Molecular Diagnostics - Practice Questions PDF

Molecular Diagnostics  Involves taking DNA or RNA, the unique genetic code found in our cells, and  It covers content areas such as molecular analyzing the sequences for red...

Molecular Diagnostics  Involves taking DNA or RNA, the unique genetic code found in our cells, and  It covers content areas such as molecular analyzing the sequences for red flags that science, molecular techniques, laboratory can pinpoint the potential emergence of a operations, and applications of molecular specific disease or disorder. testing.  The field has expanded rapidly in recent  A laboratory method that uses a sample of years. tissue, blood, or other body fluid to check for  Despite the growth of molecular diagnosis certain genes, proteins, or other molecules from the era of Hippocrates, the emergence that may be a sign of a disease or condition, of COVID-19 is still remarkable. such as cancer.  As the COVID-19 pandemic spreads  A collection of techniques used to analyze worldwide and develops new mutations, biological markers in the genome and precise diagnostic tools are becoming proteome, and how their cells express their increasingly important in managing and genes as proteins, applying molecular comprehending the disease’s epidemiology biology to medical testing.  There are multiple applications of molecular  Molecular testing encompasses a broad tests in clinical oncology category of tests that identify specific  The laboratory medicine department in 1992 sections of genetic information associated were just a few molecular diagnostic tests but with a particular disease such as cancer, to now there are molecular tests in most of the give patients an accurate diagnosis. areas of pathology  A test that sequences a patient’s DNA or  (Note: NAAT – Nucleic Acid Amplification RNA for markers of potential future diseases Test)  Areas of molecular diagnostics include infectious diseases, hematopathology, What are Examples of common genetics, solid tumor. molecular diagnostic tests?  Tests can be done to see if someone will be resistant to a specific drug  Tests are typically performed to  Involves laboratory medicine determine whether or not patients have a  You have likely heard about mail-order gene mutation associated with a specific genetic DNA testing kits by providing a saliva disease, either as an inherited or an sample, you can receive a DNA analysis of acquired mutation the particular code in your cell that makes  Inherited diseases can be tested for at you unique. the prenatal, newborn and adult stages of  This consumer-facing diagnostic test, life. referred to by one startup as a genetic “service” has taken the world by storm in  For example, a commonly inherited recent years. disease is CF.  But John Greg Howe, PhD, director of the  If a newborn is found to have two molecular diagnostics laboratory at Yale mutations in the gene associated with School of Medicine, explains that these at CF, the baby is most likely to have the home kits cannot provide the level of detail condition. and information that patients receive from a  The child can the be treated for the place like the Yale Center for Genomic disease, which can prolong his or her life. Analysis.  Doctors can perform a molecular test of  Also called MOLECULAR PATHOLOGY. a common inherited hereditary cancer MLIM [OLFU]   For Example, in breast cancer, they can which spawned the development of next- investigate for specific inherited generation sequencing. mutations in the BRCA1 and BRCA2  This has allowed DNA sequencing to be genes, which may increase the patient’s performed more efficiently and therefore risk of breast and ovarian cancer. has created lots of testing opportunities.  Acquired gene mutations can be tested  Note: DNA Sequencing refers to general for in some cases, such as for chronic laboratory techniques determining exact myeloid leukemia (CML) sequence of nucleotides. Cystic Fibrosis What’s the next for the field of Molecular Diagnostics?  An inherited condition that causes sticky mucus to build up in the lungs and  We are at the beginning stages of digestive system understanding what those changes to the  Frequent infection or difficulty in DNA sequence that indicate disease breathing mean and why they occurred.  WE have all this additional information, and nor we need to figure out what to do with it – how to best use and apply this new knowledge.  Research scientists are using this vast new amount of information to try and focus more deeply on understanding various diseases. 4 Areas of Molecular Diagnostics 1. Infectious Diseases – Microbiology and Virology 2. Hematopathology – Hematology, Microbiology, and Clinical microscopy 3. Genetics 4. Solid Tumor Why is next-generation sequencing an important scientific advancement?  Genome sequencing refers to understanding the order of the approximately 3 billion nucleotides in a genome that make up a person’s complete DNA  Following the first genetic sequencing of the human genome, there was an explosion in DNA sequencing innovation, MLIM [OLFU]  Notes:  To consider in cancer med: o Use for molecular diagnostic assay o Routinely used for personal use (?) o For personalization of ______ in a body of a human  Predicative tests o Analysis of individual targets o To choose specific cancer drugs Proteomics Genome  Large scale study of proteomes  Complete set of genetic information in an o Proteomes: Large ______ of organism. proteins produced in an organism  Provides all information which the  To determine the pattern of our genes in organism requires to function an organ Deoxyribonucleic Acid (DNA)  The molecule that carries genetic information for the development and functioning of an organism.  Located in the nucleus or nuclei of the call which makes up the body  Made up of coiling strands that wind up each other to resemble twisted ladder or double helix.  DNA bases pair up with another forming base pairs o Adenine and Thymine o Cytosine and Guanine  1 base is attached to sugar molecule and phosphate molecule, that’s why it is called sugar phosphate backbone MLIM [OLFU]  Genetic Disorders 1. Albinism a. Is an inherited condition that leads to someone having very light skin, hair, and eyes. b. Less melanin in their body c. Lack of melanocytes, too white, eyes are blue 2. Down Syndrome a. Genetic condition where a person is born with an extra chromosome. b. Affects body and brain DNA test development  Also called GENETIC TESTING c. Also known as TRISOMY 21  Medical tests that can identify mutations in our genes, chromosomes and proteins.  Not all mutations in DNA would lead to a disease sometimes it leads to normal functioning  Identify certain risks of having certain conditions or passing genetic disorder like hemophilia Ribonucleic Acid (RNA)  It is also a nucleic acid present in all living cells that has structural similarities to DNA.  Single stranded  Long chain of sugars but nucleotides only attach to one  Genetic disorders: one mutation in genome MLIM [OLFU] Structure, Function, and Replication of Adenosine with three phosphates is adenosine triphosphate (ATP). DNA and RNA (WEEK 2) Can be converted to nucleosides through GENES Hydrolysis Many contains the information to make protein o Hydrolysis = Tinatanggalan ng tubig product Notes: Difference of Nucleosides and Nucleotides other genes code for RNA products. Nucleosides = Unphosphorylated Set of DNA or Genetic information Nucleotides = Phosphorylated CHROMOSOME Pyrimidine may contain tens of thousands of genes. Cytosine 23 pairs of chromosomes per individual Thymine o 22 pairs of chromosomes Uracil o 1 pair of sex chromosome Purine Located at the nucleus Adenine Cell division can be seen on the microscope Guanine 2 MAIN TYPES OF NUCLEIC ACIDS DNA RNA DEOXYRIBONUCLEIC ACID (DNA) A nucleotide polymer in which of the monomers contains deoxyribose, a phosphate group, and one of the heterocyclic bases adenine, cytosine, GENOME guanine, or thymine. The entire genetic content of a cell This double helical structure of DNA was first described by James Watson and Francis Crick. the genetic material found in all living organisms, ranging from single-celled bacteria to multicellular mammals. It is found in the nucleus of eukaryotes and in the chloroplasts and mitochondria. Sugar = Deoxyribose Notes: GENOMICS Prokaryotes the study of genomes Nucleoid and Cytoplasm contains the DNA, DNA is free flowing since there is no envelope. NUCLEIC ACIDS Eukaryotes are large biomolecules that play essential roles in Complex all cells and viruses. Histone Portion = Chromatin Involves storage and expression of genomic information NUCLEOSIDES A nitrogen base bound to an unphosphorylated sugar is a nucleoside It is a two-subunit molecule in which a pentose sugar is bonded to a nitrogen-containing heterocyclic base Adenosine (A), guanosine (G), cytidine (C), and thymidine (T) are nucleosides. RECOMBINATION Hereditary component of all living cells Occurs through the molecular process of crossing over or physical exchange between NUCLEOTIDES molecules. If the ribose sugar is phosphorylated, the A recombinant molecule or organism is one that molecule is a nucleoside mono-, di-, or holds a new combination of DNA sequences. triphophosphate or a nucleotide. “Nasisira DNA to produce new combination” For example, adenosine with one phosphate is adenosine monophosphate (AMP). CONJUGATION Different physical states of the plasmid DNA can be resolved by distinct migration characteristics Bacteria that participate in conjugation are of during gel electrophoresis. two types, or sexes, termed F and F- Only prokaryocytes For conjugation to occur, F and F- cells must be in Duplex DNA contact with each other. o Double Stranded DNA Requirement for contact can be demonstrated by ▪ dsDNA physically separating F and F- cells. ▪ ssDNA Transfer genetic material RIBONUCLEIC ACID (RNA) It differs from DNA in the sugar moieties, having ribose instead of deoxyribose and, in one nitrogen base component, having uracil instead of thymine RNA is synthesized as a single strand rather than as a double helix. RNA species fold and loop upon themselves to take on as much of a double-stranded character as possible. TRANSFORMATION RNA can also pair with complementary single the process by which an organism acquires strands of DNA or RNA and form a double helix. exogenous DNA. a nucleic acid present in all living cells that has can occur in two ways: natural transformation structural similarities to DNA. and artificial transformation. (Artificial = Man Unlike DNA, however, RNA is most often single- made) stranded. is an important component of molecular genetics mostly involved in protein synthesis. Griffith had observed was the transfer of DNA Ribose from one organism to another without the Notes: protection of a conjugative bridge or a viral coat. Moieties = Fragment of Molecule The “transforming factor” discovered by Griffith was responsible for changing the phenotype of the avirulent rough type bacteria to that of the virulent smooth type. Avery, MacLeod, and McCarty showed that destruction of protein or RNA in the cell lysate did not affect the transforming factor, only the destruction of DNA prevented transformation. Notes: Frederick Griffith (1920) o S. Pneumoniae can be harmless to disease causing o Bacteriologist -> focus on bacterial pneumonia PRIMASE is a ribonucleic acid (RNA) synthesizing enzyme that lays down short RNA primers required for priming DNA synthesis. must work repeatedly on the lagging strand to PLASMIDS prime synthesis of each Okazaki fragment. Carry genetic information. Enzyme (6-11 Base Pairs) Due to their size and effect on the host cell, TYPES/STRUCTURE OF RNA plasmids can carry only a limited amount of Ribosomal RNA information. o rRNA is an important structural and The plasmid DNA duplex is compacted, or functional part of the ribosomes, cellular supercoiled.  organelles where proteins are synthesized o largest component of cellular RNA o One of the most important Messenger RNA o is the initial connection between the information stored in DNA and the translation apparatus that will ultimately produce the protein products responsible for the phenotype dictated by the chromosome. o Carries genetic information to start protein synthesis Transfer RNA o To transfer amino acids to match the correct mRNA codon. o Carries amino acid to ribosome TRANSCRIPTION Transcription is the copying of one strand of DNA into RNA by a process similar to that of DNA replication. the nucleolus, is the location of ribosomal RNA synthesis The polymerization of RNA from a DNA template is catalyzed by RNA polymerase. After binding to its start site in DNA, a specific sequence of bases called the promoter, RNA polymerase and its supporting accessory proteins synthesize RNA using the base sequence of the antisense strand as a guide. TRANSLATION is the process through which information encoded in mRNA directs the addition of amino acids during protein synthesis. QUALITY MANAGEMENT SYSTEM ▪ Standard within the family of ISO 8000 (QMS) First step for base level standardization Defines and establishes an organization’s quality policy and objectives ISO 9000:2000 (Quality Note: Management Systems Standards) o Quality: Katangian ng specimen to To provide customers with confidence give precise results that suppliers, contractors, ISO (International Organization for subcontractors, and vendors have documented, effective and independently Standardization) [French: Organisation verified Quality Management Systems. Internationale de Normalisation] This International Standard describes International standards ensure that the fundamentals of quality management products and services you use daily are systems, which form the subject of the safe, reliable, and of high quality. ISO 9000 family, and defines related known for its Global standards for trusted terms goods and services Also known as The New International The standards in ISO define what great Standard for Quality looks like, setting consistent benchmarks Version of ISO 9000 for businesses and consumers alike, ensuring reliability, building trust, and ISO/IEC 42001 simplifying choices International standard to establish, ISO slogan is “Making lives easier, safer implement, maintain and continuously and better.” improve AI Main goal: AIMS – Artificial Intelligence Management o Helps healthcare workers to give System accurate and precise results to IEC – International Electrotechnical patients Commission Ensures that products are safe, reliable and quality ISO/IEC 27001 It is an international non-governmental World best known standard for ISMS organization that is made up of national o Information Security Management standard bodies Systems Develop wide range proprietary industrial and commercial standards ISO 9000 Defined as a set of international standards on quality management and quality assurance developed to help companies effectively document the quality system elements needed to maintain an efficient quality system. They are not specific to any one industry and can be applied to organizations of This International Standard is any size. Applicable to the Following: Can help a company satisfy its organizations seeking advantage through customers, meet regulatory the implementation of a quality requirements, and achieve continual management system; improvement. organizations seeking confidence from Note: their suppliers that their product o ISO 9001 requirements will be satisfied. users of the products.  those concerned with a mutual ▪ Result can rule out genetic understanding of the terminology used in conditions quality management those internal or external to the Three Elements of a Quality organization who assess the quality Assurance Program: management system or audit it for the directors' qualifications conformity with the requirements of ISO on-site laboratory inspection 9001. interlaboratory comparison and those internal or external to the proficiency testing. organization who give advice or training on the quality management system Clinical & Laboratory Standards appropriate to that organization. developers of related standards. Institute (CLSI) An international, interdisciplinary, Quality Assurance and Quality nonprofit, standards developing, and Control in Diagnostic Molecular educational organization that promotes Biology the development and use of voluntary consensus standards and guidelines The newest forms of clinical diagnostic within the health care community. testing are based upon tools gleaned o Consensus from molecular biology. ▪ General agreement Manipulations of RNA and DNA from ▪ Also called unanimity various tissue sources can provide Recognized worldwide for the application valuable information about an individual's of unique consensus process in the genetic status or the basis or potential for development of standards and guidelines metastasis of a tumor. for patient testing and related health care As of 2002, over 200 laboratories in the issues. United States provide clinical services. Their processed is based on the principle Particularly when such information is to of consensus is an effective way to be used to provide genetic counseling or improve patient testing and health care determine treatment, care must be taken services. to ensure accuracy in the generation and In addition to developing and promoting reporting of results. the use of voluntary consensus standards This unit outlines the aspects of quality and guidelines, they provide an open and assurance that are important in nucleic unbiased forum to address critical issues acid-based diagnostic testing. affecting the quality of patient testing and o Nucleic Acid-based diagnostic health care. testing Publications: A document is published as ▪ Ex. mRNA vaccines a standard, guideline, or report. More specifically, it describes the three Slogan: “CLSI brings the worldwide elements of a quality assurance program. laboratory community to advance Note: common cause” o In Vivo ▪ “Within the living” Current and Future Challenges in ▪ Effects of various biological Quality Assurance in Molecular entities are tested Diagnostics Quality Assurance and Quality Control discusses how to evaluate if a particular The development and performance of genetic test is suitable for clinical testing molecular genetic assays has required o Genetic test/ DNA testing increasingly complex quality assurance in ▪ Examines a persons DNA to recent years and continues to pose new identify changes to genes, challenges. chromosomes, and o New Molecular Genetic Assay proteins ▪ NGS  Next-Generation Quality Control in the Evolving Sequencing Landscape of Molecular Testing ▪ CRISPR-Cas9 Clustered Regularly A QA/QC program for molecular Interspaced Short diagnostics should include independent Palindromic Repeats third-party controls and quality – CRISPR- management software. Associated Protein 9 A laboratory in a cutting-edge field like ▪ TALENs molecular infectious-disease testing Transcription merits an exceptional quality control Activator-like program to strengthen confidence in Effector Nucleases laboratory performance and support Quality management officers, as well as optimal patient care. academic and technical personnel are However, molecular diagnostics is not as confronted with new molecular genetic established as other diagnostics parameters, methods, changing segments, and the corresponding QC regulatory environments, questions standards and regulations are not as well- regarding appropriate validation, and defined quality control for these innovative assays Quality Assurance that are increasingly applying o Overall program to ensure final quantification and/or multiplex formats. results are accurate Yet, quality assurance and quality control Quality control guidelines are still not widely available or o Set of protocols to maintain in some circumstances have become accuracy and precision in the outdated. laboratory For these reasons, the need for solutions The rapid innovation and evolving to provide test confidence continues to regulations around molecular testing can grow. make it difficult to create and maintain an In order to integrate new test procedures effective QC program, accounting for into existing quality assurance measures, quality, efficiency, and regulatory the ISO 15189:2012 guideline can serve requirements. as an orientation. Many articles highlights relevant QC The ISO 15189:2012 guideline describes considerations for molecular diagnostics requirements for medical laboratories and presents effective QC solutions and and thus includes those performing best practices to support dynamic and molecular diagnostics. evolving molecular laboratories. o ISO 15189-2012 ▪ Develop quality Improving Laboratory management systems Performance with Effective QC Many articles gives an overview of the Practices possibilities and challenges in quality assurance of molecular parameters and Compared with other laboratory shows possible solutions. disciplines, the state of the art in quality Over the last 30 years, molecular control (QC) practices for molecular diagnostics have been implemented with diagnostic tests has fallen behind. increasing scope and speed in routine Note: clinical laboratories. o QC is done before testing Frequent Challenges specimens o Quality control QC for molecular diagnostic tests o Sample Preparation encounters many challenges. o Laboratory Contamination In the face of such issues, clinical laboratories are struggling to develop appropriate quality assurance programs  for the molecular diagnostic tests they Materials & Monitoring conduct In addition, legislation and government The laboratorians’ experience and policies threaten to increase oversight of knowledge, the availability of QC genetic tests to a level not seen before in materials, the capabilities of test laboratory medicine. systems, test costs, and regulatory The accuracy of molecular testing is requirements have an impact on current currently unknown but concerns about QC practices. quality in molecular diagnostics can only With the earliest molecular tests that be addressed once data about such were done manually, the results were accuracy are available. determined by interpreting the presence While monitoring molecular test system or absence of bands on a gel. outputs and statistical analysis provide a o Gel Electrophoresis good way to obtain data on accuracy and ▪ Loading dye precision, such traditional QC strategies ▪ Separate DNA fragments have been slow to take root in molecular according to sizes diagnostics. ▪ DNA samples are placed in Systematic quality control is a wells laboratory’s first line of defense against Such testing was often performed by potentially disastrous consequences. researchers with minimal experience in traditional QC techniques, and the QC QC Molecular Diagnostic materials consisted of previously tested Challenges: patient samples. Monitoring for systematic errors was New and rapidly evolving technologies deemed to be unnecessary because test High expectations of accuracy for once in failure was easily detected. a lifetime genetic tests o Systematic Errors Lack of quality control materials ▪ Error in measurement Lack of quantitative test system outputs Daily appearance of new genetic test Specimen Handling targets. Handling specimens according to evidence-based protocols for a particular QC Practices for Molecular Testing procedure is always the best approach to Historically, the first publication of the take. “Westgard Rules” was shown with an However, it is not always possible to know example from clinical chemistry. exactly which assays will be performed. As a result, some people believe that QC In those cases, specimens should be does not apply to other areas of testing, collected according to protocols that are like hematology, immunology, etc. nor to most likely to support the effective the new technologies and devices that maintenance of the biospecimen to most have been introduced to the laboratory in closely resemble its state prior to removal recent years. from the patient. All genetic diagnostic tests and most Having the correct cryovials, vacutainers, other molecular tests are classified as and other containers available at the high complexity and as such must comply collection site is critical, and those with the CLIA regulations for high containers should support anticipated complexity diagnostic tests downstream uses for the samples. Most complicated area in the laboratory Agents that stabilize biological samples such as anti-coagulants may be essential for some downstream applications while having a deleterious effect on others. In addition, some supplies may carry expiration dates that set a defined period of acceptable use  Every effort should be made to track expiration dates and make sure that supplies and reagents are used within the allowable time frame. Container selection is another important area of consideration in the management of patient samples. When handling specimens, universal safety precautions should always be employed. Appropriate protective clothing and personal protective items should be worn o Noxious Agents ▪ Harmful, Poisonous ▪ Destructive to living cells Contamination in Nucleic Acid Testing requiring additional resources to purify or obtain new samples. Contamination Consideration o By preventing contamination, Data Accuracy and Reliability researchers can preserve the o Contamination can introduce integrity of their samples, foreign genetic material into maximizing their utility and samples, leading to inaccurate minimizing the need for sample and unreliable results. repetition. ▪ Common contaminants: Cost and Time Efficiency RNAse o Contamination can lead to wasted RNAse is naturally resources, including reagents, occuring consumables, and time spent on o This can compromise the integrity experiments. Contaminated of research findings and samples may require repetition of potentially lead to incorrect experiments, leading to increased conclusions. costs and delays in research o By preventing contamination, progress. By implementing researchers can ensure the effective contamination accuracy and reliability of their prevention strategies, researchers data. can save valuable resources and optimize their research timelines. Experimental Reproducibility: Ethical Considerations o Contamination can significantly o Contamination can introduce impact the reproducibility of unintended genetic material into experiments. If contaminants are samples, potentially leading to present in samples, it becomes ethical concerns. In research challenging to replicate the results consistently. By implementing involving human samples or robust contamination prevention genetically modified organisms, measures, researchers can contamination can result in enhance the reproducibility of unintended consequences or their experiments, allowing for misinterpretation of results. By validation and verification of prioritizing contamination findings. prevention, researchers uphold Avoiding False Positives and False ethical standards and ensure the Negatives integrity of their research. o Contamination can result in false o Ethical = right and wrong of positive or false negative results, conduct leading to incorrect interpretations. False positives Contamination can lead to unnecessary investigations or treatments, while a process of contaminating false negatives can result in o pano nakakapasok missed diagnoses or overlooked a state of being contaminated findings. By preventing o kung gano ka contaminated ang contamination, researchers can sample minimize the occurrence of false contaminant results and ensure accurate o RNAse interpretations. Is the process of making something dirty Preserving Sample Integrity or poisonous o Nucleic acid samples are often Nucleic Acids precious and limited in quantity. Contamination can compromise are large biomolecules that play essential the integrity of these samples, roles in all cells and viruses. rendering them unusable or a major function involves the storage and expression of genomic information.  A type of Nucleic Acid, the o Continuity of Care deoxyribonucleic acid, or DNA, encodes Understanding Contamination the information cells need to make proteins. Contamination in the context of: A related type of nucleic acid, called o Biological ribonucleic acid (RNA), comes in different ▪ refers to the presence of molecular forms that play multiple unwanted or unintended cellular roles, including protein genetic material in a synthesis. sample. This can occur Are made up of nitrogen containing when foreign DNA or RNA bases, phosphate and sugar molecules molecules from other Nuclein = Nucleic Acid = DNA and RNA sources, such as environmental contaminants or other samples, are introduced into the sample being analyzed. o Amplified nucleic acids ▪ refers to the presence of unwanted or unintended genetic material in a sample. This can occur when foreign DNA or RNA Note: molecules from other RNA mas matibay sa DNA dahil sa Uracil, sources, such as environmental kinakaya ng RNA ang oxidative stress sa labas ng contaminants or other nucleus samples, are introduced Kaya nasa loob ng nucleus ang DNA dahil Madali into the sample being silang masira, unlike RNA na pwedeng lumabas analyzed. Oxidative stress yung pressure na nasa labas ng ▪ Enhanced nucleic acid/ cytoplasm mutated o Non-amplified nucleic acids Data Accuracy ▪ For non-amplified nucleic acids, contamination refers medical data accuracy is of paramount to the presence of importance in the healthcare industry due unwanted genetic material to its direct impact on patient care, safety, in a sample that has not and overall healthcare outcomes. The undergone any following are key reasons highlighting the amplification. This can importance of medical data accuracy: occur during sample o Patient Safety collection, handling, or ▪ accurate medical data storage, where external ensures that healthcare DNA or RNA molecules providers have reliable contaminate the sample information about a and may interfere with patient's medical history, downstream analysis. allergies, medications, and ▪ Unenhanced nucleic acid previous treatments. ▪ Downstream analysis = any o Clinical Decision-Making analysis done after some ▪ Healthcare professionals common procedures rely on accurate and up-to- date medical data to make informed clinical decisions. ▪ Maling diagnosis METHODS FOR MONITORING o Biosensors: These devices use CONTAMINATION, SUCH AS PCR- biological components, such as enzymes or antibodies, to detect BASED ASSAYS AND SEQUENCING contaminants. They offer rapid and TECHNOLOGIES sensitive detection and are used in PCR-based assays, such as polymerase various industries. chain reaction, are commonly used to ▪ E. coli detect and quantify specific DNA or RNA DETECTION TECHNIQUES sequences. This technique amplifies the target sequence, making it easier to Different detection techniques for detect even in low quantities. PCR-based identifying contamination assays can be designed to specifically o Microbiological Culture: This target contaminants of interest, allowing traditional technique involves for accurate and sensitive detection. They culturing samples on selective are widely used in various industries, media to grow and identify including food safety, environmental microorganisms. It allows for the monitoring, and healthcare. detection and quantification of Sequencing technologies, on the other specific contaminants, such as hand, provide a more comprehensive bacteria, fungi, or viruses. approach to contamination monitoring. o Polymerase Chain Reaction Next-generation sequencing (NGS) (PCR): PCR is a molecular biology technologies, such as Illumina technique that amplifies specific sequencing, allow for the simultaneous DNA or RNA sequences. It is sequencing of millions of DNA or RNA commonly used to detect and fragments. This enables the identification identify genetic material from of not only known contaminants but also contaminants. PCR-based assays the discovery of novel ones. NGS can can provide rapid and sensitive provide a detailed profile of the microbial detection of target organisms or community present in a sample, helping genetic markers. to identify potential sources of o Immunoassays: Immunoassays contamination. use antibodies to detect and In addition to PCR-based assays and quantify contaminants. They can sequencing technologies, other methods be designed to target specific for monitoring contamination include: proteins, toxins, or other o Immunoassays: These assays use molecules associated with antibodies to detect specific contaminants. Immunoassays are contaminants. They are commonly widely used in food safety testing, used in food safety testing and environmental monitoring, and environmental monitoring. medical diagnostics. ▪ Used for antibody detection ▪ Are bioanalytical methods o Microbiological culture: This o Next-Generation Sequencing traditional method involves (NGS): NGS technologies allow for growing microorganisms on the simultaneous sequencing of selective media to identify and millions of DNA or RNA fragments. quantify contaminants. It is widely This technique provides a used in the food and beverage comprehensive profile of the industry. microbial community present in a ▪ Bacteria sample, enabling the identification o Mass spectrometry: This of known and unknown technique can be used to detect contaminants. and quantify contaminants based o Mass Spectrometry: Mass on their mass-to-charge ratio. It is spectrometry is a technique that commonly used in environmental measures the mass-to-charge monitoring and forensic analysis. ratio of molecules. It can be used to identify and quantify  contaminants based on their water before and after handling unique mass spectra. Mass samples, using the restroom, or spectrometry is commonly used in touching any potentially environmental monitoring, food contaminated surfaces. Hand safety, and forensic analysis. sanitizers can be used as an ▪ Molecular weight of additional measure. particles o Sterilization techniques: Use o Biosensors: Biosensors are appropriate sterilization devices that use biological techniques for lab equipment, components, such as enzymes or such as autoclaving, UV antibodies, to detect irradiation, or chemical contaminants. They offer rapid and disinfection. Sterilize all reusable sensitive detection and can be materials, including glassware, designed for specific pipettes, and tools, before and contaminants or classes of after use to eliminate any potential contaminants. contaminants. o Chemical Analysis: Various ▪ Process of complete chemical analysis techniques, elimination such as chromatography or o Sample handling precautions: spectroscopy, can be employed to Follow proper techniques for identify and quantify sample handling to minimize the contaminants. These methods rely risk of contamination. Use sterile on the unique chemical properties containers, pipettes, and tips for of contaminants for detection. transferring samples. Avoid ▪ Chromatography is a touching the inside of containers process of separating or tubes with non-sterile objects. components of the mixture o Separate work areas: Maintain separate work areas for different LABORATORY PRACTICES types of experiments or samples Minimizing contamination risks in the to prevent cross-contamination. laboratory is crucial to ensure the Use dedicated equipment and accuracy and reliability of experimental tools for specific tasks and avoid results. Here are some essential sharing them between different laboratory practices to help minimize experiments or samples. contamination risks: o Regular equipment o Personal Protective Equipment maintenance: Calibrate and (PPE): Always wear appropriate maintain laboratory equipment PPE, such as gloves, lab coats, regularly to ensure accurate and safety goggles, and masks, reliable results. Malfunctioning or depending on the nature of the contaminated equipment can lead experiment. PPE acts as a barrier to erroneous data. between you and the samples, o Proper waste disposal: Dispose reducing the chances of of all waste materials, including contamination. biological, chemical, and o Clean and organized workspace: hazardous waste, according to Maintain a clean and clutter-free appropriate protocols. Use workspace. Regularly clean and designated waste containers and disinfect the laboratory benches, follow local regulations for equipment, and surfaces to disposal. prevent the buildup of ▪ 3Rs = Reduce, Reuse, and contaminants. Use designated Recycle areas for different tasks and avoid o Training and awareness: Provide cross-contamination. proper training to laboratory o Proper hand hygiene: Wash your personnel on good laboratory hands thoroughly with soap and practices, including contamination  prevention. Regularly update and Hence, alternative diagnostic methods, reinforce safety protocols and such as molecular biology techniques guidelines to ensure everyone is have been implemented. aware of the best practices. o Coproculture = culture of feces o Monitoring and quality control: ▪ Stool culture Implement regular monitoring and However, there are discrepancies in the quality control measures to reported accuracy of PCR. identify and address any potential o PCR = polymerase chain reaction sources of contamination. This ▪ PCR is a confirmatory test can include regular environmental monitoring, sample testing, and proficiency testing. ▪ Quality control Process that ensures product quality is maintained or improved By following these essential laboratory practices, you can minimize contamination risks, maintain the integrity of your experiments, and ensure the reliability of your results. Sample Integrity simply refers to the reliability or the quality of a sample being tested Note: The process involves HEAT, 65 – 98C Accuracy of Molecular Biology Techniques for the Diagnosis of Microorganisms Strongyloides stercoralis This parasite’s infection is a neglected tropical disease which can lead to severe symptoms and even death in immunosuppressed people. Unfortunately, its diagnosis is hampered by the lack of a gold standard, as the sensitivity of traditional parasitological tests is low. PROTEIN SYNTHESIS Note: 1. Initiation (Start of Transcription) Template Strand (3’ to 5’) TATA Box: This is a short DNA sequence rich in Thymine (T) and Adenine (A), found in the promoter region. Process: 1. Transcription factors (helper proteins) bind to the TATA Box. 2. This helps RNA polymerase attach to the promoter region (a section Notes: of DNA that signals "start here!"). DNA cannot withstand “OXIDATIVE 3. The DNA unwinds, and RNA PRESSURE”, so it is needed to break as polymerase is ready to begin RNA transcription. Ribosomes o Protein production 2. Elongation (Building the mRNA Strand) RNA polymerase moves along one strand Transcription of DNA (the template strand) and adds The cellular process by which DNA is RNA nucleotides (A, U, G, C) to create the copied to RNA & occurs in the nucleus. mRNA. Important rule: 3 Phases of Transcription o A (Adenine) in DNA pairs with U 1. Initiation (Uracil) in RNA (instead of T). o Begins once the promoter gene sequence is detected by the o T (Thymine) in DNA pairs with A in transcription factors called TATA RNA. box. o C (Cytosine) pairs with G o TATA box + Transcription Factors (Guanine). (RNA Polymerase) = Transcription o G (Guanine) pairs with C Initiation Complex (Cytosine). o TATA Box o A DNA sequence that This continues until the RNA polymerase indicates which species to reaches a stop sequence in the DNA. other molecules where 3. Termination (Ending Transcription) - More transcription begins. Details 2. Elongation o RNA Polymerase unwound DNA RNA polymerase keeps adding RNA bases Strand until it reaches a stop signal in the DNA. o RNA Polymerase adds the When it hits this stop sequence, it lets go complementary nucleotides to of the DNA and the newly made mRNA builds the mRNA molecule, using strand is released. complementary base pairs. The mRNA still needs some finishing 3. Termination touches before it can be used to make o RNA polymerase crosses a stop proteins. sequence in the gene Once it's fully processed, mRNA leaves o The strand is called a pre-mRNA the nucleus and goes to the ribosome to strand, help make proteins. o Pre-mRNA must be processed to a “mature mRNA”. Translation 4. Component of all cell membranes Process by which RNA transcripts are turned into proteins and peptides & occurs in the cell cytoplasm. Proteins Proteins make up all living materials. Function of Proteins 1. Help fight disease o Antibodies – fights foreign bodies 2. Build new body tissue 3. Enzymes used for digestion and other chemical reactions are proteins (Enzymes speed up the rate of a reaction) o Digestive enzymes ▪ Amylase ▪ Pepsin ▪ Lipase o Transport Enzymes ▪ Transferrin ▪ Hemoglobin o Structure ▪ Actin ▪ Collagen ▪ Keratin o Muscle Contraction ▪ Myosin o Defense ▪ Antibodies o Albumin ▪ Liver; stops fluids from going out.