PMA based real-time fluorescent LAMP for detection of Vibrio parahaemolyticus in viable but nonculturable state

Vibrio parahaemolyticus is a crucial foodborne pathogen. The viable but non-culturable (VBNC) state of V. parahaemolyticus cannot be detected by traditional culture methods. The objective of this study was to develop and evaluate a method that combines propidium monoazide (PMA) treatment with real-time fluorescent loop-mediated isothermal amplification (RF-LAMP) to detect and quantify VBNC cells of V. parahaemolyticus. Different states of cells were treated with PMA in dark for 5 min and subsequently exposed to a 650 W halogen lamp for 5 min. The cells were collected and DNA was amplified by RF-LAMP. The primers which targeted six distinct regions in the tlh gene of V. parahaemolyticus were used for the PMA-RF-LAMP assay. The results indicated that the treatment with 4 μg/mL of PMA and a further exposure to light for 5 min was suitable for PMA-RF-LAMP to distinguish viable cells from dead cells of V. parahaemolyticus. The developed assay exhibited remarkably high specificity, sensitivity and rapidity, without any cross reaction with the tested non-V. parahaemolyticus strains. There was good linear correlation between Ct values and log copy/mL in the range of 6.8–6.8 × 10⁷ copy/mL, with the reaction endpoints less than 30 cycles (30 min). It could detect as low as 14 copy/g of V. parahaemolyticus in spiked seafood samples (pomfret, shrimp, scallop, oyster and salted fish) without any interference from food matrices, dead cells and other bacteria.     

Selective turn-on fluorescence detection of Vibrio parahaemolyticus in food based on charge-transfer between CdSe/ZnS quantum dots and gold nanoparticles

Vibrio parahaemolyticus (V. parahaemolyticus) is one of the most common food-borne pathogens found along the east coast of China, Japan, and several southeast Asian countries, where marine foods are frequently consumed. Thus it is of great interest to establish a fast, sensitive and robust analytical assay for V. parahaemolyticus detection. Herein, we report a V. parahaemolyticus-specific egg yolk antibody (IgY), extracted from the egg yolks of immunized hens with inactivated V. parahaemolyticus bacteria. By immobilizing IgY onto the surface of gold nanoparticles (AuNPs) through electrostatic self-assembly, a detection system was developed based on charge-transfer quenching fluorescence, which contains core/shell CdSe/ZnS quantum dots (QDs), IgY-AuNPs and free AuNPs. In this strategy, the IgY-AuNPs were induced to aggregate quickly in the presence of target V. parahaemolyticus, resulting in a rapid fluorescence response of the QDs. More importantly, the convenient turn-on fluorescent detection system exhibited excellent selectivity over the other bacteria. Furthermore, this facile one-step method can detect V. parahaemolyticus at low concentrations of 10 cfu/mL without pre-enrichment and can be applicable for the detection of V. parahaemolyticus in real food samples. With the advantage of simplicity, rapidness, sensitivity, and specificity, this proposed approach shows promise as a successful application of V. parahaemolyticus in bioanalysis.     

Comparison of toxR and tlh based PCR assays for Vibrio parahaemolyticus

Vibrio parahaemolyticus is a Gram-negative bacterium found in marine and estuarine environments and is globally the leading cause of bacterial seafood-related illness. A real-time PCR assay for V. parahaemolyticus was developed for the marker toxR, with a large-scale and direct comparison of its applicability as a species-specific marker compared to the tlh gene carried out. Assays for tlh and toxR were used for 255 presumptive V. parahaemolyticus strains from our strain library, utilising both real-time (toxR) and conventional PCR assays (tlh). Of the 255 strains test, 254 results were in concordance; 255 strains were identified as being toxR positive (100%) and 254 strains were tlh positive (99.6%). The single discordant strain (isolate V12/023) was of interest, because it represented a presumptive V. parahaemolyticus strain, isolated from a clinical case. Whole genome sequence analysis and multi locus sequence typing of this single discordant strain was carried out, which unambiguously identified that the isolate was indeed V. parahaemolyticus. Genome analysis identified mismatches in the primer binding sites for the established tlh assay is likely responsible for the assay failing on this particular strain. The identification of false-negative results in strains that are implicated in human infections using the tlh assay and clearly highlights the relevance of the comparison with a toxR assay which showed 100% identification for the V. parahaemolyticus strains tested.     

Detection and quantification of pathogenic Vibrio parahaemolyticus in shellfish by using multiplex PCR and loop-mediated isothermal amplification assay

Vibrio parahaemolyticus is a halophilic bacterium that commonly inhabits the marine and estuarine environments. This organism is also one of the leading causative pathogen of gastroenteritis often related to consumption of raw or undercooked seafood. In this study, molluscan shellfish (bloody clams and surf clams) and crustaceans (shrimps) were monitored in wet markets and hypermarkets. Two molecular methods were employed and compared to detect total and pathogenic V. parahaemolyticus in MPN enrichments: multiplex PCR and LAMP assay. The multiplex PCR was optimized to detect the total (toxR+), tdh+ and trh+ V. parahaemolyticus. On the other hand, the LAMP assay was employed to target the pathogenic strains only, the tdh+ and trh+, respectively. Out of 232 samples examined, 229 (98.7%) were positive for V. parahaemolyticus with counts ranging from 30 to >110, 000 MPN/g. Positive samples for tdh+ V. parahaemolyticus were obtained in 77 out of 232 (33.1%) samples ranging from 30 to >110, 000 MPN/g. Meanwhile, positive samples for trh+ were identified in 16 out of 232 (6.9%) samples examined ranging from 30 to 9600 MPN/g. Detection of samples with presence of tdh+ genes did not vary between methods, but a significant difference was observed when the LAMP assay was compared to PCR to detect trh+ V. parahaemolyticus. Therefore, on occasions where the density of the targeted genes is low, the LAMP assay serves as a better alternative. Nonetheless, this study constitutes an assessment of presence of total and potentially pathogenic V. parahaemolyticus in shellfishes for domestic consumption revealing the potential risk of contracting vibriosis if precautions and safety measures are not properly managed.     

Detection of Roundup Ready soybean by loop-mediated isothermal amplification combined with a lateral-flow dipstick

LAMP–LFD, loop-mediated isothermal amplification (LAMP) combined with a lateral-flow dipstick (LFD), was developed and evaluated as a new method for the detection of Roundup Ready soybean (RRS). Biotinylated LAMP amplicons were produced by two sets of six designed primers that specifically recognized the endogenous gene (Lec1) and the event-specific 5′ -junction region (G35S) of RRS followed by hybridization with FITC-labeled probes and LFD detection. The following optimized conditions for the LAMP assay were used: deoxynucleotide triphosphate (dNTP) concentrations ranging from 0.6 to 3.2 mM, 6 mM Mg²⁺, 4 U Bst DNA polymerase and a 1:6 ratio of outer to inner primers. The LAMP–LFD results were generated within 50 min. The detection limit of LAMP–LFD was 2.4 copies of the linearized plasmid pTLH10 and was 20 times more sensitive than conventional PCR. We demonstrated the high specificity of LAMP–LFD by testing processed soybean products, genetically modified (GM) maize and Bt-cotton meal. The novel LAMP–LFD setup presented here is simple, rapid, and has the potential for future use in the detection of GM ingredients in feed and food products.     

Simultaneous detection of Vibrio cholerae,Vibrio alginolyticus,Vibrio parahaemolyticus and Vibrio vulnificus in seafood using dual priming oligonucleotide (DPO) system-based multiplex PCR assay

In this study, a rapid and reliable multiplex PCR assay for simultaneous detection of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio alginolyticus in seafood was developed using the dual priming oligonucleotide (DPO) system. Species-specific DPO primers were designed targeting the mdh, vvhA, colH and toxR genes for the discrimination of V. cholerae, V. vulnificus, V. alginolyticus and V. parahaemolyticus, respectively. Compared to conventional PCR assay, the DPO system-based multiplex PCR assay allowed a wider annealing temperature at 48 °C–68 °C to effectively amplify target genes followed an analytical detection limit of <1.5 × 10² CFU/mL (or g) for each Vibrio species in pure cultures or artificially contaminated food matrix. A total of 396 bacterial strains including 209 targets and 187 other bacterial strains were used to test the specificity of the DPO system-based multiplex PCR assay, and results showed that specific PCR product was only observed in target bacterial strains without nonspecific priming. Practical application of the assay indicated that target Vibrio species in seafood, clinical samples and foodborne outbreaks can be accurately detected. This DPO system-based multiplex PCR assay developed in this study would be a powerful tool for the rapid and reliable detection of the target Vibrio species.     

A rapid loop-mediated isothermal amplification method for detection of the modified GM cry1A gene in transgenic insect-resistant cotton and rice

Among the commercial genetically modified (GM) crops, the insect-resistant GM crops are the major cultivars that cry gene is introduced into. A cry1Ab/1Ac GM fusion gene (GFM cry1A) and a GM truncated cry1Ac gene (cry1Ac-Mon) is the key foreign gene employed for construction of GM crops by China researchers and Monsanto Technology LLC respectively. Here these two genes are entitled “GM cry1A” gene and a rapid visual loop-mediated isothermal amplification (LAMP) assay method for detection of GM cry1A in transgenic insect-resistant crops was established. The LAMP assay was performed at an optimal temperature of 65 °C for 60 min in the presence of a set of four specific primers recognized six distinct sequences of the GM cry1A gene. The rough detection limit to the GM cry1A in samples is as low as 0.01% (a weight ratio of transgenic insect-resistant rice/cotton to non-transgenic rice/cotton). Comparatively, the sensitivity of this LAMP method is 10 times over that of the conventional PCR method. Fifteen cultivars/events and five Bt strains with or without cry1A gene were analyzed using the LAMP method as well as PCR method. The results demonstrate that this LAMP method shows a distinct specificity to the GM cry1A gene comparing with PCR analysis. Therefore, this LAMP method will be a potential effective tool for screening the GM cry1A gene in GM crops which are widely plant in China and other developing countries.     

Production and characterization of a monoclonal antibody against recombinant thermolabile hemolysin and its application to screen for Vibrio parahaemolyticus contamination in raw seafood

Thermolabile hemolysin gene (tlh) is regarded as a species-specific marker for Vibrio parahaemolyticus. To assess the utility of the tlh gene product, thermolabile hemolysin (TLH), as a marker to screen for V. parahaemolyticus-contamination in raw seafood, we generated a monoclonal antibody (MAb) against recombinant TLH and established with an enzyme-linked immunosorbent assay using the MAb (TLH-ELISA). TLH-ELISA testing of broth cultures for 78 V. parahaemolyticus strains showed positive results all around. In contrast, most broth cultures of 53 non-V. parahaemolyticus species tested yielded negative results.We devised a screening method using TLH-ELISA to check for low-level contamination of V. parahaemolyticus in raw seafood within 24 h and evaluated its ability. In testing of V. parahaemolyticus-spiked raw seafood, results suggested that our screening method can detect 100 most-probable-number (MPN) of V. parahaemolyticus/g. Further, on testing 119 commercial raw seafood samples with our screening method, 117 samples were determined to contain less than 100 MPN of V. parahaemolyticus/g. All of the 117 samples were also estimated by the MPN method to contain less than 100 MPN of V. parahaemolyticus/g. Taken together, these results suggest that our screening method using TLH-ELISA is useful to check for low-level (<100 MPN/g) of V. parahaemolyticus in raw seafood.     

Event-specific analytical methods for six genetically modified maize events using visual and real-time loop-mediated isothermal amplification

Currently 138 genetically modified (GM) maize events have been authorized for commercial cultivation, comprising more than 65 per cent stacked events. With the increase in number of GM maize events globally, cost- and time-efficient diagnostics with on-site applicability are required to check for authorized GM events. Six GM maize events, namely, Bt11, GA21, MON810, MON89034, NK603 and TC1507, also present in 89 stacked events, are being widely commercialized in more than 17 countries. Visual and real-time loop-mediated isothermal amplification (LAMP) assays targeting these six GM maize events are being reported in the present study. Specificity of the developed LAMP assays was confirmed using fourteen commercialized GM maize events. Limit of detection of visual and real-time LAMP assays targeting Bt11, GA21, MON810, MON89034 and TC1507, was up to 0.01%, detecting 8 target copies, and for NK603 event-specific assays, was up to 0.1% detecting 73 target copies. Practical applicability of developed LAMP assays was verified using a set of five stacked GM maize events, namely, Bt11 × GA21, MON89034 × NK603, MON89034 × NK603 × TC1507, TC1507 × NK603 and TC1507 × MON810; and six powdered maize samples of proficiency testing. The reported LAMP assays can be efficiently employed for screening for presence of selected GM maize events in single or stacked form.     

Development of a multiplex real-time PCR method for simultaneous detection of Vibrio parahaemolyticus,Listeria monocytogenes and Salmonella spp. in raw shrimp

Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella spp. are important pathogens contaminating seafood in China. In this study, we developed an efficient multiplex real-time PCR for the simultaneous detection of V. parahaemolyticus, L. monocytogenes and Salmonella spp. in raw shrimp without a prior enrichment step. In a test using 28 target and non-target strains only the targets were detected and two calibration curves, for pure cultures and artificially contaminated samples, were used to evaluate the efficiencies of this method. Amplification efficiencies of this multiplex real-time PCR were excellent in pure cultures and artificially contaminated shrimps. The limits of detection in artificially contaminated shrimps were 112 CFU/g for V. parahaemolyticus, 158 CFU/g for L. monocytogenes and 103 CFU/g for Salmonella spp. We validated this multiplex real-time PCR method on 48 commercial samples and the results were comparable to standard culture methods. This efficient multiplex real-time PCR, where each test takes only 50 min after DNA extraction, is a useful tool for high-throughput surveillance of V. parahaemolyticus, L. monocytogenes and Salmonella spp. in seafood products.     

Simultaneous detection of Listeria species isolated from meat processed foods using multiplex PCR

Listeriosis is a foodborne disease caused by the pathogenic Listeria monocytogenes and is considered as a serious health problem due to the severity of symptoms and its high mortality rate. Listeria genus is divided into six species and especially L. monocytogenes is an important foodborne pathogen in humans and livestock. Recently, other Listeria species are reported as pathogenic strains in decayed foods and environments as well. High mortality rate of listeriosis demands for rapid methods to detect the potential presence of the food pathogens in the food industry. We have developed a multiplex PCR for rapid and simultaneous detection of six Listeria species including Listeria grayi, Listeria innocua, Listeria ivanovii, L. monocytogenes, Listeria seeligeri and Listeria welshimeri to identify specific Listeria species in processed foods. The optimized multiplex PCR in this study utilized one Listeria genus specific and each Listeria species-specific primer pairs. Each primer pair yields the products of 370-bp for Listeria genus-specific, 201-bp for L. grayi-specific, 749-bp for L. innocua-specific, 463-bp for L. ivanovii-specific, 509-bp for L. monocytogenes-specific, 673-bp for L. seeligeri-specific and 281-bp for L. welshimeri-specific. We have successfully applied multiplex PCR strategy to 93 Listeria isolates from processed meat products to determine specific Listeria species and out of which 81 strains of L. monocytogenes, 10 strains of L. innocua and 2 strains of L. welshimeri were identified. This established multiplex PCR provides rapid and reliable results and will be useful for the detection of Listeria species in contaminated food products and clinical samples.     

Application of grape seed extract in depuration for decontaminating Vibrio parahaemolyticus in Pacific oysters (Crassostrea gigas)

This study investigated the potential application of grape seed extract (GSE) in depuration to increase its efficacy in reducing Vibrio parahaemolyticus populations in Pacific oysters (Crassostrea gigas). Pacific oysters were inoculated with five clinical strains of V. parahaemolyticus to 10⁴⁻⁵ MPN/g and depurated in UV-irradiated artificial seawater (ASW) containing GSE at a level of 1.0% (1.8 mg/mL total phenolic contents as gallic acid equivalents) or 1.5% (3.1 mg/mL total phenolic contents as gallic acid equivalents) at 12.5 °C for up to 5 days. Changes of V. parahaemolyticus populations in oysters during depuration were analyzed every 24 h using the three-tube most probable number (MPN) method. The populations of V. parahaemolyticus in inoculated oysters decreased by 3.06 and 3.71 log MPN/g, respectively, after 4 and 5 days of depuration in ASW at 12.5 °C. However, populations of V. parahaemolyticus in oysters were reduced by 3.01 and 4.18 log MPN/g after 2 days of depuration at 12.5 °C in ASW containing 1.0 and 1.5% GSE, respectively. Further studies confirmed that depuration using ASW containing 1.5% GSE with 3.1 mg/mL total phenolic contents as gallic acid equivalents at 12.5 °C for two days was capable of achieving >3.52 log MPN/g reductions of V. parahaemolyticus in Pacific oysters.     

In-house validation of novel multiplex real-time PCR gene combination for the simultaneous detection of the main human pathogenic vibrios (Vibrio cholerae,Vibrio parahaemolyticus,and Vibrio vulnificus)

In recent years the incidence of vibriosis has greatly increased, raising the concern among consumers about the innocuity of certain food products. Previous studies demonstrated various advantages of molecular methods, including qPCR, for the screening of food-borne pathogens. The new method developed in the present study allows fast and reliable detection of the main human pathogenic Vibrio species (Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus). Specificity of the combination of primers and probes was successfully tested against several bacterial species and strains (44 different strains). Evaluation of the qPCR efficiency reported a value of 94% with the simultaneous amplification of the internal amplification control. The evaluation of the quality of the method was based on six parameters: relative sensitivity, specificity, accuracy, positive and negative predictive values as well as Kappa index of concordance. Each of the values obtained were higher than 96%. Additionally a very low limit of detection was determined for the developed method (less than 10 cfu/25 g). All the parameters of the method analyzed were obtained from the analysis of a wide variety of foodstuffs, water samples and reference material from proficiency tests, and compared against the culture reference method.     

Behavior of Vibrio parahemolyticus cocktail including pathogenic and nonpathogenic strains on cooked shrimp

Pathogenic Vibrio parahaemolyticus, an important foodborne bacterium, coexists with nonpathogenic strains of V. parahaemolyticus in the environment. However, current predictive models for V. parahaemolyticus usually focused on single strain without considering the pathogenic and nonpathogenic bacteria cocktail coexist situation. In this study, the behavior of four V. parahaemolyticus strains (F18: tlh⁺/trh⁺/tdh⁻, ATCC 17802: tlh⁺/trh⁺/tdh⁻, F36: tlh⁺/trh⁻/tdh⁻, ATCC 33847: tlh⁺/trh⁻/tdh⁺) and their cocktails on cooked shrimp were investigated at different temperatures (4, 7, 10, 15, 20, 25 and 30 °C). Total bacteria counts were enumerated by traditional plate count method, periodically. Results showed both V. parahaemolyticus cocktails and four single strains grew in a temperature range of 15–30 °C and died at 4–7 °C. At 10 °C, for the cocktails, it inactivated at initial 100 h and then grew rapidly; but for single strain, only one strain F18 displayed the similar growth tendency, while the others grew all the time. Compared with single strains, the primary and secondary model analysis showed that the cocktails displayed a better growth activity with higher maximum growth rate and shorter lag time. The above results indicated that modeling bacterial growth by using a cocktail of V. parahaemolyticus strains may be a better option for simulating growth in the real environment. This study will fill in the gap of predictive microbiology and improve significantly the accuracy of microbial risk assessment.     

Effects of green tea extract on reducing Vibrio parahaemolyticus and increasing shelf life of oyster meats

This study investigated effects of tea extract on growth of pathogenic Vibrio parahaemolyticus and potential utilization in post-harvest treatment to extend shelf life of Pacific oysters (Crassostrea gigas). Longjing Tea, which exhibited strong bactericidal activity against V. parahaemolyticus, was selected to use in this study. Tea extract containing equal or higher than 4.6 g/l total phenolic contents (TPC) as gallic acid equivalents (GAE) determined by Folin-Ciocalteau method could reduce a mixture of five clinical V. parahaemolyticus strains in tryptic soy broth plus 1.5% NaCl from 4.5 log CFU/ml to non-detectable level (<1 log CFU/ml) within 8 h. A treatment of shucked oysters with tea extract containing 9.1 g/l TPC as GAE for 2 h at 23 ± 1 °C with oyster/tea extract ratio of approximate 0.9 g/ml resulted in greater (p < 0.05) V. parahaemolyticus reductions (0.8 log MPN/g) compared to controls (0.2 log MPN/g). The following shelf life study indicated that green tea treatment at oyster/tea extract ratio of approximate 0.7 g/ml could enhance reducing V. parahaemolyticus while retarding the growth of total bacteria in oysters during 5 ± 1 °C storage. Therefore, green tea might be utilized as a natural antimicrobial agent to inactivate V. parahaemolyticus in oysters and extend the shelf life during refrigeration storage.     

Rapid detection of viable Bacillus cereus emetic and enterotoxic strains in food by coupling propidium monoazide and multiplex PCR (PMA-mPCR)

Bacillus cereus can cause emetic and diarrheal food poisoning. It is widespread in nature and therefore, considered a major foodborne pathogen. To develop a sensitive and reliable assay for detecting enterotoxin genes (nheA, entFM, hblD, cytK) and emetic toxin (ces), specific primers each targeting one individual gene were designed. Propidium monoazide (PMA) was coupled with the developed multiplex PCR (mPCR) for the detection of viable B. cereus. The inclusivity and exclusivity of the PMA-mPCR was confirmed using a panel of 44 strains including 17 emetic and 9 enterotoxic B. cereus reference strains and 18 non-target strains. The limit of detection (LOD) without PMA treatment in pure DNA was 2 pg/reaction tube. The LOD of mPCR assay in pure heat-killed dead bacteria was 4.0 × 10² CFU/mL. Also, the LOD on the viable bacteria with or without PMA treatment was similar (3.8 × 10² CFU/mL) showing that the PMA treatment did not significantly decrease sensitivity. Finally, the newly developed PMA-mPCR successfully detected 4.8 × 10³ and 3.6 × 10³ CFU/g of viable B. cereus F4810/72 (emetic) and B. cereus ATCC 12480 (enterotoxic) reference strains, respectively, in food samples. Hence, this study combines PMA and mPCR to detect viable B. cereus with a wide range of toxin detection (5 toxins). Thus, the novel PMA-mPCR assay developed in this study is a rapid and efficient diagnostic tool for the monitoring of viable B. cereus in food samples and potentially other samples via appropriate DNA extraction.     

Effect of temperature on pathogenic and non-pathogenic Vibrio parahaemolyticus biofilm formation

Biofilm formation is crucial for the environmental survival and transmission of Vibrio parahaemolyticus, an important food-borne pathogen in seafood. The biofilm developmental process of pathogenic (n = 22) and non-pathogenic (n = 17) V. parahaemolyticus strains on polystyrene microtiter plates under 15 °C, 25 °C and 37 °C was investigated using crystal violet staining, and validated by confocal laser scanning microscopy. The results indicated that biofilm developmental process at 15 °C and 25 °C were divergent, biofilm formation increased continuously at 15 °C, while at 25 °C biofilm formation increased gradually and peaked at 12 h. Also the biofilm formation was dramatically elevated at 25 °C in comparison with that at 15 °C and 37 °C. Additionally, pathogenic strains, on average, formed more biofilm than non-pathogenic strains at all temperatures measured. Moreover, extensive strain variability was observed during biofilm formation and indexed using the coefficient of variation (CV). This index increased with increasing temperature and this index, at all temperatures, peaked after 12 h. The results of this study provide insight into the developmental process of biofilm, which allow us to further optimize strategies to control V. parahaemolyticus biofilm in food industry.     

The detection of Atlantic cod (Gadus morhua) using loop mediated isothermal amplification in conjunction with a simplified DNA extraction process

Atlantic cod (Gadus morhua) is a commercially important species of white fish, and one of three species legally identifiable as cod in the UK. Mislabelling of G. morhua does occur, as does the substitution of G. morhua for less expensive species. Sensitive molecular tests based on PCR have been developed for this species, but they have limitations, including the need for expensive thermal cycling equipment, and complex DNA extraction procedures. A loop mediated isothermal amplification (LAMP) assay was designed for the G. morhua cytochrome b gene, which was capable of detecting 0.1% w/w G. morhua in a homogenised raw fish mix. The LAMP assay was also able to detect G. morhua DNA when a rapid sample preparation was used, involving heating 100 mg of fish in a 1 ml aliquot of water and testing the supernatant, showing a higher tolerance of amplification inhibitors than a PCR assay. The LAMP assay did not generate a positive result when challenged with a range of non-target species, including Gadus macrocephalus, and Gadus chalcogrammus, indicating a high level of specificity. Direct detection of a positive reaction using propidium iodide was also demonstrated.