Liquid Saliva Research Articles

Revealing microbial community characteristics in healthy human, cat and canine salivas and looking for species-specific microbes.

As two kinds of increasingly popular pets, the saliva of cat or canine is most likely to be left at the crime scene compared with the common types of body fluids in forensics. Accurately identifying the species of saliva samples found at the crime scene involving pets will help the investigators find available testing materials, reduce the consumption of reagents and save the investigative time of the case. Therefore, it is necessary to explore the characteristics and differences of saliva microbiomes of cat, canine and human. In this study, 16S rRNA gene amplicon sequencing technology was used to reveal microbial communities of saliva samples of healthy human, cat, and canine. Alpha diversity analyses indicated that canine saliva demonstrated the highest microbial diversity, followed by cat saliva, whereas human saliva microbial diversity was the lowest. The saliva samples of the three species all had their own unique microbial community compositions, and the dominant phyla of canine and cat salivas were Proteobacteria and Bacteroidete, while the dominant phyla of human saliva were Firmicutes and Proteobacteria. There was no significant statistical difference in the salivary microbiota obtained by the two collection methods (cotton swab and liquid saliva). The gender of cats and canines might have no effect on the salivary microbiota, but the different breeds had an impact on their saliva microbiomes. Principal coordinates analysis, non-metric multidimensional scaling analysis and random forest analysis all indicated significant differences in microbial community structures among the three species, allowing inference on the species sources of saliva samples by microbiome method. Differential microbial biomarkers for the salivas of three species were screened out using a variety of bioinformatics analyses, and the results demonstrated that Prevotella melaninogenica, Veillonella parvula, and Haemophilus parainfluenzae could be used as species-specific microbial biomarkers of human saliva. The detections of human species-specific microbes provide a potential method for determining human saliva.

Liquid saliva-based Raman spectroscopy device with on-board machine learning detects COVID-19 infection in real-time.

With greater population density, the likelihood of viral outbreaks achieving pandemic status is increasing. However, current viral screening techniques use specific reagents, and as viruses mutate, test accuracy decreases. Here, we present the first real-time, reagent-free, portable analysis platform for viral detection in liquid saliva, using COVID-19 as a proof-of-concept. We show that vibrational molecular spectroscopy and machine learning (ML) detect biomolecular changes consistent with the presence of viral infection. Saliva samples were collected from 470 individuals, including 65 that were infected with COVID-19 (28 from hospitalized patients and 37 from a walk-in testing clinic) and 251 that had a negative polymerase chain reaction (PCR) test. A further 154 were collected from healthy volunteers. Saliva measurements were achieved in 6 minutes or less and led to machine learning models predicting COVID-19 infection with sensitivity and specificity reaching 90%, depending on volunteer symptoms and disease severity. Machine learning models were based on linear support vector machines (SVM). This platform could be deployed to manage future pandemics using the same hardware but using a tunable machine learning model that could be rapidly updated as new viral strains emerge.

Addressing the alternate hypothesis: Transfer and persistence of saliva beneath fingernails

This study investigated the transfer and persistence of salivary DNA under fingernails. This was performed to address a common alternate hypothesis presented to scientists in court, asserting that a relatively large quantity of DNA detected beneath the fingernails, typically from a victim of crime, originates from innocuous transfer of saliva in a casual setting.It was determined through these studies that contact with liquid saliva was an effective way to transfer foreign DNA beneath fingernails. However, when saliva was dried, DNA did not readily transfer through casual contact.When liquid saliva was placed directly beneath fingernails the amount of DNA detected from the saliva donor twenty-four hours later was several hundred-fold lower than the amount detected when sampling occurred immediately following deposition. Furthermore, when the recipients’ hands were washed immediately following the deposition of liquid saliva beneath fingernails, the majority of foreign DNA was removed following one hand washing and all detectable foreign DNA was removed from most recipients’ hands after three or six hand washings.This study demonstrates that casual contact with wet saliva can result in the transfer of substantial quantities of DNA beneath fingernails but that it does not typically persist for extended periods of time and is mostly removed if the hands are washed soon after deposition.

Raman spectroscopic characterisation of non stimulated and stimulated human whole saliva

Human saliva is a unique biofluid which can reflect the physiopathological state of an individual. The wide spectrum of molecules present in saliva, compounded by the close association of salivary composition to serum metabolites, can provide valuable information for clinical diagnostic applications through highly sensitive vibrational spectroscopic techniques such as Raman spectroscopy. However, the nature of saliva, in terms of collection and patient-related characteristics, can be considered factors which may strongly affect the Raman spectral profile of salivary samples and disrupt the search for specific salivary biomarkers in the detection of diseases. The main objective of this study was to highlight spectral features associated with the type of collection in an intra- and inter-patient approach. Saliva was collected using both stimulated and non-stimulated approaches from 20 donors, concentrated by centrifugal filtration and further analysed using Raman spectroscopy. The methodology adopted for liquid saliva showed consistency in the qualitative analysis of the groups, confirming the reproducibility of this Raman spectroscopic approach. Using principal component analysis (PCA) and partial least squares – discriminant analysis (PLSDA), non stimulated saliva could be differentiated from stimulated saliva in both intra- and inter-patient analysis, with a classification efficiency of 77 and 87 %, respectively. The bicinchoninic acid (BCA) assay showed a similar trend in terms of total protein concentration, showing a slight increase in stimulated saliva samples. These results are valuable in the process of developing and establishing Raman spectroscopy as a novel diagnostic tool in the future as well as controlling variability, in order to determine specific spectroscopic markers related to a multifactorial disease for diagnostic or follow-up purposes.

Evaluating the use of hypoxia sensitive markers for body fluid stain age prediction

To augment DNA profiling and body fluid identification techniques efforts are being made to increase the amount of information available from a crime scene stain, which includes efforts to identify externally visible characteristics through phenotypic analysis. A key question surrounding crime scene stains is the length of time between deposition of the stain and its subsequent recovery, in that is the stain recovered related to the incident in question or from a previously deposited stain number of weeks earlier? The inability to answer this fundamental question has a detrimental effect upon the successful completion of a criminal investigation. Once a body fluid leaves the body, the oxygen concentration in the environment changes; therefore, it may be that this change could cause a change in the expression of hypoxia-sensitive biomarkers. Here, a range of bloodstains, liquid saliva and liquid semen samples were collected at 0 days, 7 days, 14 days, 21 days and 28 days of degrading at room temperature (19–22 °C), before undergoing total RNA extraction and cDNA synthesis. Blood was recovered from filter paper with 3 mm2, with saliva and semen being left in their tubes and swabbed at the appropriate times. All samples then underwent quantitative PCR targeting Vascular Endothelial Growth Factor A (VEGFA) and Hypoxia-Inducible Factor 1 Alpha (HIF1A), with B-Actin (ACTB) as a reference gene. A range of linear and quadratic correlation values was obtained from the qPCR data and used to develop a predictive model with a mean absolute deviation (MAD) of 4.2, 2.1, and 5 days for blood, saliva, and semen respectively. Blind testing indicated that a stain age prediction model based upon VEGFA with ACTB as a reference gene could be used on samples up to four weeks old with a margin of error ranging from 2 days through to 5 days. While a sizeable potential time frame exists using this model; this represents a significant step towards the target of having an accurate stain age prediction model.

Forensic application of fluorescence spectroscopy: An efficient technique to predict the presence of human saliva

In this study, we aim to use the fluorescence spectroscopic data as a preliminary forensic evidence for the detection of saliva stains from the surface of drinking glass, to see the potential of utilizing a comparatively simple method to trace the presence of saliva from inanimate objects such as cigars, papers, cloths and envelopes. Since dried stains of saliva are invisible to human eyes, detection of saliva from such sources is a challenge in forensic analysis. The fluorescence emission spectra of dried saliva samples collected from drinking glass were compared to that of undiluted liquid saliva of the same volunteers. The deposition of saliva was predicted based on the emission spectra of the enzyme amylase around 350 nm, which is present in high concentrations in saliva. The dried saliva stains and undiluted liquid saliva showed an emission peak at 350 ± 5 nm and 345 ± 5 nm, respectively. The fluorescence emission spectra obtained from the dried saliva samples confirmed well to those of undiluted liquid saliva and amylase.The fluorescence intensity and area underneath the fluorescence peak of undiluted liquid saliva as well as dried saliva were also studied. The study concludes that by correlating fluorescence emission peak, fluorescence intensity and area under the curve, saliva can be detected from dried stains. The potential of fluorescence spectroscopy is also emphasised to detect the presence of saliva from inanimate objects like that of drinking glass, by identifying the emission peak of amylase, one of the prominent saliva components. Thus by using this simple technique, the best possible samples for a detailed DNA analysis may be screened and selected, which could significantly contribute to forensic identification.

Aloe vera for Dry Mouth Denture Patients - Palliative Therapy.

Xerostomia (dry mouth) is dryness of the mouth which is due to reduced salivary flow. Lack of adequate saliva causes discomfort in denture wearing patients and decreases retention of dentures. The ability of saliva to wet the tissue surface is one of the most important properties for oral comfort and retention of complete denture in dry mouth patients. This study was conducted to evaluate and compare the wetting ability of therapeutic Aloe vera saliva substitute and commercially available Aqwet saliva substitute on heat-polymerized acrylic resin. Contact angle of liquid saliva substitute was considered as an indicator of wettability. Aloe vera liquid (Aloe vera - Group I) and Aqwet saliva substitute (Aqwet - Group II) were compared in terms of their wetting ability. Forty samples of heat-polymerized acrylic resin were fabricated and divided into two groups with 20 samples in each. Advancing, receding contact angles and angle of hysteresis were measured using contact angle goniometer and DSA4 software analysis. Mann-Whitney U test was applied for statistical analysis of the study. The mean advancing angle and receding angle of Group I (Aloe vera) was smaller than Group II (Aqwet). Mean angle of hysteresis of Group I (Aloe vera) was higher than Group II (Aqwet). Mann-Whitney U test revealed that there is no significant difference in contact angles between the two groups. Wetting ability of Group I (Aloe vera) saliva substitute was found to be better compared to Group II (Aqwet) on heat-polymerized acrylic resin.

Bioanalysis of dried saliva spot (DSS) samples using detergent-assisted sample extraction with UHPLC-MS/MS detection

Dried saliva spot (DSS) sampling is a non-invasive sample collection technique for bioanalysis that can be potentially implemented at the patient's home. A UHPLC-MS/MS assay was developed using detergent-assisted sample extraction to quantify BMS-927711, a drug candidate in development for the treatment of migraines, in human DSS. By implementing DSS sampling at the patients' home, the bioanalytical sample collection for pharmacokinetic evaluation can be done at the time of the acute migraine attack without the need for clinical visits. DSS samples were prepared by spotting 15 μL of liquid saliva onto regular Whatman FTA™ DMPK-C cards and verified with a UV lamp (at λ 254 nm or 365 nm) during DSS punching. The 4-mm DSS punches in a 96-well plate were sonicated with 200 μL of [13C2, D4]-BMS-927711 internal standard (IS) solution in 20/80 MeOH/water for 10 min, followed by sonication with 50 μL of 100 mM NH4OAc with 1.0% Triton-X-100 (as detergent) prior to liquid-liquid extraction with 600 μL EtOAc/Hexane (90:10). UHPLC-MS/MS was performed with an Aquity® UPLC BEH C18 Column (2.1 × 50 mm, 1.7 μm) on a Triple Quad™ 5500 mass spectrometer. The assay was linear with a concentration range from 2.00 to 1000 ng mL−1 for BMS-927711 in human saliva. The intra- and inter-assay precision was within 8.8% CV, and the accuracy was within ±6.7% Dev of the nominal concentration values. This UHPLC–MS/MS assay has been successfully applied to determine the drug's pharmacokinetics within a clinical study. For the first time, we observed BMS-927711 exposure in human DSS, confirming the suitability of this sampling technique for migraine patients to use at home. Detergent-assisted extraction with Triton-X-100 could be very useful in DSS or other dried matrix spot (DMS) assays to overcome low or inconsistent analyte recovery issues.

Comparative evaluation of different extraction and quantification methods for forensic RNA analysis

Since about 2005, there is increasing interest in forensic RNA analysis whose versatility may very favorably complement traditional DNA profiling in forensic casework. There is, however, no method available specifically dedicated for extraction of RNA from forensically relevant sample material.In this study we compared five commercially available and commonly used RNA extraction kits and methods (mirVana™ miRNA Isolation Kit Ambion; Trizol® Reagent, Invitrogen; NucleoSpin® miRNA Kit Macherey-Nagel; AllPrep DNA/RNA Mini Kit and RNeasy® Mini Kit both Qiagen) to assess their relative effectiveness of yielding RNA of good quality and their compatibility with co-extraction of DNA amenable to STR profiling.We set up samples of small amounts of dried blood, liquid saliva, semen and buccal mucosa that were aged for different time intervals for co-extraction of RNA and DNA. RNA quality was assessed by determination of ‘RNA integrity number’ (RIN) and quantitative PCR based expression analysis. DNA quality was assessed via monitoring STR typing success rates.By comparison, the different methods exhibited considerable differences between RNA and DNA yields, RNA quality values and expression levels, and STR profiling success, with the AllPrep DNA/RNA Mini Kit and the NucleoSpin® miRNA Kit excelling at DNA co-extraction and RNA results, respectively.Overall, there was no ‘best’ method to satisfy all demands of comprehensible co-analysis of RNA and DNA and it appears that each method has specific merits and flaws. We recommend to cautiously choose from available methods and align its characteristics with the needs of the experimental setting at hand.

Potential DNA mixtures introduced through kissing.

The use of saliva samples is an alternative to blood samples when a large number of control samples are to be compared by DNA investigations. The most convenient and safe method is by using cotton wool swabs. In this investigation the average DNA content of saliva samples taken by three different sampling techniques (i. e. cotton wool swab, filter paper, liquid saliva) was compared. In addition the possibility of a DNA mixture of saliva samples after intensive kissing was investigated by taking samples from voluntary pairs. Mixed STR patterns were found in five samples but restricted to the first sampling after kissing within max. 60 s.

Determination of phenotypes of salivary amylase in liquid saliva and saliva stains

An isoelectric focusing method is described for typing salivary amylase in liquid saliva and saliva stains. The estimated gene frequencies in a British population, calculated on the basis of three alleles operating at a single locus, were Amy 1, 0.909; Amy 2, 0.065; Amy 3, 0.026. This system may be useful in forensic investigations.

Mode of ingestion in a heteropterous insect Dysdercus koenigii (F.) (Pyrrhocoridae)

The mode of ingestion from different substrates by D. koenigii has been studied by observing the action of its stylets, by chemical analysis of the ingested food, and by measuring the rate of ingestion. When the insect feeds on a freely exposed liquid the whole tip of its beak is introduced into the substrate and the stylets hardly protrude beyond the labium. The bug does not eject any saliva into the liquid which enters the gut without any prior change. During feeding on a solid substrate like a tablet of sucrose or sucrose-starch mixture, all parts of which are ingestible, the tips of its stylets and labium advance together into the food. When it feeds on solid substrates like cotton or okra seeds, their kernels and leaves, or wire gauze-wrapped sucrose tablets, the surfaces of which are non-ingestible, its labium is held outside and only the stylets enter. If the non-ingestible surface is tough and non-porous, e.g. testa of a seed or cuticular covering of a leaf, the insect deposits an acidic ‘salivary sheath’ which provides rigidity to the labium during insertion of the stylets. Such a sheath is not formed inside the seed- or leaf-tissues nor even outside a substrate having a soft (seed-kernel), porous (wire gauze-wrapped sucrose tablet), or ingestible (sucrose tablet) surface. Inside each solid substrate the stylets move along closely placed and intercrossing tracks. Throughout their movements in the ingestible parts, drops of an alkaline ‘liquid saliva’ are ejected and immediately ingested in rapid succession at the tips of the stylets. The liquid contents of a substrate like a leaf or water-soaked seed with a high water content are sucked in as such. The solid constituents of a substrate are ingested after their liquefaction by dissolution of soluble substances and suspension of fine, dislodged, insoluble particles in the ejected liquid saliva. External digestion of solid food constituents is not necessary for their ingestion. The insects fed on cotton seed take in the yellow pigments, lipids, proteins, free amino acids, raffinose, sucrose, galactose, and traces of starch from the kernel-tissues between the pigment glands. Freely exposed liquids are ingested 8–12 times faster than the constituents of water-rich solid diets, e.g. leaves and water-soaked seeds. Ingestion from dry substrates, e.g. dry seeds and sucrose tablets, is four times slower than that from water-rich solid diets. The rate of ingestion from a solid substrate is determined by the solubility of its constituents or their capacity to form a suspension in the ejected salivary drops. The quantity of various constituents of different substrates ingested during single feeds of equal duration depends upon the action of the stylets, rate of ingestion, and concentration of food constituents.