Authentication of traditional meat products (Haleem) sold in India: a first report confirming species mislabelling.

Traditional meat products like Haleem play a pivotal role in the culinary landscapes of Indian consumers, along with high economic value and business potential. Due to anticipated gains associated with adulterating 'Haleem' and constant evasion from regulatory oversight, the susceptibility to adulteration has substantially increased. Furthermore, no reports/surveillance regarding their labelling compliance has been reported. Hence, we conducted a 2-year surveillance using 100 samples collected from Hyderabad, India, using the Chipron™ DNA macroarray analysis technique. The method was validated for sensitivity (1%), specificity, and with proficiency test samples. Following this, the surveillance samples (beef, chicken, and mutton Haleem) were tested. The surveillance revealed an alarming adulteration of 46% of the samples, with different proportions of adulterant species. Adulteration of unconventional meat like camel meat was also found. These concerning results necessitate the requirement of stricter and constant regulatory surveillance to safeguard consumer trust and preserve the authenticity of traditional meat products. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-024-05947-9.

SARS-CoV-2 in digital era: Diagnostic techniques and importance of nucleic acid quantification with digital PCRs.

Studies related to clinical diagnosis and research of SARS-CoV-2 are important in the current pandemic era. Although molecular biology has emphasised the importance of qualitative analysis, quantitative analysis with nucleic acids in relation to SARS-CoV-2 needs to be clearly emphasised, which can provide perspective for viral dynamic studies of SARS-CoV-2. In this regard, the requirement and utilization of digital PCR in COVID-19 research has substantially increased during the pandemic, necessitating the aggregation of its cardinal applications and future scopes. Hence, this meta-review comprehensively addresses and emphasises the importance of nucleic acid quantification of SARS-CoV-2 RNA with digital PCR (dPCR). Various quantitative techniques of clinical significance like immunological, proteomic and nucleic acid-based diagnosis and quantification, have been comparatively discussed. Furthermore, the core part of the article focusses on the working principle and advantages of digital PCR, along with its applications in COVID-19 research. Several important applications like viral load quantitation, environmental surveillance and assay validation have been extensively investigated and discussed. Certain key future scopes of clinical importance, like mortality prediction, viral/variant-symbiosis, and antiviral studies were also identified, suggesting several possible digital PCR applications in COVID-19 research.

Duplex real-time PCR assay with high-resolution melt analysis for the detection and quantification of Listeria species and Listeria monocytogenes in meat products.

Listeria contamination in foods of animal origin is one of the most concerning food safety issues. A duplex, SYBR green-based, real-time PCR assay was developed with high-resolution melting analysis-based differentiation of the genus Listeria and Listeria monocytogenes. The primers were designed and tested against other related foodborne pathogens. The assay was optimized for standard parameters in a non-orthogonal fashion and validated following international standards. The LODabs and LOQ of the assay were calculated to be 0.78 and 1.56 ng of the target DNA. The LODrel of the assay was found to be 1% Listeria DNA in background DNA. The assay was evaluated for applicability in artificially spiked samples, providing a 120 CFU/ml detection. The assay was validated with proficiency test samples and also with samples collected for surveillance analysis. This well-established and validated assay can be utilized as a qualitative and quantitative tool for addressing the Listeria contamination in the food safety contexts. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05695-2.

Development and comparative evaluation of droplet digital PCR and quantitative PCR for the detection and quantification of Chlamydia psittaci.

Chlamydia psittaci is a zoonotic pathogen mainly transmitted by psittacine birds and poultry. The low shedding rate of the pathogen in the apparently healthy birds and human clinical cases may result in false-negative results. In the present study, a droplet digital PCR (ddPCR) assay was developed and compared with optimized quantitative PCR (qPCR) for the detection of C. psittaci from the clinical samples. The ddPCR assay was found to be comparatively more sensitive than the qPCR, wherein the limit of detection (LOD) of ddPCR was upto 2.4 copies of the DNA template, whereas, the qPCR could detect upto 38 copies of the DNA template in the reaction mixture. Overall, the developed ddPCR assay was found to be robust, specific, and could reliably quantify up to 17.8 copies of the DNA template. Finally, the applicability of the developed ddPCR assay was tested by screening the field samples (n = 124), comprising lung tissues from dead poultry and feral birds; pooled faecal samples from the free-living birds, commercial and backyard poultry farms; pharyngeal and cloacal swabs collected from the duck farms. Of these, a total of seven samples were found to be positive by the ddPCR, whereas, three samples could be detected as positive using the qPCR. The developed ddPCR could serve as a reliable screening tool, particularly in those clinical samples wherein the shedding of C. psittaci is substantially very low.

Authentication of camel meat using species-specific PCR and PCR-RFLP.

In India and some of the African countries, one of the unconventional meats receiving the latest attention in meat adulteration is camel meat. So, the objective of this study was to develop a species-specific PCR based on mitochondrial cytochrome b (CYTB) gene and a PCR-RFLP assay of mitochondrial 12S rRNA to identify camel meat in suspected samples. Known sample of camel meat, samples suspected to be from illegally slaughtered camel and known samples of cattle, buffalo, sheep, goat, pork and chicken were used in the study. DNA were extracted from all samples following spin column method and PCR amplification were carried out using both CYTB and 12S rRNA gene primers. The CYTB gene amplification produced amplicon with a size of 435 bp without any non-specific spurious amplification towards other species studied. Further, the 12S rRNA PCR products were analysed both by sequencing and by RFLP using enzyme AluI. On BLAST analysis the 448 bp sequence obtained from suspected samples showed > 99% sequence homology to previously reported Camelus dromedaries (accession no: AM 9369251.1). On AluI digestion of the 448 bp product from both known and suspected camel samples, a specific RFLP pattern with three distinct products of 90, 148 and 210 bp size were evident, which were significantly different from the pattern of cattle, sheep, goat, chicken and buffalo. Further, after in-house validation, this cost effective and rapid method of camel meat identification is placed into practice for regular screening of vetero-legal samples in the lab.

Development of on package indicator sensor for real-time monitoring of meat quality.

AIM: The aim was to develop an indicator sensor for real-time monitoring of meat quality and to compare the response of indicator sensor with meat quality parameters at ambient temperature. MATERIALS AND METHODS: Indicator sensor was prepared using bromophenol blue (1% w/v) as indicator solution and filter paper as indicator carrier. Indicator sensor was fabricated by coating indicator solution onto carrier by centrifugation. To observe the response of indicator sensor buffalo meat was packed in polystyrene foam trays covered with PVC film and indicator sensor was attached to the inner side of packaging film. The pattern of color change in indicator sensor was monitored and compared with meat quality parameters viz. total volatile basic nitrogen, D-glucose, standard plate count and tyrosine value to correlate ability of indicator sensor for its suitability to predict the meat quality and storage life. RESULTS: The indicator sensor changed its color from yellow to blue starting from margins during the storage period of 24 h at ambient temperature and this correlated well with changes in meat quality parameters. CONCLUSIONS: The indicator sensor can be used for real-time monitoring of meat quality as the color of indicator sensor changed from yellow to blue starting from margins when meat deteriorates with advancement of the storage period. Thus by observing the color of indicator sensor quality of meat and shelf life can be predicted.