Duplex droplet digital PCR (ddPCR) for simultaneous quantification of bovine and porcine gelatin in capsules.

Detection of bovine and porcine in gelatin-based products is important as species fraud and product mislabeling may have a detrimental impact on customers who have health, ethical, and religious concerns about animal products. The duplex droplet digital PCR (ddPCR) assay using double-quenched probes has been developed for quantification and detection of porcine and bovine DNA in gelatin capsules. A DNA mixture derived from gelatin was found to have a limit of detection as low as 0.001 ng/µl for porcine samples and 0.01 ng/µl for bovine samples. DNA from 12 other distinct species was tested with the bovine and porcine probes, showing high specificity for this method. The test was validated using fifty-five commercial supplement and pharmaceutical capsules, of which 17 were positive for bovine and/or porcine DNA. This study shows that the duplex ddPCR is reliable for routine analysis in the identification of bovine and porcine origins for gelatin capsules. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01204-x.

A Simple Distance Paper-Based Analytical Device for the Screening of Lead in Food Matrices.

A simple and rapid distance paper-based analytical device (dPAD) for the detection of lead (Pb) in foods is proposed herein. The assay principle is based on competitive binding between carminic acid (CA) and polyethyleneimine (PEI) to Pb in a food sample. The paper channels were pre-immobilized with PEI, before reacting with a mixture of the sample and CA. Pb can strongly bind to the CA; hence, the length of the red color deposition on the flow channel decreased as a lower amount of free CA bound to PEI. The dPAD exhibited good linear correlation, with ranges of 5-100 µg·mL-1 (R2 = 0.974) of Pb. Although, the limit of detection (LOD) of this platform was rather high, at 12.3 µg·mL-1, a series of standard additions (8.0, 9.0, and 10.0 µg·mL-1) can be used to interpret the cutoff of Pb concentrations at higher or lower than 2 µg·mL-1. The presence of common metal ions such as calcium, magnesium, nickel, and zinc did not interfere with the color distance readout. The validity of the developed dPAD was demonstrated by its applicability to screen the contamination of Pb in century egg samples. The results obtained from the dPAD are in accordance with the concentration measured by atomic absorption spectroscopy (AAS) (n = 9). In conclusion, this proposed dPAD, combined with the standard addition method, could be applied for screening Pb contamination in food matrices. This platform is, therefore, potentially applicable for field measurements of Pb in developing countries, because it is cheap and rapid, and it requires no significant laborious instruments.

The Alterations of Erythrocyte Phospholipids in Type 2 Diabetes Observed after Oral High-Fat Meal Loading: The FTIR Spectroscopic and Mass Spectrometric Studies.

Little is known about the postprandial remodelling of erythrocytes phospholipids (PLs) in type 2 diabetics (T2DM). Therefore, this study aims to compare the alterations of erythrocyte PLs in T2DM to those of healthy subjects after ingestion of a high-fat meal. Eleven T2DM and ten healthy subjects underwent a high-fat meal loading. Erythrocytes were isolated from blood obtained after fasting and 4 h after the meal. Fourier Transform Infrared (FTIR) spectroscopy was initially used to screen erythrocyte PLs by monitoring C-H stretching vibrations. Phosphatidylcholine (PC) molecular species were further investigated by Liquid Chromatography-Electrospray Ionisation-Mass Spectrometry (LC-ESI-MS). For the control group, FTIR revealed postprandial changes in C-H stretching vibrations, particularly of the olefinic band. These findings were supported by LC-ESI-MS data, showing marked changes in PC molecular species, especially of the PC34:1 (where 34 and 1 mean the summed number of carbons and double bonds, respectively). However, similar changes of those were not apparent in the T2DM group. Our results reveal marked postprandial alterations of erythrocyte PC species in healthy subjects whereas only mild alterations are observed in T2DM. The discrepant effects of high-fat meal loading suggest abnormal PC remodelling in the diabetic erythrocyte that may affect its membrane fluidity and integrity.

Alterations of atherogenic low-density lipoproteins and serum fatty acids after 12 week moderate exercise training in sedentary Thai women.

The potential benefit of aerobic exercise upon cardiovascular disease (CVD) through an increasing high-density lipoproteins (HDLs) is acknowledged. However, its effects on low-density lipoproteins (LDLs) and their subpopulations, are unknown in Thailand. Twenty sedentary Thai women undertook a 12-week exercise training program (60% heart rate reserve) comprising 25-minute cycling followed by 10-minute warm-up/cool-down 3 times a week with a group of 20 matched sedentary subjects as control. Triacylglycerols (TGs) and cholesterol (C) of plasma lipoproteins including triacylglycerol-rich lipoproteins (TRLs), large, buoyant LDL (lb-LDL), small, dense LDL (sd-LDL) and HDLs were analyzed while serum fatty acid profiles were also assessed. It was found that plasma TGs, TRL-TGs, sd-LDL-C and sd-LDL-C/lb-LDL-C (S/L) ratio decreased significantly after 12-weeks of exercise to -9%, -8%, -17% and -19% respectively from baseline (p < 0.05). Serum fatty acid profiles remained unchanged. No alteration of any parameters was found in the control group without exercise. These findings suggest that moderate exercise training, even without a change of HDLs, impedes the shift of lb-LDL to more atherogenic sd-LDL, thus possibly preventing cardiovascular disease in healthy, sedentary Thai women.

Design of degenerate primers for multiplex nested-PCR detection of human lymphotropic herpesviruses.

To develop the rapid diagnosis and typing of human lymphotropic herpesviruses by using multiplex nested-PCR, the primary PCR (1(o) PCR) primers were redesigned as degenerate primers based on a highly conserved sequences of each DNA polymerase gene of EBV, CMV, HHV-6, HHV-7 and HHV-8. The forward degenerate primer (HHV/1+) contained 12 different sequences, whereas there were 8 different sequences in the reverse degenerate primer (HHV/ 1-). Optimization of multiplex nested-PCR assay conditions were performed to search for the appropriate amount of degenerate primers, dNTP, Taq DNA polymerase, template of secondary PCR (2(o) PCR) and annealing temperature used in 1(o) PCR reaction. Detection sensitivity was the same as described in previous report (approximately 10-100 genome copies). To ensure a true negative result, PCR detection of hepatitis B virus genome was used as internal control. Our presented results, the designed degenerate primers could be used to detect various types of HHV by multiplex nested-PCR.

Human herpesvirus infection in children with fever and maculopapular rash.

Fever with maculopapular rash is a common problem in children. Infection with human herpesviruses is one of the common etiologies in fever with rash. The aim of this study has been to examine patients presenting with fever and maculopapular rash without respiratory symptoms for human herpesviruses infection by using multiplex nested-polymerase chain reaction. A descriptive and prospective study was conducted at King Chulalongkorn Memorial Hospital, Bangkok, Thailand from June 2000 to December 2001. One hundred patients, 43 boys and 57 girls, aged between 2 months and 14 years were recruited. Human herpesvirus 6 (HHV6) was the most common (24%) whereas HHV7, Epstein-Barr virus (EBV) and cytomegalovirus (CMV) were present in 9%, 3% and 2% of the patients, respectively. Four percent of the patients simultaneously harbored HHV6 and HHV7. Only one patient had CMV, HHV6 and HHV7. Patients with HHV7 had a mean age of 4.5 years, whereas those with HHV6 had a mean age of 1.6 years. HHV6 and HHV7 were commonly found as causes of fever and maculopapular rash without respiratory symptoms. Co-infection with different herpesviruses can be found in the same patient.