caspase-3 activation in PC12 cells by MeHg. CGA eventually protected PC12 cells against MeHg-induced apoptosis. The results highlighted that CGA may exert neuroprotective effects through its antioxidant actions.

Role of M2 Macrophages in Sepsis-Induced Acute Kidney Injury.

BACKGROUND: Sepsis is a major cause of acute kidney injury (AKI), with high rates of morbidity and mortality. M2 macrophages have been shown to play important roles in the secretion of anti-inflammatory and tissue repair mediators. In this study, we investigate the role of M2 macrophages in sepsis-induced AKI by depleting these cells in vivo through the systemic administration of liposomal clodronate (LC). METHODS: Male Sprague-Dawley rats were subjected to cecal ligation and puncture (CLP) or sham surgery. Biochemical and histological renal damage was assessed. Macrophage infiltration and M2 macrophage depletion were assessed by immunohistochemistry. RT-PCR was used to investigate the expression of the inducible nitric oxide synthase (iNOS), arginase 1 (Arg-1), and found in inflammatory zone 1 (FIZZ1) mRNAs. Western blots were performed to assay the tissue levels of interleukin-10 (IL-10) and tumor necrosis factor alpha (TNF-α). RESULTS: M2 macrophages were obviously detected 72 h after sepsis-induced AKI. Kidney injury was more severe, renal function was decreased, and blood creatinine and blood urea nitrogen (BUN) levels were higher after M2 macrophage depletion. M2 macrophage depletion significantly inhibited the proliferation of tubular cells. M2 macrophage depletion also downregulated IL-10 expression and increased TNF-α secretion during sepsis-induced AKI. CONCLUSIONS: M2 macrophages attenuate sepsis-induced AKI, presumably by upregulating IL-10 expression and suppressing TNF-α secretion.

Dual FITC lateral flow immunoassay for sensitive detection of Escherichia coli O157:H7 in food samples.

A pattern of signal amplification lateral flow immunoassay (LFIA) for pathogen detection, which used fluorescein isothiocyanate (FITC) labeled antigen and antibody for dual FITC-LFIA was developed. Escherichia coli O157:H7 (E.coli O157:H7) was selected as the model analyte. In the signal amplification LFIA method, FITC was mixed with sample culture medium, with the presence of E.coli O157:H7 in the samples, the bacteria could emit a yellow-green fluorescence after incubation, creating a fluorescent antigen probe. This antigen probe was added to LFIA, which already contained E.coli O157:H7 monoclonal antibodies-FITC (McAb-E.coli O157:H7-FITC) dispersed in the conjugate pad. Another E.coli O157:H7 McAb was the test line, and goat anti-mouse IgG antibody was the control line in nitrocellulose (NC) membrane. The visual limit of detection (LOD) of the strip for qualitative detection was 10(5) CFU/mL while the LOD for semi-quantitative detection could down to 10(4) CFU/mL by using scanning reader. Signal amplification LFIA was perfectly applied to the detection of food samples with E.coli O157:H7. The LOD was substantially improved to 1 CFU/mL of the original bacterial content after pre-incubation of the bread, milk and jelly samples in broth for 10, 8 and 8h respectively. The results of this method was more sensitive by 10-fold than the conventional colloidal gold (CG) based strips and comparable to the traditional ELISA. This simple, low-cost and easy to be popularized method served as a significant step towards the development of monitoring food-borne pathogens in food-safety testing.

Simple sensitive rapid detection of Escherichia coli O157:H7 in food samples by label-free immunofluorescence strip sensor.

A simple, one-step, rapid method to detect Escherichia coli O157: H7 (E. coli O157: H7) using a label-free immunofluorescence strip sensor is presented. Fluorescein isothiocyanate (FITC) was added to the sample culture medium to prepare the fluorescent probe for the label-free strip sensor. With the presence of E. coli O157: H7 in the samples, the bacteria could emit a yellow-green fluorescence after incubation and maintain good affinity to the monoclonal antibodies (McAb) against E. coli O157: H7. The direct-type immunofluorescence strip sensor was based on the binding between fluorescent bacteria and the unlabeled McAb immobilized at the test line in nitrocellulose membrane (NC membrane) reaction zone. The visual limit of detection (LOD) of the strip for qualitative detection was 10(6)cells/mL while the LOD for semi-quantitative detection could go down to 10(5)cells/mL by using scanning reader. The LOD was substantially improved to 1cells/mL of the original bacterial content after pre-incubation of the bread, milk and jelly samples in broth for 10, 10 and 8h respectively, which was competitive to some current rapid E. coli O157: H7 detection methods. Besides the obvious advantages, including reduced detection time and operation procedures, the results of this method meet the various detection requirements for E. coli O157: H7 and are comparable to the traditional enzyme-linked immunosorbent assay (ELISA) and double antibody sandwich gold-labeled strips. This is the first report of semi-quantitative immunofluorescence strip for directly detecting foodborne pathogen using only one unlabeled antibody. All detections could be achieved in less than 5min. In addition, this simple, low-cost and easy to be popularized method served as a significant step towards the development of monitoring foodborne pathogens in food-safety testing.

A new spot quality control for protein macroarray based on immunological detection.

Protein macroarray is a new, simple and multiple biochemistry detection system, in which the test spots are more than 1mm diameter and results directly visible and instrumentation is not necessary. This technology, however, possesses recognized problems with spot quality and uniformity, issues that can limit its application. Previous methods have been developed for spot quality control, but they are complicated or require specific instrumentation. Therefore, we have developed a spot quality control buffer supplement with Ponceau S (SQCB-PS) as a direct and visible means of monitoring spot quality on nitrocellulose membrane prior to hybridization. In this report, 1% (w/v) Ponceau S and 10% (v/v) glycerol in spotting buffer were found to be optimal for spot quality control and food-borne pathogens multiple detection. Furthermore, the sensitivity and specificity of the protein macroarray were not compromised by spotting buffer with Ponceau S. Under optimal conditions, this visible spot quality control method makes the detection more reliable, and should enhance the wider use of the technique.