Rapid visual nucleic acid detection of Vibrio alginolyticus by recombinase polymerase amplification combined with CRISPR/Cas13a.

Vibrio alginolyticus (V. alginolyticus) is a common pathogen in the ocean. In addition to causing serious economic losses in aquaculture, it can also infect humans. The rapid detection of nucleic acids of V. alginolyticus with high sensitivity and specificity in the field is very important for the diagnosis and treatment of infection caused by V. alginolyticus. Here, we established a simple, fast and effective molecular method for the identification of V. alginolyticus that does not rely on expensive instruments and professionals. The method integrates recombinase polymerase amplification (RPA) technology with CRISPR system in a single PCR tube. Using this method, the results can be visualized by lateral flow dipstick (LFD) in less than 50 min, we named this method RPA-CRISPR/Cas13a-LFD. The method was confirmed to achieve high specificity for the detection of V. alginolyticus with no cross-reactivity with similar Vibrio and common clinical pathogens. This diagnostic method shows high sensitivity; the detection limit of the RPA-CRISPR/Cas13a-LFD is 10 copies/µL. We successfully identified 35 V. alginolyticus strains from a total of 55 different bacterial isolates and confirmed their identity by (Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF MS). We also applied this method on infected mice blood, and the results were both easily and rapidly obtained. In conclusion, RPA-CRISPR/Cas13a-LFD offers great potential as a useful tool for reliable and rapid diagnosis of V. alginolyticus infection, especially in limited conditions.

The Clinical Significance of sICAM-1 in Differentiating Benign Breast Lesions from Breast Cancer.

OBJECTIVE: Breast cancer is a common type of malignant tumour worldwide and the second leading cause of death in women. The present study aims to investigate the clinical significance of serum soluble intercellular adhesion molecule-1 (sICAM-1) in differentiating benign breast lesions from breast cancer. METHODS: Plasma samples were obtained from 200 breast cancer patients, 47 patients with benign breast lesions and 50 age- and sex-matched healthy individuals as controls. Plasma levels of sICAM-1 were measured in all the samples using commercially available enzyme-linked immune-sorbent assay (ELISA) kits. The serum levels of carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA15-3) were detected by the UniCel® DxI 800 Immunoassay System with matched kits. RESULTS: The plasma levels of CEA and CA15-3 were 1.22±0.2 (ng/mL) and 6.39±1.5 (ng/mL) in the healthy control group, 1.40±0.3 (ng/mL) and 5.81±2.1 (ng/mL) in the benign breast lesion (BBL) group, and 5.29±0.6 (ng/mL) and 27.08±5.7 (ng/ mL) in the breast cancer (BC) group. Plasma levels of CEA and CA15-3 in the BC group were significantly higher than those in the BBL and healthy control groups (all P<0.05), but the plasma levels of CEA and CA15-3 were not significantly different between the BBL group and the healthy control group, P=0.548 and P=0.2976, respectively. The diagnostic sensitivity and specificity were 13.4% and 98.0% for CEA and 22.2% and 100.0% for CA15-3. For plasma sICAM-1, the diagnostic sensitivity and specificity were 98% and 94% at a cut-off value of 20.0 (ng/mL), with an area under the receiver operating characteristic (ROC) curve of 0.99, which could be used to distinguish between healthy controls and the BC group; the diagnostic sensitivity and specificity were 95.5% and 94.0% at a cut-off value of 20.0 (ng/mL) with an area under the ROC curve of 0.98, which could be used to distinguish between healthy controls and the BBL group; and the diagnostic sensitivity and specificity were 44.6% and 94.1% at a cut-off value of 40.0 (ng/mL), with an area under ROC curve of 0.68, which could be used to distinguish between the BBL group and the BC group. The plasma levels of sICAM-1 were 15.43±2.3 (ng/mL) in healthy controls, 29.8±3.5 (ng/ mL) in the BBL group, and 50.07±12.2 (ng/mL) in the BC group. The plasma level of sICAM-1 in the BC group was the highest among all three groups (all P<0.05). CONCLUSIONS: The CEA, CA15-3 and sICAM-1 levels were increased in breast cancer patients, especially in those with node and/or organ metastasis. After diagnosis, CEA, CA15-3 and sICAM-1 levels are closely related to tumour metastasis. sICAM-1 has great potential value in the clinical diagnosis of benign breast lesions and breast cancer.

Clinical Significance of let-7a-5p and miR-21-5p in Patients with Breast Cancer.

OBJECTIVE: To investigate the clinical significance of serum let-7a-5p and miR-21-5p in the diagnosis of breast cancer. METHODS: We examined 32 healthy people and 30 patients with benign breast lesions as controls and 90 breast cancer patients as study subjects. The expression of let-7a-5p and miR-21-5p were detected in all subjects' samples, and Cel-miR-39-3p was used as a spike-in reference. Serum miRNAs were extracted by the TRIzol method, and reverse transcription was performed with specific primers for let-7a-5p, miR-21-5p and Cel-miR-39-3p, and 2-µL reverse transcription products were used as PCR templates. A SLAN-96P fluorescent quantitative PCR instrument was used for quantitative PCR detection. RESULTS: (1) The serum levels of carcinoembryonic antigen (CEA)and carbohydrate antigen 15-3 (CA15-3) in the breast cancer group were higher than those in the healthy controls and patients with benign breast lesions; (2) The expression level of let-7a-5p in the serum of the breast cancer group was lower than that in the healthy control group (P<0.05), but there was no significant difference compared to the breast benign lesion group (P>0.05); (3) The serum expression level of miR-21-5p in the breast cancer group was lower than that in the healthy control group (P<0.05) but was not significantly different from that in the patients with benign breast lesions (P>0.05). CONCLUSION: Reduced expression of Let-7a-5p and miR-21-5p levels is of little value for early diagnosis of breast cancer; however reduced expression of Let-7a-5p and miRNA-21-5p may serve as non-invasive biomarkers for the diagnosis of breast cancer metastasis, and combination of these markers with CEA and CA15-3 can help to distinguish benign breast lesions from breast cancer.

The Distribution Frequency of Interferon-Gamma Receptor 1 Gene Polymorphisms in Interferon-γ Release Assay-Positive Patients.

Tuberculosis is caused by mycobacterium, a potentially fatal infectious bacterium. In recent years, TB cases increased in the whole world. WHO statistics data shows that the world's annual tuberculosis incidence was 8~10 million with about 3 million deaths. Several studies have shown that susceptibility to tuberculosis may be associated with IFNGR1 gene polymorphisms. Here, we report the distribution frequency of IFNGR1 gene polymorphisms in 103 cases of IGA-negative patients and 100 cases of IGA-positive patients from China by sequencing the IFNGR1 proximal ~750 bp promoter region. We found a total of 5 types of site mutations: -611 (G/A), -56 (T/C), -255 (C/T), -359 (T/C), and -72 (C/T). The two main types of gene polymorphisms among the IGA-negative and IGA-positive groups were -611 (G/A), with mutation rates of 88.3% and 78.4%, respectively, and -56 (T/C), with mutation rates of 84.5% and 83.8%, respectively, which had no statistical significance, and there was no correlation with the incidence of tuberculosis.