Guidelines for sample preparation procedures in GMO analysis

This document aims at providing guidelines for a correct sample preparation in GMO analysis on food, feed, seed, plants and propagating material samples. It has been developed by the European Network of GMO Laboratories (ENGL), specifically by the ENGL working group “sample preparation procedures” upon a mandate of the ENGL steering committee. The structure of the document is based on that of an existing ISO standard (ISO 6498:2012), dealing with sample preparation for feed; starting from this, the procedures were adapted to the needs of GMO detection and the scope concerning matrices was widen to food, feed and seeds. These guidelines cover the key steps of the sample preparation, i.e. the size of the laboratory sample, the mass reduction techniques as well as techniques for comminution and mixing; considerations about the test portion are included and some performance tests to be applied at different steps of the process are provided. While sample preparation, falling after sampling and before analytical testing for GMOs, has suffered from a lack of guidance and harmonization, this document provides detailed and practical hints on this important step, thus contributing to foster testing harmonisation.JRC.I.3-Molecular Biology and Genomic

La composición de los ácidos grasos en los productos cárnicos crudocuroados tradicionales de Croacia y Eslovenia

Cilj ovog rada bio je istražiti sastav masnih kiselina tradicionalnih suhomesnatih proizvoda iz Hrvatske i Slovenije. Uz analize osnovnih kemijskih parametara šest vrsta tradicionalnih suhomesnatih proizvoda (tri iz Hrvatske i tri iz Slovenije), analiziran je i uspoređen sastav masnih kiselina s obzirom na skupine zasićenih (SFA), mononezasićenih (MUFA) i polinezasićenih (PUFA) masnih kiselina, kao i omjeri n-6/n-3 te PUFA/SFA. Oleinska kiselina (C18:1n9c) se općenito pokazala kao najzastupljenija masna kiselina u rasponu od 36,23±0,19% u Kraškom zašinku do 47,44±0,36% u istarskoj kobasici i sličnoj vrijednosti od 47,41±0,24% u Zgornjesavinjskom želodecu. Palmitinska kiselina (C16:0) bila je najzastupljenija SFA određena u rasponu od 23,68±0,16% do 27,50±0,17%, a druga po zastupljenosti bila je stearinska (C18:0), u rasponu od 12,21±0,21% do 18,61±0,15%. Po skupinama, MUFA se pokazala kao glavni sastavni dio hrvatskih proizvoda istarske kobasice (50,07%) i Dalmatinskog pršuta (49,92%) te slovenskih proizvoda Zgornjesavinjskog želodeca (51,97%) i Kraškog pršuta (48,03%), dok su SFA bile najznačajnije u buđole (49,40%) i Kraškog zašinka (45,86%). Dobivene vrijednosti omjera n-6/n-3 bile su u rasponu od 7,52 do 25,30 te za PUFA/SFA od 0,19 do 0,31, u skladu sa ranije objavljenim podacima za druge europske tradicionalne suhomesnate proizvode, za koje su također određene više vrijednosti omjera u odnosu na preporuke međunarodnih zdravstvenih organizacija. Uzimajući u obzir preporuke za spomenute nutritivne omjere, tradicionalni suhomesnati proizvodi iz Hrvatske i Slovenije ne bi se nalazili unutar poželjnih vrijednosti, no istodobno je važno imati na umu činjenicu da se ovi proizvodi proizvede i konzumiraju kao specijaliteti i samim time uobičajeno u umjerenim količinama.The aim of this study was to investigate the fatty acids composition of traditional dry-cured meat products from Croatia and Slovenia. With analyses of basic chemical properties in six types of representative traditional meat products (three from Croatia and three from Slovenia) fatty acids composition were determined and compared according to groups of saturated (SFA), monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids, as also according to ratios n-6/n-3 and PUFA/SFA. Oleic acid (C18:1n9c) was found generally in the highest amounts, ranging from 36.23±0.19% in “Kraški zašink” to 47.44±0.36% in Istrian sausage and similar value of 47.41±0.24% in “Zgornjesavinjski želodec”. Palmitic acid (C16:0) was the major SFA found from 23.68±0.16% to 27.50±0.17%, and then stearic acid (C18:0) ranged from 12.21±0.21% to 18.61±0.15%. According to groups, MUFA were the major constituents of Croatian product Istrian sausage (50.07%) and Dalmatinski pršut (49.92%) as also of the Slovenian product Zgornjesavinjski želodec (51.97%) and Kraški pršut (48.03%) whereas SFA were the major constituents of Buđola (49.40%) and Kraški zašink (45.86%). Obtained values of n-6/n-3 ranged from 7.52 to 25.30 and of PUFA/SFA from 0.19 to 0.31, were in agreement with earlier published data for other European traditional dry-cured meat products that also shown the higher ratios than suggested by international health organizations. Taking into account recommendations for these nutritional ratios, traditional dry-cured meat products from Croatia and Slovenia would not be within the desirable limits, but at the same time it is important to take into account the fact that these products are produced and consumed as specialties and hence usually in moderate quantities.Ziel dieser Arbeit war es, die Zusammensetzung der Fettsäuren in traditionellen Rohwurstprodukten aus Kroatien und Slowenien zu untersuchen. Neben der Auswertung der wichtigsten chemischen Parameter in sechs Sorten traditioneller Rohwurstprodukte (drei aus Kroatien und drei aus Slowenien), wurde auch die Zusammensetzung der Fettsäuren in Bezug auf die Gruppen der gesättigten Fettsäu- ren (SFA), einfach gesättigten Fettsäuren (MUFA) und der mehrfach ungesättigten Fettsäuren (PUFA) ausgewertet und verglichen sowie das Verhältnis zwischen n-6/n-3 und PUFA/SFA untersucht. Es zeigte sich, dass die Ölsäure (C18:1n9c) im Allgemeinen die am meisten vertretene Fettsäure ist, deren Werte sich in einem Bereich von 36,23±0,19% im Kraški zašinek (luftgetrockneter Schweinenacken) bis 47,44±0,36% in der istrischen Wurst und 47,41±0,24% im Zgornjesavinjski želodec bewegen. Die Palmitinsäure (C16:0) war die am meisten vertretene gesättigte Fettsäure mit Werten von 23,68±0,16% bis 27,50±0,17%, gefolgt von der Stearinsäure (C18:0), mit Werten von 12,21±0,21% bis 18,61±0,15%. Nach Gruppen betrachtet zeigten sich die einfach gesättigten Fettsäuren als der wichtigste Bestandteil der kroatischen istrischen Wurst (50,07%) und des dalmatinischen Schinkens (49,92%) sowie der slowenischen Produkte Zgornjesavinjski želodec (51,97%) und Kraški pršut (48,03%), während die gesättigten Fettsäuren am meisten in der Budjola (49,40%) und im Kraški zašink (45,86%) vertreten sind. Beim Verhältnis von n-6/n-3 ergaben sich Werte von 7,52 bis 25,30 und beim Verhältnis PUFA/SFA von 0,19 bis 0,31, was den bereits verö&entlichten Angaben für andere traditionelle europäische Rohwurstprodukte entspricht, bei denen auch höhere Werte im Vergleich zu den Empfehlungen internationaler Gesundheitsorganisationen festgestellt wurden. Berücksichtigt man die Empfehlungen für die genannten nutritiven Verhältnisse, würden die Werte traditioneller Rohwurstprodukte aus Kroatien und Slowenien nicht zu den wünschenswerten Werte zählen; zugleich darf man aber nicht außer Acht lassen, dass diese Produkte als Spezialitäten hergestellt und verzehrt werden, somit gewöhnlich auch in gemäßigten Mengen.In questo studio è stata analizzata la composizione degli acidi grassi nei salumi insaccati tradizionali della Croazia e della Slovenia. Oltre ad aver analizzato i fondamentali parametri chimici di sei tipi di salume tradizionale (tre della Croazia e tre della Slovenia), è stata anche analizzata ed equiparata la composizione degli acidi grassi rispetto ai gruppi degli acidi grassi saturi (SFA), monoinsaturi (MUFA) e polinsaturi (PUFA) anche rispetto al rapporto n-6/n-3 e al rapporto PUFA/SFA. L’acido oleico (C18:1n9c) è risultato, in generale, l’acido grasso più rappresentato secondo un range compreso tra il 36,23±0,19% del Kraški zašinek e il 47,44±0,36% della salsiccia istriana (valori simili - 47,41±0,24% - sono stati riscontrati anche in ordine allo Zgornjesavinjski želodec). L’acido palmitico (C16:0) s’è rivelato l’acido grasso saturo (SFA) più rappresentato secondo un range compreso tra il 23,68±0,16% e il 27,50±0,17%, seguito dall’acido stearico (C18:0) secondo un range compreso tra il 12,21±0,21% e il 18,61±0,15%. Considerati per gruppo, gli acidi grassi monoinsaturi (MUFA) si sono dimostrati l’elemento principale di parte dei due salumi croati salsiccia istriana (50,07%) e prosciutto crudo di Dalmazia (49,92%) e dei salumi sloveni Zgornjesavinjski želodec (51,97%) e Kraški pršut (48,03%), mentre gli acidi grassi saturi (SFA) sono stati riscontrati in prevalenza nella buđola (49,40%) e nel Kraški zašinek (45,86%). Circa il rapporto n-6/n-3, sono stati ottenuti valori compresi tra il 7,52 e il 25,30, mentre, per quanto riguarda il rapporto PUFA/SFA, sono stati ottenuti valori compresi tra lo 0,19 e lo 0,31, in sintonia con i dati precedentemente pubblicati e riferiti ad altri salumi tradizionali europei, anch’essi con valori superiori rispetto alle raccomandazioni delle organizzazioni sanitarie internazionali. Prendendo in considerazione le raccomandazioni circa i summenzionati rapporti nutritivi, i salumi tradizionali della Croazia e della Slovenia non possono essere annoverati tra i prodotti nella norma (i cui valori corrispondono a quelli desiderati). Tuttavia, è importante anche aver presente il fatto che i prodotti in oggetto si producono e consumano come specialità tipiche regionali e quindi, di norma, in piccole quantità.El objetivo de la investigación fue examinar la composición de los ácidos grasos en los productos cárnicos crudo-curados tradicionales de Croacia y Eslovenia. Junto con el análisis de los parámetros básicos de seis tipos de productos cárnicos crudo-curados tradicionales (tres de Croacia y tres de Eslovenia), también fueron hechos el análisis y la comparación de la composición de los ácidos grasos teniendo en cuenta los ácidos grasos saturados (SFA), ácidos grasos monoinsaturados (MUFA), los ácidos grasos poliinsaturados (PUFA) y las proporciones n-6/n-3 PUFA/SFA. El ácido oleico (C18:1n9c) se mostró como el ácido graso generalmente más común comprendido de 36,23±0,19% en zašink de Karst a 47,44±0,36% en la salchicha de Istria, con valores similares de 47,41±0,24% en Zgornjesavinjski želodec. El ácido palmítico (C16:0) fue el SFA más común entre 23,68±0,16% y 27,50±0,17%,, seguido por el ácido esteárico (C18:0) comprendido de 12,21±0,21% a 18,61±0,15%. Entre grupos, el MUFA se mostro como el componente principal de los productos croatas como la salchicha de Istria (50,07%) y el jamón de Dalmacia (49,92%), pero también de los productos eslovenos Zgornjesavinjski želodec (51,97%) y el jamón de Karst (48,03%), mientras los SFA eran los más importantes en buđola (tipo de embutido) (49,40%) y en zašink de Karst (45,86%). Los valores obtenidos en proporciones n-6/n-3 estaban comprendidas entre 7,52 y 25,30 y 0,19 a 0,31 para los PUFA/SFA, de acuerdo con los datos publicados anteriormente para otros productos cárnicos crudo-curados tradicionales europeos, para los cuales fueron determinados los valores de las proporciones en vista de las recomendaciones de las organizaciones internacionales de salud. Tomando en cuenta las recomendaciones para las proporciones nutritivas mencionada, los productos cárnicos crudo-curados tradicionales de Croacia y Eslovenia no están entre las valores deseables, pero hay que recordar que estos productos están producidos y se consumen con la intencion de ser especialidades y es común consumirlos en cantidades moderadas

Tkivna kultura bolhača (Tanacetum cinerariifolium) in z njo povezana okužba z mikroorganizmi

Microbial contamination was observed on several subcultures of Pyrethrum (Tanacetum cinerariifolium) (Trevir.) Schultz-bip. callus lines. The presence of microorganisms was detected by isolation of contaminants in pure culture from 7 out of 34 callus lines and direct amplification of eubacterial 16S rDNA in the pyrethrum callus and plants and isolated bacteria. Altogether 16 contaminants were further analyzed, observing their morphology on several media and restriction of amplified 16S rDNA. Analysis revealed presence and persistence of morphologically and genetically diverse bacteria in pyrethrum tissue culture. Due to cross reactivity of 16S rDNA primers with DNA of plant origin, no conclusions could be drawn on the origin of contaminants.Po vzpostavitvi kalusnih linij bolhača (Tanacetum cinerariifolium) (Trevir.) Schultz-bip. smo v kulturah opazili okužbo z mikroorganizmi. Z različnimi izolacijskimi postopki in gojitvenimi metodami smo iz 7 od 34 kalusnih linij izolirali posamezne seve mikroorganizmov. Nobeden od izolatov ni bil izoliran iz več kot dveh kalusnih linij. S pomnoževanjem eubakterijske 16S rDNA in nadaljnjo restrikcijsko analizo z encimom Mn/I smo analizirali kalusno tkivo, rastline bolhača in izolirane bakterije. Analiza je pokazala morfološko in genetsko raznolikost bakterij prisotnih v tkivni kulturi bolhača. Zaradi navzkrižne reaktivnosti uporabljenih 16S rDNA oligonukleotidnih začetnikov, na prisotnost podobnih bakterijskih sevov v kalusnem tkivu in steblih bolhača, ni bilo mogoče sklepati na izvor okužb

Robust saliva-based RNA extraction-free one-step nucleic acid amplification test for mass SARS-CoV-2 monitoring

Early diagnosis with rapid detection of the virus plays a key role in preventing the spread of infection and in treating patients effectively. In order to address the need for a straightforward detection of SARS-CoV-2 infection and assessment of viral spread, we developed rapid, sensitive, extraction-free one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) tests for detecting SARS-CoV-2 in saliva. We analyzed over 700 matched pairs of saliva and nasopharyngeal swab (NSB) specimens from asymptomatic and symptomatic individuals. Saliva, as either an oral cavity swab or passive drool, was collected in an RNA stabilization buffer. The stabilized saliva specimens were heat-treated and directly analyzed without RNA extraction. The diagnostic sensitivity of saliva-based RT-qPCR was at least 95% in individuals with subclinical infection and outperformed RT-LAMP, which had at least 70% sensitivity when compared to NSBs analyzed with a clinical RT-qPCR test. The diagnostic sensitivity for passive drool saliva was higher than that of oral cavity swab specimens (95% and 87%, respectively). A rapid, sensitive one-step extraction-free RT-qPCR test for detecting SARS-CoV-2 in passive drool saliva is operationally simple and can be easily implemented using existing testing sites, thus allowing high-throughput, rapid, and repeated testing of large populations. Furthermore, saliva testing is adequate to detect individuals in an asymptomatic screening program and can help improve voluntary screening compliance for those individuals averse to various forms of nasal collections