Reverse dot blot hybridization assay— An expeditious diagnostic tool for drug resistant TB

Tuberculosis (TB) is one of the leading infectious diseases that is highly transmissible. It is reported to cause approximately 10 million infections and 1.4 million deaths globally in the year of 2019.¹ Prompt detection and treatment of TB are essential to intercept the proliferation of the disease. In the case of drug- resistant TB, failure to detect the resistance of anti TB drugs may give rise to extensively drug- resistant tuberculosis (XDR-TB). Thus, the rapid detection of drug resistance is vital;, however, the current standard drug susceptibility tests (DST) may take up to 12 weeks raising an alarming concern.² A study was done in China by Li Wan et al, on the accuracy of the reverse dot blot hybridization assay (RDBH) for rapidly detecting the resistance of four anti TB drugs (rifampicin (RIF), isoniazid (INH), streptomycin (SM) and ethambutol (EMB)) in mycobacterium tuberculosis (MTB) isolates.³ Continuous...

Hybridoma Screening by Antigen Capture: Reverse Dot Blot

A dot blot is widely used to determine the productivity of a given hybridoma. This assay can also be used to screen a fusion or subclone plate for productive hybridoma clones. First, a nitrocellulose membrane is coated with an affinity-purified goat or rabbit anti-mouse immunoglobulin and then incubated with hybridoma tissue culture supernatant. Monoclonal antibodies in the supernatant are then “captured” on the coated nitrocellulose membrane surface and detected by screening with horseradish peroxidase (HRP).

A Cross-Sectional Study for Evaluation of KRAS and BRAF Mutations by Reverse Dot Blot, PCR-RFLP, and Allele-Specific PCR Methods Among Patients with Colorectal Cancer

Background: KRAS and BRAF genes are the biomarkers in Colorectal Cancer (CRC) which play prognostic and predictive roles in CRC treatment. Nowadays, the selection of rapid and available methods for studying KRAS and BRAF mutations in anti-EGFR therapy of patients suffering from CRC plays a significant role. In this study, the mutations of these two oncogenes were evaluated by different methods. Methods: This study was performed on 50 Formalin-Fixed Paraffin-Embedded (FFPE) tissue blocks of patients diagnosed with colorectal cancer. After DNA extraction, KRAS and BRAF gene mutations were evaluated using reverse dot blot, and results were compared with PCR-RFLP and allele-specific PCR for KRAS and BRAF mutations, respectively. Results: KRAS gene mutations were detected in 42% of patients, of which 30% were in codon 12 region, and 12% in codon 13. The most frequent mutations of KRAS were related to G12D and 10% of patients had BRAF mutated genes. The type of KRAS gene mutations could be evaluated by reverse dot blot method. In general, the results of PCR-RFLP and allele-specific PCR were similar to the findings by reverse dot blot method. Conclusion: These findings suggest that PCR-RFLP and allele-specific PCR methods are suitable for screening the presence of the mutations in KRAS and BRAF oncogenes. In fact, another method with more sensitivity is needed for a more accurate assessment to determine the type of mutations. Due to higher speed of detection, reduced Turnaround Time (TAT), and possible role of some KRAS point mutations in overall survival, reverse dot blot analysis seems to be an optimal method.

Hemophagocytic syndrome associated with Mycobacterium bovis in a patient with X-SCID: a case report

Background Mycobacterium bovis could infect patients with immunodeficiency or immunosuppressive conditions via Bacillus Calmette-Guérin (BCG) vaccination. Tuberculosis-related hemophagocytic syndrome (HPS) is reported, but not HPS caused by Mycobacterium bovis in children. Case presentation A 4-month Chinese boy presented fever and cough. The initial laboratory investigation showed the lymphocyte count of 0.97 × 109/L, which decreased gradually. HPS was diagnosed based on the test results that fulfilled the HLH-2004 criteria. In addition, Mycobacterium tuberculosis complex was detected from his peripheral blood via metagenomic next-generation sequencing (mNGS) and M. bovis was identified by polymerase chain reaction-reverse dot blot (PCR-RDB). Thus, the patient was treated with Isoniazid, Rifampin, and Pyrazinamide, but not improved. However, parents refused to accept further therapy, and was discharged on the day 12 of admission. To confirm the pathogenesis, genetic analysis was performed. Mutation in the interleukin-2 receptor subunit gamma gene: Exon 6: c.854G > A; p. Arg285Gln was detected in the patient and the mother, which could underlie X-linked severe combined immunodeficiency. Conclusions A boy with X-SCID was diagnosed with M. bovis-associated HPS, emphasizing that X-SCID should be considered when M. bovis is detected in a male infant with low lymphocyte counts.

Rapid Molecular Detection for Differentiation of Homozygous HbE and ß0-Thalassemia/HbE in Samples Related With HbE >80% and Variable HbF Levels

Objective To validate a novel rapid molecular testing method for differentiation of homozygous hemoglobin (Hb)E and HbE/β 0-thalassemia genotypes using multiplex melt curve combined with high-resolution melt (HRM) analysis in a single test tube. Methods All 10 genotypes contained (β N/β N; n = 95), (β N/β 3.5-kb; n = 71), (β N/β 45-kb; n = 28), (β N/β E; n = 10), (β E/β 3.5-kb; n = 6), (β E/β 45-kb; n = 4), (β E/β 41/42; n = 28), (β E/β 17; n = 9), (β E/β IVSI#1; n = 6), and (β E/β E; n = 76) were recruited for validation. A proposed strategy for rapid differentiation of β 0-thalassemia/HbE disease and homozygous Hb E in specimens with HbE greater than 80% and variable HbF levels was demonstrated. Results In the validation method, all genotypes showed 100% concordance, compared with the conventional reverse dot blot (RDB) and gap–polymerase chain reaction (PCR) methods. Conclusions Our newly developed method could be useful in routine laboratory settings. The method is rapid, simple, and cost effective; does not require a post-PCR step; and can be applied in routine settings.

Hoàn thiện quy trình phát hiện đồng thời 14 vi khuẩn gây bệnh đường ruột bằng kĩ thuật PCR-Reverse Dot Blot (PCR-RDB)

Ngộ độc thực phẩm, với một trong những nguyên nhân chính do nhiễm khuẩn vẫn luôn là mối lo ngại, mang tính toàn cầu, được Tổ chức Sức khỏe Thế giới rất quan tâm. Việc xác định chính xác đối tượng vi khuẩn nhiễm vẫn luôn là một nhu cầu cấp thiết của các labo lâm sàng. Nghiên cứu trước đây của chúng tôi đã được công bố với việc thành công bước đầu trong việc xây dựng một quy trình dựa trên kĩ thuật PCR-Reverse Dot Blot (PCR-RDB) nhằm xác định đồng thời 12 vi khuẩn gây bệnh đường ruột ch yếu, bao gồm Bacillus cereus, Clostridium botulinum, Clostridium perfringen, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, Salmonella spp., Shigella spp.., Vibrio cholerae, Vibrio parahaemolyticus, Yersinia enterocolitica và Brucella spp. Chúng tôi tiếp tục phát triển nghiên cứu này nhằm hoàn thiện quy trình PCR-RDB bằng việc thiết kế bổ sung trên màng các loại mẫu dò nhằm làm chứng dương, chứng âm, chứng màu và chứng kiểm tra tín hiệu nền. Bên cạnh đó, xét nhu cầu lâm sàng, việc bổ sung hai loại mẫu dò đề dò hai vi khuẩn Campylobacter jejuni và Yersinia enterocolitica O:8 gây bệnh đường ruột chủ yếu ở trẻ em, là cần thiết. Quy trình PCR-RDB được hoàn thiện trong nghiên cứu này vì vậy có khả năng phát hiện đồng thời 14 vi khuẩn gây bệnh đường ruột, một cách đặc hiệu, với độ nhạy đạt 10^2 bản sao/ml, đã được thử nghiệm trên 30 mẫu phân, ghi nhận kết quả hoàn toàn khớp với kít thương mại PowerCheckTM 20 Pathogen Multiplex Real-time PCR Kit (Korea).