A Rapid and Sensitive One Step-SYBR Green Based Semi Quantitative Real Time RT-PCR for the Detection of peste des petits ruminants Virus in the Clinical Samples

A sensitive and rapid single step real time （rt） RT-PCR was standardized using one-step Brilliant SYBR Green kit for detection and semi-quantitation ofpeste des petitis ruminants virus （PPRV） using the virus RNA and matrix （M） protein gene-specific primers and compared with established conventional RT-PCR and TaqMan RT-PCR. The assay amplifies a 124 bp fragment of the PPRV M gene with Tm of 78.28 to 78.50. The assay was linear within a range of 50 ng to 0.5 fg total virus RNA with a detection limit （sensitivity） of 0.5 fg. Based on the serial dilution of the live-attenuated PPR vaccine virus, the detection limit was -0.0001 cell culture infectious dose 50% units （TCID50）. Additionally, swab materials spiked with known titre of vaccine virus were equally well detected in the assay. The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the field and experimental clinical samples. The assay detected the PPRV nucleic acid as early as 3 day post infection （dpi） and up to 20 dpi in swab materials from the experimental samples. The assay was rapid and more sensitive than TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the PPR suspected clinical samples of sheep and goats. Therefore, the established, simplified SYBR green rt RT-PCR is an alternative test to the already existing various diagnostic assays and could be useful for rapid clinical diagnosis with advantage in reducing risk of contamination.

Assessment of Appropriateness of Diagnostic Lymph Node Tissue Collection from the Operating Theater and by Interventional Radiologist. A Clinical Audit

Background: Appropriate sample requesting, collecting and timely dispatch to the appropriate laboratory is essential in establishing diagnosis of pathologies with lesions. Much time and effort may be wasted if this is not done according to certain standards. We conducted this study to assess the route of lymph node samples from requests to reaching the laboratories. Methods: We conducted an audit over a period from 4th June until 10th Aug 2023. Data for all the procedures performed over this period on lymph node samples (was entered into and analysed using Excel. Results: A total of eighteen samples for sixteen patients were obtained during this period. Median age of the patients was 34 years (19 - 73) with a M:F ratio of 5:11. Among the IR samples, nine samples were from the neck, three from inguinal area and one from axilla. Seven samples (53.8%) were tru-cut biopsies, six samples (46.15%) were FNA. All samples were sent to the pathology laboratory fixed in formalin. Samples for TB were sent only for five cases (31.25%) and for only two cases (12.5%) were samples sent for bacterial culture. For the OR samples, none were sent for either bacterial culture or TB. Overall, eight patients (50%) were not investigated for any infectious etiologies like brucella, toxoplasmosis, CMV, EBV plus other possible causes. Repeat sampling was required for 25% of patients (within and out of the audit period). Conclusions: to avoid delays in making diagnoses, it is paramount to consider infectious etiologies as possible diagnosis for lymphadenopathy and request appropriate investigations. This requires liaising with infectious diseases/clinical microbiology experts to guide regarding types of samples, types of media and timely dispatch to the correct laboratory.

Rapid detection of Mycoplasma Pneumoniae in Clinical Samples by the Polymerase Chain Reaction Technique.

The polymerase chain reaction (PCR) technique was used to detect mycoplasma pneumoniae in clinical samples

Sample size re-estimation without un-blinding for time-to-event outcomes in oncology clinical trials

Sample size re-estimation is essential in oncology studies. However, the use of blinded sample size reassessment for survival data has been rarely reported. Based on the density function of the exponential distribution, an expectation-maximization（EM） algorithm of the hazard ratio was derived, and several simulation studies were used to verify its applications. The method had obvious variation in the hazard ratio estimates and overestimation for the relatively small hazard ratios. Our studies showed that the stability of the EM estimation results directly correlated with the sample size, the convergence of the EM algorithm was impacted by the initial values, and a balanced design produced the best estimates. No reliable blinded sample size re-estimation inference can be made in our studies, but the results provide useful information to steer the practitioners in this field from repeating the same endeavor.

Dual-miRNA-Propelled Three-Dimensional DNA Walker for Highly Specific and Rapid Discrimination of Breast Cancer Cell Subtypes in Clinical Tissue Samples

Accurate discrimination of cell subtypes at the molecular level is especially important for cancer diagnosis,but no current method allows rapid and precise detection of breast cancer subtypes.Herein,we developed an elegant DNA walker for direct and rapid differentiation of breast cancer cell subtypes via detection of dual-miRNAs in clinical tissue samples.This DNA nanomachine can be specifically initiated by endogenous miR-21 and miR-31,and the sensitivity was dramatically improved due to the DNAzyme-mediated signal amplification.This DNA walker enabled rapid detection of double miRNA characteristics in different breast cell lines and also distinguished the fluctuations in a single cell.Applications of this DNAzyme-based nanomachine in vivo and in clinical samples were demonstrated for efficient detection of breast cancer subtypes,making the method generally applicable for precise management of cancers.

Advances in the Detection of Mycoplasma hyopneumoniae by Polymerase Chain Reaction (PCR) Technology

Mycoplasma hyopneumoniae is an important pathogen causing Mycoplasmal pneumonia of swine, which generally causes secondary infections and mixed infections, thus seriously threats the development of swine industry and resulting in huge economic losses. Using PCR technology has very important significance to the correct diagnosis of Mycoplasmal pneumonia at the early stage. In this paper, specific target genes of Mycoplasma hyopneumoniae, methods for clinical sample collection, key technical factors of DNA sample processing method, and the research progress, main advantages and disadvantages, and application of general PCR technology, multiple PCR technology, nested-PCR technology, real-time fluorescence quantitative PCR technology, gene chip detection technology and loop-mediated isothermal amplification in detection of Mycoplasma hyopneumoniae were summarized, which provided convenience for the effective diagnosis and prevention of Mycoplasmal pneumonia of swine.

Integrative Analysis of Genome,3D Genome,and Transcriptome Alterations of Clinical Lung Cancer Samples

Genomic studies of cancer cell alterations,such as mutations,copy number variations(CNVs),and translocations,greatly promote our understanding of the genesis and development of cancers.However,the 3D genome architecture of cancers remains less studied due to the complexity of cancer genomes and technical difficulties.To explore the 3D genome structure in clinical lung cancer,we performed Hi-C experiments using paired normal and tumor cells harvested from patients with lung cancer,combining with RNA sequenceing analysis.We demonstrated the feasibility of studying 3D genome of clinical lung cancer samples with a small number of cells(1×10^(4)),compared the genome architecture between clinical samples and cell lines of lung cancer,and identified conserved and changed spatial chromatin structures between normal and cancer samples.We also showed that Hi-C data can be used to infer CNVs and point mutations in cancer.By integrating those different types of cancer alterations,we showed significant associations between CNVs,3D genome,and gene expression.We propose that 3D genome mediates the effects of cancer genomic alterations on gene expression through altering regulatory chromatin structures.Our study highlights the importance of analyzing 3D genomes of clinical cancer samples in addition to cancer cell lines and provides an integrative genomic analysis pipeline for future larger-scale studies in lung cancer and other cancers.

Molecular recognition triggered aptazyme cascade for ultrasensitive detection of exosomes in clinical serum samples

Exosomes have attracted widespread interest due to their inherent advantages in tumor diagnosis and treatment monitoring.However,it is still a big challenge for highly sensitive and specific detection of exosome in real complexed samples.Herein,a molecular recognition triggered aptazyme cascade strategy was developed for ultrasensitive detection of cancer exosomes in clinical serum samples.In this design,one target exosome could capture a large quantity of aptazymes for the first-step signal amplification.And then the captured aptazyme was activated and recycled to release the fluorophore-labelled substrate strand for a cascaded signal amplification.Notably,the activation of aptazyme only occurs whenithas bound with target exosome,ensuring a low background.The experimental results show that the limit of detection(LOD)and the limit of quantification(LOQ)are 3.5×10^(3) particles/μL and 1.7×10^(4) particles/μL,respectively,which is comparable to the results of most existed fluorescence-based exosome probes.Moreover,this assay possesses high specificity to distinguish exosomes derived from other cell lines.Furthermore,this fluorescence probe was utilized in cancer patient and healthy serum samples successfully,suggesting its great potential for clinical diagnosis and biological studies.

The methodology flaws in Hinman's acupuncture clinical trial, Part Ⅲ: Sample size calculation

In the October 2014 publication of JAMA,Dr.Hinman and colleagues published the study＂Acupuncture for Chronic Knee Pain：A Randomized Clinical Trial,＂in which the authors concluded that＂in patients older than50 years with moderate or severe chronic knee pain,neither laser nor needle acupuncture conferred benefi t over sham for pain or function.Our fi ndings do not support acupuncture[1]