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Bacterial infections are common causes of morbidity and mortality worldwide; therefore, environmental contamination by bacterial pathogens represents a global public health concern. Consequently, a selective, rapid, sensitive, and in-field detection platform for detecting significant bacterial contamination is required to ensure hygiene and protect public health. Here, we developed a fast and simple platform for the selective and sensitive detection of bacteria by measuring adenosine triphosphate (ATP) bioluminescence following targeted photothermal lysis mediated by antibody-conjugated gold nanorods. This method employed both targeted photothermal lysis of bacteria by near-infrared (NIR) irradiation and highly selective detection of the lysed bacteria via ATP bioluminescence within 36 min (incubation, 30 min; NIR irradiation, 6 min). The use of the proposed method allowed limits of detection in pure solution of 12.7, 70.7, and 5.9 CFU for Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes, respectively. Additionally, bacteria were successfully detected on artificially inoculated plastic cutting boards. Furthermore, this method was highly specific, without cross-reaction among pathogenic bacteria. We believe that the proposed method has significant potential as an on-site diagnostic tool for applications associated with public health and environmental pollution monitoring.

Here, we report an enhanced colorimetric method using enzymatic amplification with nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) precipitation for the ultrasensitive detection of Escherichia coli O157:H7 through immunomagnetic separation-selective filtration. Biotinylated anti- E. coli O157:H7 antibody and streptavidin-alkaline phosphatase were conjugated to the surface of magnetic nanoparticles, and E. coli O157:H7-conjugates complexes remained on the membrane filter surface. The resultant light brown spots on the membrane filter were amplified with NBT/BCIP solution to yield enzyme-catalyzed precipitation, which increased with an increasing E. coli O157:H7 concentration. E. coli O157:H7 was detected in pure samples with limits of detection of 10 and 6.998 colony-forming units (CFU)/mL through visual observation and measurement of optical density, respectively. The proposed method was applied to a lettuce sample inoculated with selective E. coli O157:H7, which was detected within 55 min without cross-reactivity to non-target bacteria. This enhanced colorimetric method has potential for on-site detection of food contaminants and environmental pollutants.

Salmonella enterica is one of the most encountered causative pathogens of food-borne illnesses. Outbreaks of such diseases are commonly associated with the consumption of fresh fruits and vegetables. In this study, we report a simple colorimetric strategy for the detection of S. enterica Serovar Typhimurium in fresh-cut lettuce leaves based on polymerase chain reaction (PCR) products generated by gene-specific primers integrated with the horseradish peroxidase-mimicking DNAzyme (HRPzyme). The HRPzyme sequence was integrated at the 5′ end of the forward and reverse primers specific to 16S rRNA of S. enterica Typhimurium. At the end of the PCR reaction, unamplified HRPzyme-integrated primers were folded into G-quadruplex structure in the presence of hemin and then, they catalyzed the oxidation of 2,2′-azinobis (3-ethylbenzothiazolinesulfonic acid) (ABTS) with H2O2. The intensity of oxidized ABTS colorimetric signal was linearly and inversely related to S. enterica Typhimurium concentration. The latter relationship demonstrated diagnostic potential of this rapid, simple, highly sensitive, and selective colorimetric platform for S. enterica Typhimurium detection.

Worldwide, norovirus is one of the most associated causes of acute gastroenteritis, which leads to nearly 50 000 child deaths every year in developing countries. Therefore, there is great demand to develop a rapid, low-cost, and accurate detection assay for the foodborne norovirus infection to reduce mortality caused by norovirus. Considering the importance of norovirus, we have demonstrated a highly sensitive and specific colorimetric detection method for analysis of human norovirus genogroups I and II (HuNoV GI and GII) in oyster samples. This is the first report to employ colorimetric HRPzyme-integrated polymerase chain reaction (PCR) for direct norovirus detection from the real shellfish samples. We found that the HRPzyme-integrated PCR method is more sensitive than the gel electrophoresis approach and could detect the HuNoV GI and GII genome up to 1 copy/mL. The specificity of the proposed method was successfully demonstrated for HuNoV GI and GII. Further, we performed testing HuNoVs in the spiked oyster samples, and the HRPzyme-integrated PCR method proved to be an ultrasensitive and selective method for detecting HuNoVs in the real samples. By integration of the proposed method with the portable PCR machine, it would be more reliable to improve food safety by detecting HuNoVs in the different types of shellfish, such as oyster and mussel, at the production field.

We have developed a novel strategy for the colorimetric detection of PCR products by utilizing a target-specific primer modified at the 5′-end with an anti-DNAzyme sequence. A single-stranded DNAzyme sequence folds into a G-quadruplex structure with hemin and shows strong peroxidase activity. When the complementary strand binds to the DNAzyme sequence, it blocks the formation of the G-quadraduplex structure and loses its peroxidase activity. In the presence of the target gene, PCR amplification proceeds, and anti-DNAzyme sequence modified primers present in the reaction mixture form a double strand through primer extension. Therefore, it does not block the DNAzyme sequence. Further, a colorimetric signal is generated by the addition of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and H2O2 at the end of the reaction. We have successfully detected a single copy of the HIV type 1 gag gene in buffer and 10 copies in human serum. The strategy developed could be used to detect DNA and RNA in complex biological samples by simple primer designing that includes DNAzyme and a DNA extended primer.

We identified a peel-specific expressed gene in Citrus unshiu fruits by differentially expressed gene (DEG) analysis, which showed a homology with carotenoid isomerase-like genes identified from other plants and, therefore, designated as CuCRTISO-like. Here we determined the promoter sequence of CuCRTISO-like and analyzed histochemical GUS activity using transgenic Arabidopsis plants harboring CuCRTISO-like promoter-GUS gene constructs (pCRTL-Prom1~pCRTL-Prom5 lines). The promoter activity of CuCRTISO-like was detected in the cotyledon at 5 and 10 days after germination (DAG), young leaf, and anther, but not in the cotyledon at 15 DAG and mature leaf. Several cis-acting elements involved in hormones and abiotic stresses are located on the CuCRTISO-like promoter. Salicylic acid and ethylene treatments induced the GUS activity in the pCRTL-prom1 and pCRTL-Prom4 line, respectively. Treatment of drought and wounding stress induced the GUS activity in the pCRTL-Prom4 and pCRTL-Prom3 line, respectively. Heat stress treatment induced GUS activity more strongly as the promoter length decreased except for no GUS activity in the pCRTL-Prom5 line. The CuCRTISO-like expression during fruit maturation of C. unshiu showed a peel-specific expression pattern. Our results suggest that CuCRTISO-like promoter activity is regulated in a developmental and organ-specific manner, and responds to hormones and abiotic stresses.

Carotenoid isomerase (CRTISO) catalyzes the isomerization of prolycopene to all-trans-lycopene in the carotenoid biosynthetic pathway. We isolated a promoter region of CuCRTISO from Citrus unshiu. We determined whether the promoter encoded organ-specific or developmental-specific expression and identified possible cis-acting promoter elements. The promoter and two truncated versions were fused to the β-glucuronidase (GUS) gene and transformed into Arabidopsis thaliana. Transgenic lines expressing the 2501-bp promoter (pCiso-Prom1) and truncated promoters (pCiso-Prom2 and pCiso-Prom3) showed the same developmental and organ-specific activity. GUS expression was detected in the cotyledon and root at 5 and 10 days after germination, mature leaf, and anther. The CuCRTISO promoter contained several cis-acting elements involved in hormonal and environmental stress. Drought stress or abscisic acid treatment did not induce GUS expression in any transgenic lines. Heat stress induced GUS expression in the pCiso-Prom1 line; this promoter construct contains the heat stress-responsive element. Ethylene and cold stress treatments induced GUS expression only in the pCiso-Prom3 line, although all transgenic lines contained the same cis-acting ethylene and low-temperature response elements, which could indicate the existence of unknown repressor element(s) in the CuCRTISO promoter. The CuCRTISO expression of Citrus unshiu during fruit maturation showed that in the green stage, it was highly expressed and then subsequently decreased after the stage. These studies indicate that CuCRTISO promoter activity is regulated in a developmental and organ-specific manner that responds to heat, cold, and ethylene. These results provide new insights into the role of cis-acting element(s) in CuCRTISO promoter activity.

A highly sensitive localized surface plasmon resonance (LSPR) aptasensor for detection of adenosine triphosphate (ATP) has been developed. The sensor utilizes two split ATP aptamers, one (receptor fragment) being covalently attached to the surface of a gold nanorod (GNR) and the other labeled with a random DNA sequence and TAMRA dye (probe fragment). In the presence of both ATP and the probe fragment, a significant shift takes place in the wavelength of the LSPR band. This phenomenon is a consequence of the fact that the split fragments assemble into an intact folded structure in the presence of ATP, which brings about a decrease in the distance between the GNR surface and TAMRA dye and an associated LSPR wavelength. By using this sensor system, concentrations of ATP in the range of 10 pM-10 μM can be determined. In addition, by taking advantage of its denaturation properties, the LSPR aptasensor can be reused by simply subjecting it to quadruple salt-addition/2M NaCl washing steps. That the new method is applicable to biological systems was demonstrated by its use to measure ATP concentrations in E. coli and, thus to determine cell concentrations as low as 1.0×10(3) CFU.

Neural stem cells are mobile, are attracted to regions of brain damage, and can migrate a considerable distance to reach a glioma site. However, the molecular basis of the progression of gliotropism to malignant gliomas remains poorly understood. With the use of clinically and histologically assessed glioma cells, we have assessed their protein and gene profiles via proteomics and microarray approaches, and have identified candidate genes from human glioma tissues. This research is expected to provide clues to the molecular mechanisms underlying the migration of neural stem cells (F3 cell) to glioma sites. The expression of 16 proteins was shown to have increased commonly in human glioma tissues. Among them, the expression of annexin A2, TIMP-1, COL11A1, bax, CD74, TNFSF8, and SPTLC2 were all increased in human glioma cells, as confirmed by Western blotting and immunohistochemical staining. In particular, annexin A2 effects an increase in migration toward F3 and glioblastoma cells (U87 cell) in a Boyden chamber migration assay. An ERK inhibitor (PD98057) and a CDK5 inhibitor (rescovitine) inhibited 50% and 90% of annexin A2-induced migration in F3 cells, respectively. A similar chemotactic migration was noted in F3 and U87 cells. These results demonstrated that 7 candidate proteins may harbor a potential glioma tropism factor relevant to the pathology of malignant glioma. These results reveal that this novel molecular approach to the monitoring of glioma may provide clinically relevant information regarding tumor malignancy, and should also prove appropriate for high-throughput clinical screening applications.