A specific hybridisation internalisation probe (SHIP) enables precise live-cell and super-resolution imaging of internalized cargo

Facilitated by the advancements in microscopy, our understanding of the complexity of intracellular vesicle traffic has dramatically increased in recent years. However, distinguishing between plasma membrane-bound or internalised ligands remains a major challenge for the studies of cargo sorting to endosomal compartments, especially in small and round cells such as lymphocytes. The specific hybridization internalisation probe (SHIP) assay, developed for flow cytometry studies, employs a ssDNA fluorescence internalisation probe and a complementary ssDNA quenching probe to unambiguously detect the internalized receptors/cargo. Here, we adopted the SHIP assay to study the trafficking of receptor/ligand complexes using B lymphocytes and B cell receptor-mediated antigen internalization as a model system. Our study demonstrates the potential of the SHIP assay for improving the imaging of internalized receptor/ligand complexes and establishes the compatibility of this assay with multiple imaging modalities, including live-cell imaging and super-resolution microscopy.

Clinical evaluation of a novel point-of-care assay to detect Mycoplasma pneumoniae and associated macrolide resistant mutations

The recent increase in macrolide-resistant Mycoplasma pneumoniae (M. pneumoniae) in Asia has become a continuing problem. A point-of-care testing method that can quickly detect M. pneumoniae and macrolide-resistant mutations (MR mutations) is critical to proper antimicrobial use. Smart Gene TM (MIZUHO MEDY Co., Ltd. Tosu-City, Saga, Japan) is a compact and inexpensive fully automatic gene analyzer that combines amplification with polymerase chain reaction (PCR) and the quenching probe method to specify the gene and MR mutations simultaneously. We performed a clinical evaluation of this device and its reagents on pediatric patients with M. pneumoniae-suspected respiratory infections and evaluated the impact of the assay on antimicrobial selection. Using real-time PCR as a comparison control, the sensitivity of Smart Gene TM was 97.8% (44/45), its specificity was 93.3% (98/105) and its overall concordance rate was 94.7% (142/150). The overall concordance rate of Smart Gene TM diagnosis of MR mutations in comparison with sequence analysis was 100% (48/48). The ratio of MR mutations was significantly higher at high-level medical institutions than at a primary medical clinic (P = 0.023), and changes in antibiotic therapy to drugs other than macrolides was significantly more common in patients with MR mutations (P = 0.00024). Smart Gene TM demonstrated excellent utility in the diagnosis of M. pneumoniae and the selection of appropriate antimicrobials for MR mutations at primary medical institutions, which play a central role in community-acquired pneumonia care. The use of this device may reduce referrals to high-level medical institutions for respiratory infections, thereby reducing the medical and economic burden on patients.

Discrimination of a single nucleotide polymorphism in the haptoglobin promoter region, rs5472, using a competitive fluorophore-labeled probe hybridization assay following loop-mediated isothermal amplification

ABSTRACT Personalized peptide vaccination, which involves activation of the host immune system against cancer cells using personalized peptide vaccines (PPVs), can improve overall survival in multiple cancer types. However, the clinical efficacies of PPVs vary for unknown reasons. Recently, a single nucleotide polymorphism (NG_012651.1:g.4461_5460[4960A>G]) in the haptoglobin promoter region, rs5472, was significantly associated with clinical response of PPV. Therefore, rs5472 is expected to be a predictive biomarker for PPV therapy. Here, we described a single nucleotide discrimination method for rs5472 analysis by combining the loop-mediated isothermal amplification and quenching probe methods. In evaluation of saliva samples, this method showed high concordance with the results of Sanger sequencing (100%, n = 36). Importantly, this method did not require calculation of melting temperature for single nucleotide discrimination and could therefore be carried out on a simple instrument. Accordingly, this method may be more robust and applicable to near-patient testing.