Loop-mediated isothermal amplification is known for its high sensitivity, specificity and tolerance to inhibiting-substances. In this work, we developed a device for performing real-time colorimetric LAMP combining the accuracy of lab-based quantitative analysis with the simplicity of point-of-care testing. The device innovation lies on the use of a plastic tube anchored vertically on a hot surface while the side walls are exposed to a mini camera able to take snapshots of the colour change in real time during LAMP amplification. Competitive features are the rapid analysis (< 30 min), quantification over 9 log-units, crude sample-compatibility (saliva, tissue, swabs), low detection limit (< 5 copies/reaction), smartphone-operation, fast prototyping (3D-printing) and ability to select the dye of interest (Phenol red, HNB). The device’s clinical utility is demonstrated in cancer mutations-analysis during the detection of 0.01% of BRAF-V600E-to-wild-type molecules from tissue samples and COVID-19 testing with 97% (Ct < 36.8) and 98% (Ct < 30) sensitivity when using extracted RNA and nasopharyngeal-swabs, respectively. The device high technology-readiness-level makes it a suitable platform for performing any colorimetric LAMP assay; moreover, its simple and inexpensive fabrication holds promise for fast deployment and application in global diagnostics.

BACKGROUND Decisions on which pesticide to use in agriculture are expected to become more difficult, as the number of available chemicals is decreasing. For Tetranychus urticae (T. urticae), a major pest for which a number of candidate markers for pesticide resistance are in place, molecular diagnostics could support decision‐making for the rational use of acaricides. RESULTS A suite of 12 TaqMan qPCR assays [G314D (GluCl1), G326E, I321T (GluCl3), G119S, F331W (Ace‐1), H92R (PSST), L1024V, F1538I (VGSC), I1017F (CHS1), G126S, S141F, P262T (cytb)], were validated against Sanger‐sequencing, and subsequently adapted for use with the ddPCR technology. The concordance correlation coefficient between the actual and ddPCR measured mutant allelic frequencies was 0.995 (95% CI = 0.991–0.998), and no systematic, proportional, or random differences were detected. The achieved Limit of Detection (LoD) was 0.1% (detection of one mutant in a background of 999 wild type mites). The ddPCR assay panel was then assessed in terms of agreement with phenotypic resistance, through a pilot application in field populations from Crete, with strong correlation and thus predictive and diagnostic value of the molecular assays in some cases (e.g., etoxazole and abamectin resistance). Molecular diagnostics were able to capture incipient resistance that was otherwise missed by phenotypic bioassays. The molecular and phenotypic resistance screening of T. urticae field populations from Crete, revealed both multi‐resistant and susceptible populations. CONCLUSION The highly sensitive T. urticae molecular diagnostic platforms developed in this study could prove a valuable tool for pesticide resistance management. © 2021 Society of Chemical Industry.

Breast cancer (BC) continues to affect the lives of millions of women worldwide. Several members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) subfamily are involved in tumor progression. Notably, the CEACAM subfamily harbors the already established cancer biomarker CEA, as well as other potential molecular markers. CEACAM19, a recently identified gene belonging to CEACAM subfamily, was discovered and cloned by members of our research group. The present study analyzes, quantitatively, the expression of CEACAM19 and evaluates its clinical relevance in BC. Total RNA was extracted from 143 cancerous and 89 normal adjacent breast tissue specimens. Following reverse transcription, quantitative analysis of CEACAM19 mRNA expression levels was performed via real-time PCR and the comparative Ct (2-∆∆Ct) method. CEACAM19 expression and detailed clinicopathological data were used for extensive biostatistical analyses. CEACAM19 was found to be overexpressed in breast cancer tissue specimens compared to normal tissue counterparts (p=0.013). CEACAM19 mRNA expression status was also associated with clinicopathological features indicative of aggressive behavior and poor prognosis in BC, such as high tumor grade (p=0.031) and high Ki67 proliferative index (p=0.038). A significant negative association was documented between CEACAM19 expression and tumor ER status (p=0.018) as well as patients' menopausal state (p=0.016). Our results suggest that CEACAM19 mRNA expression represents a promising, novel and clinically useful tissue biomarker for breast cancer management.

High throughput methodologies have revealed the existence of an unexpectedly large number of long noncoding RNAs (lncRNAs). The unconventional role of lncRNAs in gene expression regulation and their broad implication in oncogenic and tumor suppressive pathways have introduced lncRNAs as novel biological tumor markers. The most prominent example of lncRNAs application in routine clinical practice is PCA3, a FDA-approved biomarker for prostate cancer. Regarding digestive system malignancies, the oncogenic HOTAIR is one of the most widely studied lncRNAs in the preclinical level and has already been identified as a potent prognostic marker for major malignancies of the gastrointestinal tract. Here, we provide an overview of recent findings regarding the emerging role of lncRNAs not only as key regulators of cancer initiation and progression in colon, stomach, pancreatic, liver, and esophageal cancers, but also as reliable tumor markers and therapeutic tools. lncRNAs can be easily, rapidly, and cost-effectively determined in tissues, serum, and gastric juice, making them highly versatile analytes. Taking also into consideration the largely unmet clinical need for early diagnosis and more accurate prognostic/predictive markers for gastrointestinal cancer patients, we comment upon the perspectives of lncRNAs as efficient molecular tools that could aid in the clinical management.

Examining for new BC biomarkers has proven that kallikrein-related peptidase (KLK) family members represent promising serum and/or tissue molecular tools for early diagnosis, effective prognosis, and treatment monitoring of patients. The aim of this study was to investigate, the previously unexplored, prognostic significance of KLK8 in BC. KLK8 mRNA expression was quantitatively analyzed in 150 cancerous and 100 corresponding normal breast tissue specimens via a SYBR Green-based RealTime PCR methodology. Expression data and patients' clinicopathological parameters were used for extensive biostatistical analyses, including internal validation. KLK8 mRNA expression was significantly downregulated in the cancerous tissue part relative to the non-cancerous counterpart (P \ 0.001), in the majority of the paired breast tissue samples. KLK8 expression was associated with advanced TNM stage (P = 0.019) and positive nodal status involvement (P = 0.044). Triple negative (TNBC) and HER2 overexpressing tumors exhibited higher KLK8 expression levels (P \ 0.001), compared to Luminal A and B molecular subtypes. Kaplan-Meier survival curve analysis revealed that BC patients with high KLK8 expression had significantly shorter disease-free survival (DFS) intervals (P \ 0.001) compared to those belonging in the KLK8-low expression group. Cox univariate analysis confirmed the association between KLK8 expression, analyzed as a continuous variable, and poor patients' outcome (Hazard ratio [HR] = 3.28, P \ 0.001). Most importantly, multivariate analysis showed that KLK8 expression is a strong and independent predictor of adverse DFS in BC ([HR] = 2.74; P = 0.002). Our results show that KLK8 mRNA expression is associated with aggressive tumor characteristics and it can serve as a novel independent biomarker of unfavorable prognosis for BC patients. Keywords