Assessment of crustacean allergen detection methods: cross reactivity with edible insect samples.

Increased interest in consumption of insects in recent years has led to an increased focus on associated food safety concerns, and allergy is one of the most relevant. In the United States, crustacean shellfish are regulated as a major allergenic food group per the Food, Drug, and Cosmetic (FD&C) Act. Insects and crustacean shellfish are both arthropods, and clinical cross-reactivity between the two groups has been demonstrated. The goal of this work was to establish whether that clinical cross-reactivity translates into analytical cross-reactivity with detection assays targeting crustacean shellfish allergens. Edible insect samples were analyzed using four different crustacean allergen detection methods: Multi-Analyte Profiling Food Allergen Detection Assay (xMAP FADA), enzyme-linked immunosorbent assay (ELISA), western blot, and real-time polymerase chain reaction (PCR). Results indicate that the immunoassay-based xMAP FADA, ELISA, and western blot were susceptible to cross-reactivity, while the DNA-based PCR methods had minimal reactivity with insect samples. These results confirm that edible insects show analytical cross-reactivity with the immunoassays which may result in false positive detection of crustacean allergens in insect samples. Confirmation using DNA-based PCR, which shows little to no cross-reactivity, clarifies ambiguous results.

Optimised multiplex droplet digital PCR is more precise, but not more sensitive, than real-time PCR for the detection of allergenic peanut.

The United States requires labelling of food products containing major allergens, such as peanut, through the Food Allergen Labeling and Consumer Protection Act. Accurate labelling requires sensitive, specific and robust detection methods, and PCR-based techniques have proven highly effective. This article describes the transition of a previously developed multiplex real-time PCR assay for allergenic peanut to a droplet digital PCR format. The triplex droplet digital PCR assay was developed in a probe mixing format and directly compared to the established real-time PCR assay. Data are provided for thorough optimisation in the digital format, including the effects of primer and probe concentration, cycle number and annealing/extension time. Optimisation parameters influenced relative location and separation of droplet clusters but not final copy number. The droplet digital PCR assay was linear over five orders of magnitude; its lower limit of detection was 0.05 pg DNA per reaction, more sensitive than published digital PCR allergen assays. It was more precise, but not more sensitive, than the previously established real-time PCR assay.

Comparison of real-time PCR and ELISA for the detection of crustacean shellfish allergens.

Food allergies are a significant public health concern, and crustacean shellfish represent one of the major FDA regulated food allergens. Allergic individuals must avoid foods containing crustaceans, and this necessitates highly sensitive and accurate detection methods. Two of the major methods used are protein-based ELISA and DNA-based real-time PCR. In order to properly compare these very different methodologies, we used identical split samples for a side-by-side comparison and analysed them using four different real-time PCR methods and two different commercial ELISA kits. Three real-time PCR assays targeting the mitochondrial 12S genes of shrimp, crab, and lobster were compared to a commercial ELISA assay for total crustacean protein. A fourth real-time PCR assay targeting the tropomyosin gene of shrimp was compared to an ELISA assay for shrimp tropomyosin. All comparisons were carried out in two different food matrices: Manhattan clam chowder and fish sauce. PCR assays had a more broad dynamic range (0.1-106 mg/kg) as compared to ELISA (200-4000 mg/kg) and did not show matrix interference like ELISA. In cases where the ELISA assays did not have matrix interference, there was good qualitative agreement between PCR and ELISA.

A method to detect allergenic fish, specifically cod and pollock, using quantitative real-time PCR and COI DNA barcoding sequences.

BACKGROUND: Fish are one of eight major allergens defined in the US Food Allergen Labeling and Consumer Protection Act, and cod and pollock are two of the major fish allergens. This paper describes development and validation of a method to detect cod and pollock in complex food matrices using real-time polymerase chain reaction (PCR). Mitochondrial cytochrome oxidase I (COI) sequences obtained through DNA barcoding were used to design a single set of primers and probe which detected three species in the genus Gadus: Atlantic cod, Pacific cod, and walleye pollock. RESULTS: Cod spiked into three different food matrices (cooking oil, clam chowder, and hushpuppy mix) yielded high linearity, dynamic range spanning six orders of magnitude, and lower limits of detection at 1-10 ppm (ppm; mg kg-1 ). Frying had an adverse effect on the lower limit of detection, but not on linearity. CONCLUSIONS: This work shows that COI DNA barcoding sequences can be used to effectively design real-time PCR assays for detection of food allergens in complex matrices. While full-length DNA barcodes distinguish individual species, the PCR assay designed here detected three different species. This is likely because real-time PCR assays are tolerant to basepair mismatches and do not utilize the full length of the DNA barcode. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

Development and Evaluation of a Real-Time PCR Multiplex Assay for the Detection of Allergenic Peanut Using Chloroplast DNA Markers.

Peanut is one of the most commonly consumed allergy-causing foods in the United States. Prevention of accidental consumption by allergic individuals is assisted by methods that effectively identify the presence of peanut in food, even at trace levels. This study presents a multiplex real-time polymerase chain reaction (PCR) assay that uses chloroplast markers ( matK, rpl16, and trnH-psbA) to specifically detect peanut in three types of foods: baked goods, chocolate, and tomato sauces. Food matrices were spiked with raw peanut at concentrations ranging from 0.1 to 105 ppm. The assay was evaluated with respect to linear range and reaction efficiency. High reaction efficiencies were generally obtained across 6-7 orders of magnitude. Limits of detection were between 0.1 and 1 ppm, and reaction efficiencies were mostly within the preferred range of 100 ± 10%. Our results indicate that real-time PCR assays using chloroplast markers can be a valuable tool for peanut detection.

Application of Multiantigen Profiling To Detect Pecan.

A problem often encountered in the detection and identification of undeclared tree nut food allergens is the lack of analytical methods. This problem is accentuated by the current trend, whereby the primary methods used to detect food allergens are antibody-based enzyme-linked immunosorbent assays (ELISAs) and the development of analyte-specific antibodies takes months. The recently developed xMAP food allergen detection assay (xMAP FADA) has the ability to generate multiantigen profiles with tree nuts, thereby providing a potential solution to this problem. The xMAP FADA includes 22 antibodies targeting peanut, soy, and nine tree nuts. The high number of antibodies to a diverse group of tree nuts and legumes and the propensity of tree nuts to cross-react have enabled the development of multiantigen profiling, whereby an analyte reacts with the various antibodies to generate a profile. Recently, a question arose regarding the possible presence of pecan dust at a manufacturer of pecan products that also stored fresh produce. The lack of suitable pecan ELISAs created an analytical challenge that was resolved using multiantigen profiling with the xMAP FADA. Pecan was detected on swab samples by using multiantigen profiling and confirmed by DNA analysis. The use of multiantigen profiling provided an analytical capability beyond what was possible with an analyte-specific analytical method.

A group-specific, quantitative real-time PCR assay for detection of crab, a crustacean shellfish allergen, in complex food matrices.

A real-time PCR assay was developed for detection of crab, a crustacean allergen, in food products. Group-specific primers and probes were developed to detect numerous species of crab. Method validation included tests of detection in complex food matrices, evaluation of commercial food products, and cross-reactivity testing on a wide variety of crustaceans. The method was able to detect several species of crab spiked into complex food matrices at levels ranging from 0.1 to 105 parts per million (weight/weight), worked equally well on different platforms, exhibited high specificity for crab over other types of crustaceans, and yielded much higher signals from commercial food products listing crab as an ingredient than from those containing other crustaceans.

Quantitative multiplex real-time PCR assay for shrimp allergen: comparison of commercial master mixes and PCR platforms in rapid cycling.

Real-time PCR has been used widely in numerous fields. In food safety, it has been applied to detection of microbes and other contaminants, including food allergens. Interest in rapid (fast) cycling real-time PCR has grown because it yields results in less time than does conventional cycling. However, fast cycling can adversely affect assay performance. Here we report on tests of commercial master mixes specifically designed for fast real-time PCR using a shrimp allergen assay we previously developed and validated. The objective of this work was to determine whether specialized commercial master mixes lead to improved assay performance in rapid cycling. Real-time PCR assays were carried out using four different master mixes and two different rapid cycling protocols. Results indicated that specialized master mixes did yield quality results. In many cases, linear ranges spanned up to 7 orders of magnitude, R(2) values were at least 0.95, and reaction efficiencies were within or near the optimal range of 90 to 110%. In the faster of the two rapid cycling protocols tested, assay performance and PCR amplification were markedly better for the shorter PCR product. In conclusion, specialized commercial master mixes were effective as part of rapid cycling protocols, but conventional cycling as used in our previous work is more reliable for the shrimp assay tested.

Two quantitative real-time PCR assays for the detection of penaeid shrimp and blue crab, crustacean shellfish allergens.

Food allergen detection methods must be able to specifically detect minute quantities of an allergenic food in a complex food matrix. One technique that can be used is real-time PCR. For the work described here, real-time PCR assays were developed to detect penaeid shrimp and blue crab, crustacean shellfish allergens. The method was tested using shrimp meat and crab meat spiked into several types of foods, including canned soups, deli foods, meat, seafood, and prepared seafood products. Foods were spiked with either shrimp or crab at levels ranging from 0.1 to 106 parts per million (ppm) and analyzed either raw or cooked by a variety of methods. Real-time PCR data were used to generate linear standard curves, and assays were evaluated with respect to linear range and reaction efficiency. Results indicate that both assays performed well in a variety of food types. High reaction efficiencies were achieved across a linear range of 6-8 orders of magnitude. Limits of detection were generally between 0.1 and 1 ppm. Cooking methods used to simulate thermal processing of foods had little effect on assay performance. This work demonstrates that real-time PCR can be a valuable tool in the detection of crustacean shellfish.

SYTO dyes and EvaGreen outperform SYBR Green in real-time PCR.

BACKGROUND: Real-time PCR can be carried out using either probes or DNA dyes. SYBR Green has been used the most, but it suffers from several drawbacks. Numerous other DNA dyes are commercially available, but with limited structural information. Dye behavior in real time PCR is difficult to predict, so empirical data are needed. In the work described here, a panel of 23 different DNA dyes--including green, orange, and red SYTO dyes, EvaGreen, and SYBR Green--were evaluated with respect to their performance in real time PCR. FINDINGS: Data were analyzed for reaction inhibition, effects on amplicon melting temperature, fluorescent signal strength, and reaction efficiency. This is the first report of reaction efficiency using alternatives to SYBR Green. Results indicated substantial variation in performance even within the SYTO dye family. EvaGreen and the SYTO dyes 13, 16, 80, and 82 performed better than SYBR Green in general, and high reaction efficiencies can be achieved using these dyes. CONCLUSIONS: Empirical data were generated for 23 DNA dyes. This paper confirms and extends previous findings that among commercially available DNA dyes, EvaGreen and certain SYTO dyes are the most desirable alternatives to the commonly used SYBR Green in real-time PCR.

Molecular indications of protein damage in adenoviruses after UV disinfection.

Adenoviruses are resistant to monochromatic, low-pressure (LP) UV disinfection--but have been shown to be susceptible to inactivation by polychromatic, medium-pressure (MP) UV--when assayed using cell culture infectivity. One possible explanation for the difference between UV lamp types is that the additional UV wavelengths emitted by MP UV enable it to cause greater damage to viral proteins than LP UV. The objective of this study was to examine protein damage in adenoviruses treated with LP and MP UV. Results show that MP UV is more effective at damaging viral proteins at high UV doses, though LP UV caused some damage as well. To our knowledge, this study is the first to investigate protein damage in UV-treated adenovirus, and the overview presented here is expected to provide a basis for further, more detailed work.

UV disinfection of adenoviruses: molecular indications of DNA damage efficiency.

Adenovirus is a focus of the water treatment community because of its resistance to standard, monochromatic low-pressure (LP) UV irradiation. Recent research has shown that polychromatic, medium-pressure (MP) UV sources are more effective than LP UV for disinfection of adenovirus when viral inactivation is measured using cell culture infectivity assays; however, UV-induced DNA damage may be repaired during cell culture infectivity assays, and this confounds interpretation of these results. Objectives of this work were to study adenoviral response to both LP and MP UV using (i) standard cell culture infectivity assays and (ii) a PCR assay to directly assess damage to the adenoviral genome without introducing the virus into cell culture. LP and MP UV dose response curves were determined for (i) log inactivation of the virus in cell culture and (ii) UV-induced lesions per kilobase of viral DNA as measured by the PCR assay. Results show that LP and MP UV are equally effective at damaging the genome; MP UV is more effective at inactivating adenovirus in cell culture. This work suggests that the higher disinfection efficacy of MP UV cannot be attributed to a difference in DNA damage induction. These results enhance our understanding of the fundamental mechanisms of UV disinfection of viruses-especially double-stranded DNA viruses that infect humans--and improve the ability of the water treatment community to protect public health.

Potentiation of neurite outgrowth and reduction of apoptosis by immunosuppressive agents: implications for neuronal injury and transplantation.

OBJECT: Immunosuppressive agents are believed to play a role in recovery from spinal cord injury, but the underlying mechanisms by which neuronal function is improved by these agents are poorly understood. In this study, the authors evaluate the effect of immunosuppressive medications on neurite outgrowth and cell survival after a pharmacologically induced injury. METHODS: Differentiated human neuroblastoma SH-SY5Y cells were injured using the calcium agonist thapsigargin. After cellular injury, neurite outgrowth in the presence or absence of immunosuppressive agents was measured. Apoptosis was quantified with the aid of a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay. Neurite outgrowth was severely restricted following thapsigargin injury. Outgrowth was potentiated, however, by the addition of concentrations of 1 and 10 mM cyclosporin A in a dose-dependent fashion. Similarly, addition of 10 nM FK506 increased the percentage of neurites in the 20- to 40-micron range. A low dose (1 mM) of dexamethasone did not have a significant effect on neurite outgrowth, but a higher dose (10 mM) increased the percentage of neurites in the 10- to 45-micron range. These agents also lessened the degree of thapsigargin-induced apoptosis. CONCLUSIONS: Immunosuppressive agents such as cyclosporin A, FK506, and dexamethasone can potentiate neurite outgrowth and protect against apoptotic cell death in a human postmitotic neuronal cell line. Such effects may have implications for lessening neuronal injury after neurotrauma, stroke, or neurodegeneration.

Hemoglobin conformation couples erythrocyte S-nitrosothiol content to O2 gradients.

It is proposed that the bond between nitric oxide (NO) and the Hb thiol Cys-beta(93) (SNOHb) is favored when hemoglobin (Hb) is in the relaxed (R, oxygenated) conformation, and that deoxygenation to tense (T) state destabilizes the SNOHb bond, allowing transfer of NO from Hb to form other (vasoactive) S-nitrosothiols (SNOs). However, it has not previously been possible to measure SNOHb without extensive Hb preparation, altering its allostery and SNO distribution. Here, we have validated an assay for SNOHb that uses carbon monoxide (CO) and cuprous chloride (CuCl)-saturated Cys. This assay is specific for SNOs and sensitive to 2-5 pmol. Uniquely, it measures the total SNO content of unmodified erythrocytes (RBCs) (SNO(RBC)), preserving Hb allostery. In room air, the ratio of SNO(RBC) to Hb in intact RBCs is stable over time, but there is a logarithmic loss of SNO(RBC) with oxyHb desaturation (slope, 0.043). This decay is accelerated by extraerythrocytic thiol (slope, 0.089; P < 0.001). SNO(RBC) stability is uncoupled from O(2) tension when Hb is locked in the R state by CO pretreatment. Also, SNO(RBC) is increased approximately 20-fold in human septic shock (P = 0.002) and the O(2)-dependent vasoactivity of RBCs is affected profoundly by SNO content in a murine lung bioassay. These data demonstrate that SNO content and O(2) saturation are tightly coupled in intact RBCs and that this coupling is likely to be of pathophysiological significance.

Effect of hyperoxia on interleukin-8 expression in premature versus term rabbit lung explants.

To examine whether prematurity significantly changes the lung inflammatory response to oxygen, rabbit lung explant cultures were exposed to 95% or 5% oxygen for 24 hours. Interleukin (IL)-8 protein concentrations from homogenates of the premature lung rose significantly after hyperoxia (6.8 +/- 1.8 in 5% O2 to 45.7 +/- 21.3 pg/microg protein in 95% O2) but not in the term lung (15.9 +/- 6.7 to 20.4 +/- 4.3 pg/microg protein). There was no change in IL-8 mRNA after hyperoxia in either age group. Preterm lungs demonstrated higher IL-8 levels by fluorescence-activated cell sorting (FACs) analysis and immunohistochemistry. This model may help determine why premature lungs are more susceptible to oxygen-induced disease.