Advantages of immunoglobulin Y for the detection of Staphylococcal enterotoxin A in a double‐antibody sandwich enzyme‐linked immunosorbent assay

To determine the amounts of staphylococcal enterotoxin A (SEA), a novel and sensitive enzyme‐linked immunosorbent assay (ELISA) was developed. Protein A, which is produced by Staphylococcus aureus, interferes with the reaction between SEA and anti‐SEA immunoglobulin G (IgG), resulting in a false‐positive reaction. Chicken IgY was introduced as a capture antibody in the sandwich ELISA system, as IgY binds less efficiently to protein A. When the anti‐SEA IgG antibody was used as the capture and detection antibodies (IgG‐IgG ELISA), the background levels of protein A increased, thus resulting in a false‐positive reaction. A 0.01 ng mL^?1 concentration of protein A significantly increased the absorbance value of the blank wells. When the anti‐SEA IgY antibody was used as the capture antibody, 1000 ng mL^?1 of protein A did not affect the absorbance value. The ELISA system using anti‐SEA IgY as a capture antibody and anti‐SEA IgG as a detection antibody (IgY‐IgG ELISA) showed a detection limit of <0.25 ng mL^?1 and a creditability of R² = 0.98. These findings demonstrate the advantage of chicken IgY for the detection of SEA by means of double‐antibody sandwich ELISA.     

Discrimination between Bacillus and Alicyclobacillus Isolates in Apple Juice by Fourier Transform Infrared Spectroscopy and Multivariate Analysis

Alicyclobacillus is a causative agent of spoilage in pasteurized and heat‐treated apple juice products. Differentiating between this genus and the closely related Bacillus is crucially important. In this study, Fourier transform infrared spectroscopy (FT‐IR) was used to identify and discriminate between 4 Alicyclobacillus strains and 4 Bacillus isolates inoculated individually into apple juice. Loading plots over the range of 1350 and 1700 cm^?1 reflected the most distinctive biochemical features of Bacillus and Alicyclobacillus. Multivariate statistical methods (for example, principal component analysis and soft independent modeling of class analogy) were used to analyze the spectral data. Distinctive separation of spectral samples was observed. This study demonstrates that FT‐IR spectroscopy in combination with multivariate analysis could serve as a rapid and effective tool for fruit juice industry to differentiate between Bacillus and Alicyclobacillus and to distinguish between species belonging to these 2 genera.     

Adsorptive Removal of Patulin from Apple Juice Using Ca‐Alginate‐Activated Carbon Beads

This study aimed to investigate the adsorption of patulin from apple juice by Ca‐alginate‐activated carbon (Ca‐alginate‐AC) beads. The capacity of patulin was determined by high‐performance liquid chromatography. The results showed that Ca‐alginate‐AC beads have significant ability to reduce patulin from contaminated apple juice. Furthermore, the adsorption process did not affect the quality of apple juice. The effects of contact time, initial patulin concentration, adsorbent dose, and temperature were assessed. The removal percentage of patulin increased with contact time, adsorbent dose, and temperature. A reduction was also noted to bind patulin at increased levels of contamination. The equilibrium data were fitted to Langmuir, Freundlich, and Temkin isotherm models and the isotherm constants were calculated at different temperatures. The adsorption equilibrium was best described by the Freundlich isotherm (R² > 0.990). The pseudo 1st‐order model was found to describe the kinetic data satisfactorily. Thermodynamic parameters such as standard Gibbs free energy (ΔG??), standard enthalpy (ΔH?), and standard entropy (ΔS?) were evaluated. The results showed that the adsorption was spontaneous and endothermic nature.     

A rapid detection of Escherichia coli O157:H7 by competition visual antigen macroarray

Pathogenic bacterial contamination is a serious problem for the food industry and in public health. Rapid, accurate and affordable testing for pathogenic bacterial strains is desirable. In this study, a competition visual antigen macroarray (CVAM) for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7) has been developed. This array was able to utilize an HRP‐labeled anti‐E. coli O157:H7 MAb at a concentration of 1:20000 while having a similar sensitivity of 10 ⁵ CFU/ml for E. coli O157:H7 detection as double antibody sandwich array and conventional ELISA. The detection time of CVAM was short as 2 h and can be examined directly by the naked eye. Moreover, there was no cross‐reactivity in the detection of tested in this study. Application of CVAM for the detection of E. coli O157:H7 artificially contaminated milk samples was feasible that showed the antigen array could be applied in the detection of food pathogen infections.     

Phylogenetic and spectroscopic analysis of Alicyclobacillus isolates by 16S rDNA sequencing and mid-infrared spectroscopy

Alicyclobacillus spp. are a group of thermophilic, acidophilic, spore-forming bacteria, some of which cause spoilage in commercially pasteurized fruit and vegetable juice products including apple, orange, tomato and carrot juice. In this study, we characterized seven isolates of Alicyclobacillus (14-2, KF, A-Gala 2-1, C-Fuji 6, Gala 9-2, 1016, 18-1) and a reference strain A. acidiphilus DSM 14558 by a 16S rDNA sequencing technique and Fourier transform infrared (FT-IR) spectral features. The degree of similarity between Alicyclobacillus isolates based upon phylogenetic analysis of sequence data and multivariate statistical analysis of FT-IR spectral data was determined. Results from both methods were consistent, suggesting that a combination of methods targeting both 16S rDNA and mid-infrared spectroscopic signatures may be a suitable and more accurate approach to either identify or discriminate Alicyclobacillus isolates from fruit and vegetable juice products.     

Characterisation of malolactic conversion by Oenococcus oeni to reduce the acidity of apple juice

The high concentration of malic acid is responsible for the acidity and sourness in apple juice. Bio‐conversion of malic acid to lactic acid through malolactic conversion (MC) in apple juice using Oenococcus oeni was investigated. When apple juice was inoculated with O. oeni (1 × 10⁶ CFU mL^?1), over 90% of malic acid was converted into lactic acid within 96 h at room temperature. When pH of apple juice was adjusted to 4.1 prior to inoculation, MC was completed within 60 h. MC was enhanced at a higher temperature (30°C) when compared with room temperature. The rate of MC was directly proportional to the number of bacteria added and MC was completed within 24 h at 1 × 10⁹ CFU mL^?1 initial cell density. MC occurred equally under aerobic and anaerobic conditions. The sensory analysis of partial MC‐applied juice when compared against control revealed potential for use of MC for manufacture of low‐acid apple juice.     

A Simple and Green Route for Room‐Temperature Synthesis of Gold Nanoparticles and Selective Colorimetric Detection of Cysteine

Gold nanoparticles (AuNPs) were synthesized at room temperature following a simple, rapid, and green route using fresh‐squeezed apple juice as a reducing reagent. The optimal AuNPs, based on the particle color, stability, and color change suitable for colorimetric detection of cysteine (Cys), are synthesized using 5 mL of 10% apple juice, 1 mL of 10 mM gold precursor solution, and 1 mL of 0.1 M NaOH. Under this set of parameters, the AuNPs are synthesized within 30 min at room temperature. The average size (11.1 ± 3.2 nm) and ζ potential (–36.5 mV) of the AuNPs synthesized were similar to those of AuNPs prepared via the conventional citrate‐reduction method. In the presence of Cys, unlike with any other amino acid, the AuNPs aggregated, possibly due to the gold–sulfur covalent interaction, yielding red‐to‐purple color change of the sample solution. The red‐shift of the localized surface plasmon resonance peak of the AuNPs responsible for the color change was recorded by UV‐vis spectrometer. The effect of other potential interferents such as glucose, ascorbic acid, K⁺, Na⁺, Ca²⁺, Zn²⁺, Ag⁺, Ni²⁺, Cu²⁺, Co²⁺, and Hg²⁺ were also examined. The results show that AuNPs can be used to selectively detect and measure Cys with a linear dependency in the range of 2 to 100 μM and a limit of detection (signal‐to‐noise ratio > 3) of 50 nM. The results suggest that the green‐synthesized AuNPs are useful for simple, rapid, and sensitive colorimetric detection of Cys, which is an essential amino acid in food and biological systems.     

Growth inhibition and inactivation of Alicyclobacillus acidoterrestris endospores in apple juice by hyperbaric storage at ambient temperature

Control of endospores of Alicyclobacillus acidoterrestris in pasteurized apple juice using hyperbaric storage at 18 to 23 °C was compared to storage at atmospheric pressure and 18 to 23 °C, as well as refrigeration at ~4 °C for up to 30 days. The juice samples were inoculated with approximately 1 × 10⁵ CFU/mL spores. The juice spoiled quickly at atmospheric pressure and ambient temperature, while under refrigeration spore levels remained unchanged for 30 days. Hyperbaric storage of inoculated apple juice at 25, 50 and 100 MPa at 18 to 23 °C resulted in spore inactivation at more rapid rates as pressure magnitudes increased, reaching levels below the detection limit of 10 CFU/mL at 50 and 100 MPa. In highly acid foods such as apple juice, hyperbaric storage at pressures ≤100 MPa and ambient temperature was effective in inactivating spores of A. acidoterrestris for periods up to 30 days.These results indicate hyperbaric storage at ambient temperature as a clearly more efficient preservation procedure to control the development of A. acidoterrestris endospores, compared to ambient temperature and refrigerated storage, in highly acidic foods as apple juice.     

Identification and haplotype distribution of Alicyclobacillus spp. from different juices and beverages

Alicyclobacillus spp. is an important thermoacidophilic, spore-forming spoilage bacterium that is a major concern for beverage and juice industries. In order to develop effective control strategies and adequately address the prevalence of contamination sources, it is necessary to characterize Alicyclobacillus' ecology in fruit, juice and beverage production and processing environments.Alicyclobacillus spp. isolates were collected from juice, beverage, ingredients, and environmental samples over a period of ten years. A total of 141 isolates were characterized as Alicyclobacillus spp. by 16S rRNA analysis and the most frequently isolated species was found to be Alicyclobacillus acidoterrestris (45%), A. acidocaldarius subsp. acidocaldarius (30%), and A. acidocaldarius (11%).The majority of thermotolerant sporeformers isolated from apple juices and concentrates was found to be A. acidoterrestris (24 out of 36 total apple isolates); while A. acidoterrestris was most frequently associated with citrus, citrus concentrates, and their associated environments, isolated by University of Florida (UF) (15 out of total 28 UF citrus isolates). However, A. acidocaldarius and subsp. acidocaldarius were frequently isolated by Cornell University (CU) (29 out of 35 CU citrus isolates), from citrus juices made from concentrate. Four major haplotypes of Alicyclobacillus spp. were identified based on the 16S rRNA gene sequencing from the 141 isolates tested. The Allelic Types (ATs) matched the phylogenetic analysis grouping of the different Alicyclobacillus spp. based on the isolation source.Our results suggest a predisposition for certain ATs of Alicyclobacillus spp. depending on juice or ingredient isolation source.     

Effects of 1‐methylcyclopropene and post‐controlled atmosphere air storage treatments on fresh‐cut Ambrosia apple slices

BACKGROUND: The effect of 1‐methylcyclopropene (1‐MCP) treatment and two different post‐controlled atmosphere air storage (PCAAS) durations on the quality and chemistry of fresh‐cut Ambrosia apple slices was studied. RESULTS: PCAAS for 1 or 2 weeks prior to slicing had an overall positive effect on the resultant quality of fresh‐cut apple slices. The most significant responses to PCAAS were the suppression of both phenolic and o‐quinone accumulation in slices, and this was related to the significantly lower browning potential values obtained for slices from PCAAS‐treated apples. Polyphenol oxidase (PPO), peroxidase (POX) and ascorbate peroxidase (APOX) activities were not affected by 1‐MCP or PCAAS treatments. PPO and POX activities were almost completely inhibited by a 50 g L^?1 calcium ascorbate anti‐browning dip of apple slices from all treatments. CONCLUSION: The most dramatic effect of the PCAAS treatments was to reduce the accumulation of soluble phenolics, which is likely the reason that o‐quinone accumulation was also inhibited in treated fruits. The consequent reduction in browning potential may be the explanation as to why PCAAS treatment has been shown to reduce fresh apple slice browning in previous work. Copyright © 2012 Society of Chemical Industry     

Isolation and identification of Alicyclobacillus acidocaldarius by 16S rDNA from mango juice and concentrate

In this study we investigate the spoilage of ultra high temperature UHT mango juice as well as a carbonated fruit juice blend to identify organisms contributing to the spoilage. The mango concentrate, the final product, as well as the other ingredients used during manufacturing, were tested for the presence of Alicyclobacillus by polymerase chain reaction (PCR) and sequencing analyses. Microbiological examination of the mango pureé and spoiled fruit juices, using YSG agar [yeast extract 2 g, glucose 1 g, soluble starch 2 g, pH 3.7 (adjust with 2N H₂SO₄), H₂O 1000 mL, bacto agar 15 g] incubated at 55 °C, detected sporeforming, acid dependent and thermotolerant bacteria. The hyper variable region of the 16S rDNA was amplified. The nucleotide sequence of the PCR fragments was determined using the ABI Prism 310 automated DNA sequencer and the collected sequencing data were analysed and compared with the non‐redundant database using NCBI‐BLAST. Alicyclobacillus acidocaldarius were isolated and identified by 16S rDNA gene sequences analyses. The results indicated that the mango purèe, as well as the final product of mango juice and the fruit juice blend, were positive for Alicyclobacillus. The preventative measures of low pH, pasteurization of mango juice and the subsequent use of aseptic packaging were not regarded as sufficient to prevent the outgrowth of Alicyclobacillus spoilage organisms.     

Development of a sensitive and group‐specific polyclonal antibody‐based enzyme‐linked immunosorbent assay (ELISA) for detection of malachite green and leucomalachite green in water and fish samples

BACKGROUND: Malachite green (MG) is widely used in fishery, since it is easily adsorbed by fish during waterborne exposure and is rapidly metabolised into leucomalachite green (LMG). However, both MG and LMG are potential carcinogens, teratogens and mutagens. In this study the LMG derivative bearing an amino group on the phenyl ring was synthesised and coupled to carrier proteins. An LMG polyclonal antibody‐based enzyme‐linked immunosorbent assay (ELISA) was developed and characterised. RESULTS: The ELISA standard curve was constructed with concentrations of 0.1–100 ng mL^?1. The IC₅₀ value for nine standard curves was in the range 0.9–2.6 ng mL^?1 and the limit of detection at a signal‐to‐noise ratio of 3 was 0.02–0.10 ng mL^?1. The cross‐reactivity values of the LMG antibody with MG, crystal violet and leucocrystal violet were 95.25, 29.07 and 212.38% respectively, while less than 0.2% cross‐reactivity was found with eight other compounds. For LMG‐spiked water and fish samples, recoveries were 76.2–95.0% and the correlation coefficient of ELISA with high‐performance liquid chromatography (HPLC) was 0.9752 (n = 7). For (LMG + MG)‐spiked fish samples the results of ELISA were similar to those of HPLC. CONCLUSION: The proposed ELISA can be utilised as an analytical tool for detecting the sum of MG and LMG in water and fish muscle samples. Copyright © 2009 Society of Chemical Industry     

Magnetoelastic biosensor for the detection of Salmonella typhimurium in food products

In this article, a magnetoelastic sensor immobilized with polyclonal antibody for the detection of Salmonella typhimurium in food products is described. The remote query nature of magnetoelastic sensors enables the detection of bacterial species in sealed and opaque containers. Bacterial binding to the antibody on the sensor surfaces changed the resonance parameters, and these changes were quantified by the shift in the sensor’s resonance frequency. Response of the sensors to increasing concentrations (5 × 10¹–5 × 10⁸ cfu/ml) of S. typhimurium in three different food products (water, fat-free milk and apple juice) was studied and similar responses were observed. These results were also further ascertained by Scanning Electron Microscopy (SEM) studies. A detection limit of 5 × 10³ cfu/ml, with a sensitivity of 139 Hz/decade was obtained for the sensors tested in water samples, as compared to 129 Hz/decade in apple juice and 127 Hz/decade in fat free milk. A 2 × 0.4 × 0.015 mm sensor was employed in all the investigations. The dissociation constant K _d and the binding valencies for S. typhimurium spiked in water samples was 435 cfu/ml and 2.33 respectively; as compared to 309 cfu/ml and 2.38 for apple juice; and 1389 cfu/ml and 1.85 for fat free milk samples. Bacterial binding was specific and a divalent binding was observed.     

Determination of Iriflophenone 3‐C‐β‐d‐Glucoside From Aquilaria spp. by an Indirect Competitive Enzyme‐linked Immunosorbent Assay Using a Specific Polyclonal Antibody

Polyclonal antibody against iriflophenone 3‐C‐β‐d ‐glucoside (IP3G), a major compound from the leaves of Aquilaria spp., was produced for the development of an enzyme‐linked immunosorbent assay (ELISA). The results showed that the antibodies were specific for IP3G. The produced antibody has low cross reactivity with iriflophenone 3,5‐C‐β‐d ‐diglucopyranoside (13%), genkwanin 5‐O‐β‐primeveroside (3.55%) and no cross reactivity found in other compounds. The range of ELISA assay extends from 100 to 1560 ng/mL with coefficient of variation (CV) 1.19% to 2.07% for intra‐assay and 3.76% to 7.15% for inter‐assay precision levels. The recovery rates of IP3G in the leaves of Aquilaria spp. were in the range of 96.0% to 99.0% with CV 4.50% to 5.32%. A correlation between ELISA and high‐performance liquid chromatography methods was obtained when analysis of IP3G in the plant samples (R² = 0.9321). These results suggest that the developed ELISA method can be applied to determine IP3G content with high specificity, rapidity, and simplicity. The developed immunosorbent assay in this study provides a useful tool for the analysis of IP3G in plant samples and products.     

The effects of different technologies on<Emphasis Type="Italic"> Alicyclobacillus acidoterrestris</Emphasis> during apple juice production

In this study, the effects of various processing steps applied during apple juice processing on Alicyclobacillus acidoterrestris were investigated. Raw apple juice was inoculated with A. acidoterrestris spores at two inoculum levels of 1×10³ cfu/ml and 1×10⁶ cfu/ml. Following enzymatic treatment, the raw juice was processed into clear juice using either conventional clarification or ultrafiltration. The number of A. acidoterrestris spores in the final product was determined to be dependent on both initial contamination level and processing conditions. Increasing temperature to 50 °C during depectinization resulted in a higher spore counts at both inoculum levels. Even if the ultrafiltration process was found to be much more convenient for the retention of A. acidoterrestris when compared to conventional clarification, the spores could penetrate the ultrafiltration membranes having both 20 and 50 kDa. Increasing membrane pore size and initial spore counts in the feed solution also increased the number of spores penetrating the membrane during ultrafiltration.     

Fate of Escherichia coli in apple and reduction of its growth using the postharvest biocontrol agent Candida sake CPA‐1

BACKGROUND: Generally, acidic fruits and fruit juices are considered ‘safe’ from a microbiological point of view. However, some outbreaks of foodborne illnesses have been linked to the consumption of unpasteurised cider. The aim of this work was to study the survival of Escherichia coli in apple juice, wounds and flesh and on apple surfaces at different temperatures and to determine the effect of the fungal biocontrol agent Candida sake CPA‐1 against the colonisation of apple by E. coli. RESULTS: Trials were conducted with a mixture of five strains of E. coli isolated from apples. E. coli was unable to grow in apple juice at 5, 15 and 25 °C but survived. At 10 °C and above, E. coli thrived in fresh‐cut apple and wounds. At 5 °C it survived in apple wounds after 27 days of storage and after 21 days in fresh‐cut apples. When E. coli was inoculated in apple wounds together with the yeast antagonist C. sake, its growth was reduced by approximately 1 log cfu wound^?1 at 25 °C. At 5 °C no effect of the biocontrol agent was observed. CONCLUSION: Despite the low pH of apple, a rapid increase in the bacterial population is possible if the temperature is not kept low enough. The biocontrol agent C. sake, developed to prevent fruit decay during storage, could also reduce E. coli growth in wounded apples at abusive temperatures. This would represent an additional benefit of using this biocontrol agent when applied to control postharvest diseases. Copyright © 2009 Society of Chemical Industry     

Characterization of Osmotolerant Yeasts and Yeast‐Like Molds from Apple Orchards and Apple Juice Processing Plants in China and Investigation of Their Spoilage Potential

Yeasts and yeast‐like fungal isolates were recovered from apple orchards and apple juice processing plants located in the Shaanxi province of China. The strains were evaluated for osmotolerance by growing them in 50% (w/v) glucose. Of the strains tested, 66 were positive for osmotolerance and were subsequently identified by 26S or 5.8S‐ITS ribosomal RNA (rRNA) gene sequencing. Physiological tests and RAPD‐PCR analysis were performed to reveal the polymorphism of isolates belonging to the same species. Further, the spoilage potential of the 66 isolates was determining by evaluating their growth in 50% to 70% (w/v) glucose and measuring gas generation in 50% (w/v) glucose. Thirteen osmotolerant isolates representing 9 species were obtained from 10 apple orchards and 53 target isolates representing 19 species were recovered from 2 apple juice processing plants. In total, members of 14 genera and 23 species of osmotolerant isolates including yeast‐like molds were recovered from all sources. The commonly recovered osmotolerant isolates belonged to Kluyveromyces marxianus, Hanseniaspora uvarum, Saccharomyces cerevisiae, Zygosaccharomyces rouxii, Candida tropicalis, and Pichia kudriavzevii. The polymorphism of isolates belonging to the same species was limited to 1 to 3 biotypes. The majority of species were capable of growing within a range of glucose concentration, similar to sugar concentrations found in apple juice products with a lag phase from 96 to 192 h. Overall, Z. rouxii was particularly the most tolerant to high glucose concentration with the shortest lag phase of 48 h in 70% (w/v) glucose and the fastest gas generation rate in 50% (w/v) glucose.     

Preparation of anti‐danofloxacin antibody and development of an indirect competitive enzyme‐linked immunosorbent assay for detection of danofloxacin residue in chicken liver

BACKGROUND: Danofloxacin is used widely as both a clinical medicine for humans and a veterinary drug in animal husbandry. In this study a polyclonal anti‐danofloxacin antibody was prepared for the first time and a simple and rapid indirect competitive enzyme‐linked immunosorbent assay (cELISA) method based on the antibody was developed to monitor danofloxacin residue in chicken liver. RESULTS: The prepared antibody showed high sensitivity, with an IC₅₀ value of 2.0 ng mL^?1 towards danofloxacin, and good specificity, with significant cross‐reactivity only towards pefloxacin (22%) and fleroxacin (21%) among commonly used (fluoro)quinolones evaluated in the study. The developed cELISA test kit had a detection limit of 0.8 ng mL^?1, and satisfactory results were obtained when it was applied to chicken liver spiked with various levels of danofloxacin. The cELISA test kit was also used to detect danofloxacin in chicken liver samples purchased from a local food market, and the results were confirmed by liquid chromatography/mass spectrometry. CONCLUSION: The anti‐danofloxacin antibody prepared in this study exhibits excellent quality, with high sensitivity and good specificity. The cELISA test kit based on the antibody has a very low detection limit and is suitable for use as an efficient screening method to detect danofloxacin residue in foods and food products. Copyright © 2009 Society of Chemical Industry     

Chemometrics coupled with ultraviolet spectroscopy: a tool for the analysis of variety,adulteration, quality and ageing of apple juices

This study demonstrates the use of UV spectroscopy (UV) in combination with chemometrics as a simple and feasible approach for analysis of variety, adulteration, quality and ageing of apple juice. The results show that PCA‐UV is adequate to differentiate apple juice varieties and adulteration. The percentage of the adulterant can be detected by PLSR‐UV with RMSE < 0.7783% and R² > 0.9980. For the evaluation of juice quality, PLSR‐UV (RMSE = 0.2555–2.3448; R² = 0.7276–0.9816) is recommended for the prediction of soluble solids, ascorbic acid, total flavonoids, total sugar and reducing sugar, whilst PCR‐UV (RMSE = 0.0000–2.7426; R² = 0.7073–1.0000) is adequate for the prediction of pH and antioxidant activity. In addition, PLSR‐UV may be used to predict the storage time with RMSE = 0.4681 day and R² = 0.9832. Therefore, UV coupled with chemometrics has potential to be developed as a portable tool for the detection of variety, adulteration, quality and ageing of not only apple juices, but also other fruit and vegetable juices.     

Screening and construct‐specific detection methods of transgenic Huafan No 1 tomato by conventional and real‐time PCR

Genetically modified (GM) tomatoes have been approved for commercialization in many countries since the first GM tomato FLAVR SAVR was permitted for planting in 1994. In China, GM tomato Huafan No 1 with a character of long shelf‐life was the first GM plant which was approved for commercialization in 1996. To meet the requirement of the GM tomatoes labeling policy that has been actualized in China since 2001, screening and construct‐specific PCR detection methods for detecting the universal elements transformed into tomato, such as cauliflower mosaic virus 35S (CaMV35s) promoter and the nopaline synthase (NOS) terminator of Agrobacterium tumefaciens, and the specifically inserted heterologous DNA sequence between CaMV35s promoter and anti‐sense ethylene‐forming enzyme (EFE) gene were set up. To make the detection methods normative, a novel single copy tomato gene LAT52 was also used as an endogenous reference gene in the PCR detection systems. The limit of detection of screening and construct specific detection methods for Huafan No 1 was 68 haploid genome copies in conventional PCR detection, and three copies in TaqMan real‐time PCR detection. The limit of quantitation of screening quantitative PCR assays for Huafan No 1 was three copies and was 25 copies for construct‐specific quantitative PCR. Two samples with known Huafan No 1 tomato content were detected using the established conventional and real‐time PCR systems, and these results also indicated that the established Huafan No 1 screening and construct‐specific PCR detection systems were reliable, sensitive and accurate. Copyright © 2005 Society of Chemical Industry