Nucleic Acid Amplification Based Testing in Indian Clinical Laboratories
Nucleic acid amplification test (NAAT) is a rapid and reliable molecular technique for quantitative and qualitative assessment of infectious and non-infectious markers for various diseases. PCR, the first ever nucleic acid amplification, invented by Kary B. Mullis in the 1980s, serves as the backbone for NAAT. This was developed to detect virtually every clinically relevant pathogen and identify pertinent DNA or RNA target sequences. Developed nations were able to utilize this relevant advancement by the late 1990s for the qualitative detection of highly prevalent diseases. Two decades later, the developing nations got their hands on this revolution through adoption in reference laboratories. NAAT was able to overcome the shortcomings of tradidional appraoches, leading to remarkable sensitivity, overwhelming specificity, and previously unattainable turnaround time. NAAT turns out to be the most accurate detection method against multiple infectious agents, leading to its widespread adoption in laboratories throughout the globe. Limitations to the PCR amplification technique do exist but did not halt the progression of NAAT and various novel amplification chemistries have been devised. Constraints like preventing crossover contamination due to increased sensitivity, presence of inhibitors in clinical specimens and nucleic acid degradation in NAAT are yet to be eradicated. The possibility of limiting such problems can be achieved through meticulous protocols. The headway made in NAAT leads to its operation over international borders to keep a check on pandemic diseases, screen or monitor infections, genetic and oncological diseases, and donor-recipient compatibility in solid organ transplant (SOT) and hematopoietic stem cell transplantation (HSCT). Timely detection of the molecular diagnostic markers will be lucrative for expeditious patient management. The present study is designed to look into the revolutionary leap brought by NAAT in the field of clinical diagnostics.
Keywords: PCR, DNA, RNA, Molecular techniques
|CAGR||Compound Annual growth Rate|
|CAP||College of American Pathologist|
|CB-NAAT||Cartridge Basedd Nucleic Acid Amplification|
|cDNA||Complementary Deoxyribonucleic Acid|
|dPCR||Digital Polymerase Chanin Reaction|
|ddPCR||Droplet Digital Polymerase Chain Reaction|
|dsDNA||Double Stranded Deoxyribonucleic Acid|
|ELISA||Enzyme-Linked Immunosorbent Assay|
|FDA||Food and Drug Administration|
|FRET||Fluorescence Resonance Energy Transfer|
|HBV||Hepatitis B Virus|
|HCV||Hepatitis C Virus|
|HIV||Human Immunodeficiency Virus|
|HVAC||Heating, Ventilation, and Air Conditioning|
|IVD||In Vitro Diagnostic Device|
|LAMP||Loop Mediated Isothermal Amplification|
|LCR||Ligase Chain Reaction|
|MDA||Multiple Displacement Amplification|
|NAAT||Nucleic Acid Amplification Test|
|NASBA||Nucleic Acid Sequence Based Amplification|
|NABL||National Accreditation Board for Testing and Calibration Laboratories|
|PCR||Polymerase Chain Reaction|
|RT-PCR||Real-Time Polymerase Chain Reaction|
|RCA||Rolling Circle Amplification|
|SDA||Strand Displacement Amplification|
|SDS-PAGE||Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis|
|USD||United States Dollar|
|WGS||Whole Genome Sequencing|
The emergence of primitive NAAT technology can be traced back to the discovery of Polymerase Chain Reaction (PCR) by Kary B Mullis in the 1980s  . PCR, the most significant headway of the time for molecular biology research  is a DNA amplification method capable of attaining millions of target copies from a single strand, thus laying the foundation for NAAT. The development of thermal-cycler instrument by PerkinElmer in 1985 aided the previously tedious manual thermal cycling process, with the commercial version built by Cetus, in collaboration with Kodak, made available in 1986  . The real-time PCR (RT-PCR) was a triumph for amplification technology ; it possessed simultaneous amplification and amplicon detection ability,   leading to widespread acceptance due to reduced carryover contamination, improved sensitivity, specificity, and reproducibility. Modification of RT-PCR to amplify and detect RNA led to a broad range of pathogen detection capabilities. Clinical microbiology was the first to adopt PCR and RT-PCR due to its edge over conventional techniques like electrophoresis and cell culture. Further advancements in research lead to numerous alternative nucleic acid amplification methods such as Loop-Mediated Isothermal Amplification (LAMP), Nucleic Acid Sequence Based Amplification (NASBA), Strand Displacement Amplification (SDA), Multiple Displacement Amplification (MDA) and many more, possessing unique distinguishable properties besides common characteristics . However, many of these are not currently economically viable and technically suited for versatile real-world applications. Multitude of NAAT based screening and diagnostic assays are being developed; CB-NAAT, a self-contained cartridge based semi-quantitative nested RT-PCR test for Tuberculosis diagnosis  is one such example from the present.
The completion of the Human Genome Project outset a revolution that lapsed three decades in the field of medical diagnostics in addition to biomedical sciences, clinical practices, and healthcare technologies. The enormous sequenced data of humans, as well as microorganisms, laid the framework to create medicines, engineering of new molecular markers for infectious agent detection, diagnosis of genetic disorders and non-infectious diseases, development of vaccines and other diagnostic tests . This emanated molecular method like nucleic acid-based testing for the detection and characterization of microorganisms and has remodeled diagnostic microbiology which is an integral part of routine specimen processing in clinical laboratories . Laboratories throughout the developed countries were switching to culture independent diagnostic testing (CIDTs) as a means for efficient and rapid detection of pathogens. This shift required large investments and new infrastructure guidelines to ensure a unidirectional workflow; separate rooms for reagent preparation, sample preparation, and amplification and product visualization, biosafety cabinets, and various instruments along with consumable items. Developing nations came across substantial monetary investment requirements for NAAT instrumentation, reagents, training facility and faculty and hence faced a major stumbling block in its routine use in most diagnostic laboratories . This delayed the adoption of NAAT technology by nearly a decade before its ultimate implementation in developing countries through reference laboratories.
Currently, molecular genetic methods in Indian clinical diagnostic laboratories are still in its infancy. Besides that, India is lagging behind when it comes to imbibing molecular genetics in clinical practice when comparing to the West or Far Eastern nations. India with over 100,000 diagnostic laboratories of which 70 percent offer pathology services and 30 percent offer radiology and imaging services , sustains a colossal network of unchecked and unregulated scattered diagnostic laboratories, only 795 of which are accredited by NABL - a national level quality council board of India . Only the top private corporates in India adopt these rapid technologies for their minuscule advantages, disregarding the constraints that are applicable to a developing country. Worldwide there are 121 different NAAT based tests for diagnosis of human diseases and 327 microbial tests for various parameters, approved and listed by the Food and Drug Administration (FDA) , yet few of these made it into the developing diagnosis markets.
The most significant application of NAAT is in clinical laboratories for diagnosis of infectious agents, pathological and oncological studies. In case of pathology, NAAT serves as a specific, sensitive and rapid    tool for identification of organisms for proper diagnosis, prognosis, and treatment of most diseases; while in virology, it further aids in identifying genotypes and subtypes, for determining antibiotic resistance, and measuring viral load. It is also being extensively used to scan pathogens and to reduce transfusion-transmitted infections because of the inability of serological methods for early detection. Other implementations include the field of transplantation where specific genes related to surface antigens are typed to predict patient-donor compatibility, in oncology by identifying genetic variations and predicting predisposition to cancer and screening genetic disorders by analyzing mutations which leads to various syndromes and also facilitating the field of pharmacogenomics to characterize gene expression and develop personalized medicine.
Objectives of the Research
● To asses downstream processing in molecular diagnostic laboratory
● To analyze state-of-the-art extraction, quantification, amplification and detection platforms employed in nucleic acid amplification testing.
● To evaluate key drivers and restraints within the Indian clinical diagnostic labs
- To analyse downstream molecular diagnosis being in one of the major Indian clinical laboratory, SRL Ltd and evaluating the key drivers and restraints, as well as the challenges faced by the Indian diagnostic/clinical sector.
Disease and diagnosis began with Egyptian medicine, the crucial sources being Edwin Smith Papyrus (17th century BC) and Papyrus Ebers (about 1550 BC). However, despite the many thousands of ritualistic and painstaking embalming during nearly 5,000 years of successive Egyptian dynasties, these surviving papyri contain only a slender body of information on pathological anatomy and the Egyptians developed very little systematic knowledge of these phenomena. It was not until the last three centuries BC that the Alexandrian Greeks, heavily influenced by Hippocrates, made lasting contributions to anatomy and pathology. The industrial and scientific revolution in the late 1800′s led to the wide use of microscopes to visualize microbes directly and to their classification into broad categories. These considerable attempts made microscopes a driving force to propel pathology forward while witnessing other new technology that remolded Pathology. The microscope was a driving force, but it was not the only force propelling medicine forward. The role of microscopy in pathology became evident in a kind of competition between Carl von Rokitansky (1804–1878) and his one-time pupil Rudolf Virchow (1821–1902). The latter came to use the microscope routinely in his autopsy studies. The microscope totally changed concepts of disease and shifted the focus from whole organs down to cells; it enabled the practice of histopathology and spawned numerous attendant advances in techniques necessary for modern practice and forged links between bacteriology and infectious disease .
Over the past 100 years, diagnostic testing has become a critical feature of standard medical practice . Entering the twentieth century, the pace of research in pathology alongside molecular biology palpably accelerated, the number of discoveries grew almost exponentially. From the basic features of histopathology and exemplifying the primacy of the microscope in pathological research and diagnosis, to blood typing which had great implication in fields of blood transfusion, and eventually tissue transplantation alongside the basis of modern understanding of nucleic acids and various chemistries for cloning, amplifying and typing of these biomolecules entangled with modern day genotypic approach for diagnosis.
The rudimentary diagnostic was based on interpreting various parameters by culturing organisms . Further conventional microbiology testing has generally included microscopy, biochemical profiling, differential staining techniques, antigen detection and serology. Microscopy has had an important role in the rapid detection of bacterial, fungal and protozoal diseases  . In the case of viruses, electron microscopy is required but this is rarely practical for clinical purposes. Antigen detection techniques usually rely on monoclonal antibodies directed against specific antigens, and modalities include ELISA and tagged immune-microscopy. While these tests are usually quite specific, their sensitivity is often low and a high false positive result is clinically unhelpful. Rapid detection and identification of microorganisms without culture was made possible by the development of immunoassays. Serology testing is based on the detection of host response to infection and is very specific in some cases (such as with HIV infection) while in others, cross-reactivity and nonspecific antibodies result in high rates of false positives. Perhaps most importantly, time taken for host seroconversion to the infecting agent rarely helps in acute disease diagnosis  thus serology can be ineffective for early diagnosis of infection. On the other hand requirements for standardization, quality, efficiency, and reduced labor costs have led to the introduction of automated systems into the microbiology laboratory for the isolation and identification of microorganisms. Continuously monitored blood culture systems such as BACTEC 9000 (BD Diagnostics, Sparks, MD) or BacT/ALERT are standard laboratory equipment today. For identification and drug-susceptibility testing, both manual and automated systems are well established. All systems are based on the miniaturization of conventional methods to reduce the volume of reagents, increase the user-friendliness, and shorten the time for obtaining a result . Generally speaking, when cultures are positive they represent a ‘gold standard’ diagnosis. However, occasionally, phenotypic testing alone proves challenging when combined with poor sensitivity, increased resource consumption, and contamination probability, false negatives, prolonged incubation time and expertise required for culture-based testing .
Both culture-based identification and immunological assays use the phenotypic characteristics of the microorganism. However, identification criteria such as colony morphology or production of certain antigens can change or be influenced by nutritional or environmental conditions and may lead to misinterpretation of results. These drawbacks were filled by the genotypic based methodologies starting with the evolution of the study of microbial genetics which has led to new frontiers to recognize pathogenic microbes in-situ within the host and in vitro without cultivation of the organism. These technologies can be applied to the recognition of the organism, its DNA/RNA, its antigenic components, specific virulence genes, and antibiotic resistance genes. Molecular methods that rely upon nucleic acids are classified into hybridization, amplification, and sequencing. Hybridization makes use of the complementary binding property of nucleic acid along with the ability to form various DNA and RNA hybrids. Amplification by PCR marked the birth of molecular diagnosis  and is done by increasing nucleic acid copy number exponentially to detectable limits . The development of nucleic acid-based amplification technologies (NAATs) including polymerase chain reaction (PCR) has enabled the detection of microbial and host genetic sequences with high sensitivity and specificity , along with the ability for microbial characterization. Tropheryma whipplei and Hepatitis C virus are examples of uncultivable organisms first detected through molecular methods. In addition to the detection and quantification of many bacterial, viral and fungal pathogens they have revolutionized the development of drugs for the treatment of HIV, HBV and HCV infections. The discovery of PCR brought enormous benefits and scientific developments such as genome sequencing, gene expressions in recombinant systems, the study of molecular genetic analyses, including the rapid determination and diagnosis of infectious diseases . PCR chemistries gained the title ‘gold standard’ for various viral and bacterial-induced infections replacing traditional culture based methods . On the other hand, multiple amplification technologies emerged over time to overcome the minor shortcomings of the PCR technology , but many of these did not gain acceptance as the original amplification method. One popular platform is the GeneXpert MTB/RIF, which is a ubiquitous NAAT based assay that is embedded throughout the globe as the first line for tuberculosis screening as recommended by WHO. These methods detect TB early, accurately and play a crucial role in reducing the burden of drug-resistant tuberculosis. There are considerable advantages for the programmatic management of drug-resistant TB such as speed, standardization of testing, potentially high throughput and reduced laboratory biosafety requirements. India is desperate to adopt modern, rapid, molecular tools with point-of-care test for eradication of high burden diseases in the country .
The present-day diagnosis of various infectious and non-infectious diseases involves some form or the other of nucleic acid based testing. Nucleic acid-based diagnostics are capable of detecting a multitude of pathogens through the identiﬁcation of their genomic sequences and are exclusively performed in centralized laboratories using high end instrumentation and skilled personnel. They have shown promise to overcome most limitations of conventional testing methods and a possibility of rapid phase adoption in clinical diagnostics as the point of care testing when adaptable devices are available commercially . The understanding of the bigger picture was attained when the sequencing technologies like pyrosequencing and next generation sequencing came into existence possessing the ability to directly read the base pairs which build the genome comprising of nucleic acids . Whole Genome Sequencing (WGS) gave the microbial profile as it stands in situ in combination with the computational systems that perform the analysis of data to allow for rapid turnaround time. WGS has been used for identification from cultured isolates or directly from clinical specimens. Sequencing is also used to demonstrate antibiotic resistance and identify known or new mutations. At this point, the approach is restricted to reference laboratories because of the complexity and cost. The next generation sequencing (NGS) provides increased accuracy and is less labor-intensive but requires extensive bioinformatics for data interpretation. For diagnostic testing, compact NGS systems with a small footprint and fast turnaround time have been developed. These features make NGS highly competitive with any currently available technology including conventional methods of in vitro cultivating bacteria, while potentially replacing complex multifaceted conventional microbiological identification procedures for phenotype identification in addition to detecting genetic markers of virulence and antibiotic resistance. Additionally, for diagnostic utility, the NGS system is easy, concise, low-cost data management and interpretative software and access to commercially available vetted databases . The ability to identify single base changes or single gene acquisitions associated with novel clinical syndromes is of particular significance in immune-compromised patients. All these make NGS preferable as a developing gold standard replacement in molecular diagnosis leading to reliable, swift and better patient management.
MATERIAL AND METHODOLOGY
Nucleic Acid Extraction
There are various methods used for nucleic acid extraction depending on the downstream processing technologies, chemistries and platforms. The most commonly used, ecnomical, TAT-efficent and high-end quality techniques are as follows :
i) Silica-Based Technology
This is a widely employed method in current kits. Nucleic acids adsorb specifically to silica membranes/beads/particles in the presence of certain salts and at a particular pH. The cellular contaminants are removed by wash steps followed by DNA elution in a low salt buffer or elution buffer. Chaotropic salts are included to aid in protein denaturation and the extraction of DNA. This method can be incorporated in spin columns and microchips are cost-effective, has a simpler and faster procedure than the organic extraction, and is suitable for automation. Kits based on this method include Purelink Genomic DNA extraction kit (Invitrogen) and DNeasy Blood and Tissue Kit (Qiagen), OIAmp DNA min and RNeasy mini (Qiagen).
ii) Magnetic Separation
This method is based on reversibly binding DNA to a magnetic solid surface/beads/particles, which have been coated with a DNA binding antibody or a functional group that interacts specifically with DNA. After DNA binding, beads are separated from other contaminating cellular components, washed and finally, the purified DNA is eluted using ethanol extraction. This method is rapid and can be automated. However, it can be more costly than other methodologies. Examples are Agencourt DNAdvance Kit (Beckman Coulter) and Magnetic Beads Genomic DNA Extraction Kit (Geneaid).
Quantification of Nucleic Acid
Quantification is done to ensure that an adequate amount of DNA or RNA is present to meet the required nucleic acid for amplification by specific instruments. This procedure is only done for non-infectious diagnosis as in the case of transplantation, genetic disorder, oncological screening, etc. Since downstream testing is very sensitive and expensive, human DNA is always quantified prior to analyzing. The DNA concentration can be assessed using four different methods: absorbance (optical density), agarose gel electrophoresis, fluorescent DNA-binding dyes, and a luciferase-pyrophosphorylation-coupled quantitation system. At SRL, the automated fluorometric method is used due to its convince and advantages when compared with other methods.
i) Fluorescent Dyes
It works on the principle of nucleic acid specific dye which emits extreme low fluorescence until they bind to the target molecules; the binding of dye is usually by intercalation between the bases leading to intense fluorescence. At a specific amount of the dye, the amount of fluorescence signal from this mixture is directly proportional to the concentration of DNA in the solution, even in the presence of other biomolecules. The signal is picked up by the instrument and is converted to a DNA concentration measurement by referring to DNA probes of known concentration provided by the sample calibration process. It then uses this relationship to calculate the concentration of a sample. Qubit Fluorometer by life technologies is a commonly used instrument.
Pre-Amplification Reaction Setup
Nucleic Acid Amplification
- Polymerase Chain Reaction
Polymerase chain reaction, a technique discovered by Kary B Mullis in the 1980s and developed by researchers at the Cetus Corporation is a simple laboratory technique to obtain exponential copies of specific DNA fragments even from samples containing minute quantities of DNA. The essential components of PCR are:
i) Template DNA- a DNA strand that contains the target sequence of interest needed to be amplified
ii) Primers- It consists of a designed pair (forward and reverse) of synthetic oligonucleotide which are complementary to the 3’ ends of each of the two strands of DNA target region.
iii) DNA polymerase- enzyme which catalyses the template dependent synthesis of DNA, the commonly used enzyme is the thermostable Taq polymerase (originally isolated from thermophilic bacterium Thermus aquaticus)
iv) Divalent cations- which are the cofactors and catalyzes the polymerase enzyme, usually used Mg 2+.
v) Deoxynucleoside triphosphates (dNTPs)- Equimolar amounts of each dNTP (dATP, dCTP, dGTP, dTTP), which are the building blocks used by the DNA polymerase enzyme to synthesize a new strand of DNA.
vi) Buffer-used to maintain suitable ionic environment for optimum activity and stability of polymerase enzyme.
One PCR cycle consists of three steps:
i) Denaturation- The first step of PCR where the sample is heated to separate or denature the two strands of the DNA (>90°C).
ii) Annealing- Following the denaturation step, the reaction temperature is lowered (usually 3-5°C below the Tm of primer) to allow the oligonucleotide primers to bind to the single strands of the template DNA.
iii) Extension- The final step of the PCR where the temperature is raised, typically to 72°C, allowing specific polymerase enzymes to synthesize a new DNA strand complementary to the DNA template.
The PCR process is commonly performed with the in small reaction tubes of volume 10-200 µl placed in a thermocycler instrument which heats and cools the reaction tube to set cycle parameters. Typically this step is repeated 25- 50 times. Each cycle theoretically doubles the amount of the target DNA present in the sample. Post PCR analysis can be done by staining DNA on agarose gel, capillary electrophoresis, solid phase or solution hybridization, high performance liquid chromatography, Southern blotting or using the gold standard DNA sequencing.
PCR do have few shortcomings such as non-specific amplification of DNA targets, carry over contamination amplification, interference of inhibitors etc., attempts were made to overcome this by devising various new chemistries based on the same principle.
- PCR Chemistries
i) Real time PCR (RT-PCR) - The Real Time PCR method employs simultaneous amplification and detection and quantitation of an amplified PCR product in ‘real time.’ This approach of PCR is based on the incorporation of intercalating fluorescent dye, SYBR green, dual labeled probes (TaqMan), hybridization probes (Light-Cycler), Molecular Beacons, or Scorpions as means of detecting amplification (increase in fluorescence signal, generated during the PCR, is in direct proportion to the amount of the PCR product). This modification avoids the requirement of a separate amplicon detection step. Here, the fluorescent molecules added to the PCR mixture produce fluorescent signals which are detected simultaneously with the progress in amplification. RT-PCR offers many advantages such as increased sensitivity, rapidity, broader dynamic range, elimination of post amplification handling steps and higher throughput. Use of a closed system minimizes carry-over contamination and melting curve analysis, which allows the amplification product to be discriminated from nonspecific products or primer-dimer turnaround time, dynamic range of target detection, and feasibility for quantitation are a few of the advantages of this method.
ii) Reverse transcriptase PCR (rt-PCR) - In this variant, a strand of RNA is initially reverse transcribed into its complementary DNA or cDNA using the reverse transcriptase enzyme. The resulting cDNA is further amplified by PCR. The reverse transcription step can be performed either in the same tube with PCR (one-step PCR) or in a separate one (two-step PCR) depending on the properties of the reverse transcriptase enzyme used. The RT-PCR is used for detection of RNA viruses in clinical samples and in gene expression studies.
iii) Nested PCR - Nested PCR is a conventional method involving two successive PCRs, where the amplification product from the first PCR reaction is used as the template for the second PCR. Either one of the primers (semi-nested PCR) or both primers (nested PCR) used in the second PCR may be different from the primers used in the first PCR. It has been employed to detect pathogens present in low copy numbers in specimens, and has the benefits of enhanced sensitivity and specificity but can be problematic due to carryover contamination from the first reaction to the second. Nested PCR has also been used for the detection of the 16s and 23s rRNA genes from a variety of bacteria and provides multiple amplicons for accurate sequencing of these genes.
iv) Multiplex PCR - Here multiple selected target regions in a sample can be amplified using different primers pairs. Since it can be used to detect multiple genes of interest in one specimen, it can minimize the number of separate reactions and help conservation of time, reagents and samples that are of limited volume.
v) In-situ PCR - The PCR amplification reaction takes place within the cell which is often fixed on a slide. It can be employed for the detection of nucleic acid in small tissue samples. The PCR master mix is directly applied onto the sample on a slide, and then both are covered using a coverslip, and the latter is subjected to amplification in a thermocycler with a slide adaptor.
vi) Digital PCR- This is a refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify nucleic acids including DNA, cDNA or RNA. The key difference between dPCR and traditional PCR lies in the method of measuring nucleic acid amounts, with the former being a more precise method than traditional PCR, though also more prone to error in the hands of inexperienced users. PCR carries out one reaction per single sample. dPCR also carries out a single reaction within a sample; however, the sample is separated into a large number of partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid amounts. The method has been demonstrated as useful for studying variations in gene sequences – such as copy number variants and point mutations – and it is routinely used for clonal amplification of samples for “next-generation sequencing.”
The amplicon can be visualized either separately after amplification (endpoint detection) or during the process (real-time detection). End point is a longer detection process that requires less complex instrumentation while providing simpler outputs for interpretation. Real time methods integrate amplification with detection, and are superior for quantitative analyte detection with a wide dynamic range. In all cases, detection methods are preferable that can differentiate target specific amplicons from non-specific amplification products which minimize the risk of carry over contamination.
- Traditional Detection Methods:
i) UV Spectroscopy
This technique works on the property of UV absorbance by biomolecules and is utilized to estimate the concentration and purity of DNA and RNA. The amount of DNA in a sample can be estimated by looking at its absorbance at a wavelength of 260nm or 280nm in the UV region.
This was the most ubiquitous detection method for amplified nucleic acid, depending on the type and weigh of nucleic acid gel matrix of varying concentration are selected. Agarose gel with 0.8-2% concentration is normally used for DNA and RNA detection alongside polyacrylamide gels for RNA. Electrophoresis is often combined with fluorescent or regular dyes to visualize the nucleic acid bands after the separation over the gel. EtBr is commonly used as a fluorescent dye for nucleic acid staining and functions by binding as well as intercalating with nucleic acid on the major and minor grooves while giving orange fluorescence under UV radiation from 500 – 590 nm. Usually EtBr is added to the agarose gel before casting. EtBr molecules bind with nucleic acids in the electrophoresing gel and gives out fluorescence under UV light.
- Real Time Analyzing Chemistries:
The real time fluorescence detection for PCR or isothermal amplification reaction can be mediated by intercalating dyes, by oligonucleotide probes that are cleaved during the reaction, or by using a probe or primer that change conformation upon target amplification.
i) SYBR Green
An asymmetric cyanine fluorescent dye that binds to the minor groove of dsDNA, developed as a safer and more sensitive alternative to ethidium bromide. Unbound SYBR green barely fluorescent in solution, but binding with DNA increases fluorescence intensity by 100- fold by undergoing conformational changes, absorbing blue light (λmax = 497 nm) and emitting green light (λmax= 520 nm). This chemistry provides a simple and inexpensive approach to all well-established real time amplification by aiding detection of PCR product quick and efficiently without causing inhibition or interference in PCR process. The drawback of this dye is that it binds to any double stranded DNA present in the reaction, includes primer dimer and other nonspecific reaction products.
ii) TaqMan Probes
These are target specific fluorogenic-labeled oligonucleotide probe with a fluorescent reporter dye on the 5' end and a quencher dye on the 3' end. When the probe is intact, the proximity of the quencher dye greatly reduces the fluorescence emitted by the reporter dye by fluorescence resonance energy transfer (FRET). When bound to target sequence, the 5’ nuclease activity of taq DNA polymerase cleaves of reporter dye facilitating quencher dye to fluorescence in each extension cycle of the PCR. Resulting in an increase in fluorescence intensity proportional to the amount of amplicon produced.
Amplicon sequencing is a highly precise target based approach which enables the identification of allele level genotypic variation. This ultra-deep sequencing of amplified products allows efficient variant identification and characterization. This method uses oligonucleotide probes designed to target and capture regions of interest, followed by next-generation sequencing (NGS). It is primary employed for the discovery of rare mutations or while dealing with highly complex samples (such as tumors mixed with germline DNA) and where conventional detection methodologies fail. The practicality of employing this technique in routine detection is restricted due to the tedious workflow and expense required. However this method of detection proves to screen genetic variants with a highly targeted approach even in difficult-to-sequence areas, such as GC-rich regions along with multiplexing amplicon capabilities and attainting high coverage, reduced turnaround time and cost compared to approaches like whole genome sequencing.
Storage is classified based on the duration of preservation. The extracted nucleic acid is stored in deep freezer (-20°C) for optimum stability of nucleic acid for 6 – 38 months period and long terms, above 3 years, ultra deep freezer (-80°C ) preservation is used to minimize degradation of nucleic acid and preserve integrity of the genetic material.
RESULTS AND OBSERVATIONS
The following observations and inferences were made during my training period at SRL, Gurgaon. I have divided my observations under the following categories, supported by the results.
- Biosafety Guidelines: Safety guidelines by NABL and CAP were strictly implemented in the laboratory. The lab is engineered as a Biosafety level III space and practices grade III in accordance to NABL issued criteria. Every sample received for analysis was considered as pathogenic and was opened in highly equipped biosafety level II rooms; however, the practice followed was biosafety level III, throughout. The procedure like sample pipetting and transfer were strictly carried inside Biosafety class II cabinets at individual workstations for prevention of infection, cross contamination and operator protection. All instruments were biohazard labeled, being operated for handling or processing clinically suspected infectious samples. All of the precautions and Safety measure strictly followed by lab personals were defined under ‘Laboratory Safety Manual’ produced by SRL. Inspection and decontamination of instruments were properly done by dedicated trained staff at specific intervals depending on the requirement, as specified by the manufacturer, (which can be daily, monthly, quarterly, biannually or annually, specific for each instrument).
- Laboratory Space Arrangement: The observed molecular NAAT laboratory contained separate functional work areas: A pre-amplification space, an amplification room and a post-amplification area. Dedicated supplies and equipment were placed at required sites. An optimum utilization of space was achieved by dividing it into four physically separate rooms :
i) Reagent Preparation Room: This room is designated for preparation and storage of reagents. Steps for master mix preparation and aliquoting were done here. It was seen that reagents must be aliquoted into smaller volume to avoid repeated freezing and thawing and also to prevent cross contamination. Contamination deterrent measures such as wiping with ethanol before and after the use of the workbench, use of separate sets of adjustable pipettes with plugged aerosol-barrier tips, laboratory coat, disposable gloves and minimal movement of personals during processing were practiced.
ii) Sample Preparation Room: Nucleic acid extraction and purification, preparation of positive and negative controls were performed here, under the negative pressure to keep aerosol contamination contained inside.
iii) Pre-amplification Room: All procedures like addition of positive, negative and internal controls, master mix inclusion and other essentials for amplification reaction setup were carried out inside biosafety cabinets. The prepared sample is then moved to amplification room.
iv) Amplification Room: This room is designated for amplification instruments like thermocyclers of various types which are classified as conventional, real-time and automated real-time PCR platforms. Stringent measures to prevent amplicon cross contamination were followed by restricting it only to dedicated and trained staff to operate the machines only. The advanced instruments like automated real time PCR machines were capable of simultaneous extraction, amplification and detection of the desired targets.
- Equipment in PCR laboratories: Instruments to carry out routine tasks are placed at designated workspace with individual work areas having their own separate set of equipment, reagents, pipette tips and racks, etc. to be used specifically in that location. Centrifuges for plate and various tubes including microfuge, vortex machines, calibrated pipet set with aerosol barrier tips, refrigerators, deep freezers and consumables like gloves, protective gear, reagents, tubes, enzymes, primers and probes, cartridges were placed near appropriate workstations under recommended manufacturer guidelines.
- Manpower: Trained personnel on working methodology that covers amplification and recombinant DNA theory and practices. Along with knowledge including biosafety, quality management, troubleshooting instruments, waste disposal, emergency procedures in a molecular laboratory. Hands-on training for each technique under the supervision of experienced personnel with individual ability to demonstrate testing procedure and result interpretation successfully by testing confirmed positive and negative control samples before being allowed to analyze diagnostic samples.
- Workflow: A unidirectional flow of the sample was followed in the laboratory for adequate contamination free processing, with individual steps being performed at separate isolated spaces having segregated workstations, supplies and equipment. The nucleic acid extraction room is equipped with a buffer space at the entrance and the inside is maintained under negative pressure by the HVAC system to prevent contaminated air from leaking out.
- Specimen: NAAT were performed on various types of samples, commonly used were blood, plasma, serum, cerebrospinal fluid, sputum, sterile body fluids, bone marrow, tissue, stool, urine, and pus. These samples were collected in sterile containers and sealed, cooled to 4°C while transporting to the testing facility. Whole blood in EDTA / heparin and plasma samples usually transported in 2-4°C , which was stored at -4°C after its arrival at the center and was processed within 48 hours from collection. The excess samples were stored at -20°C for 7 days post the release of test report.
- Nucleic Acid Extraction: The QiAamp silica matrices by Qiagen were used for DNA or RNA extraction. This semi-automatic method is preferred because of its higher yield, ergonomic nature and better economical properties as compared to the other conventional or commercially available methods for processing high sample volumes with accuracy and efficiency while yielding high quality nucleic acid.
|PCI METHOD||ENZYMATIC METHOD||SILICA COLUMN BASED|
|QUANTITY OF DNA||900-100 ng||700-800 ng||100-400 ng|
|PURITY OF DNA||-1.8-1.90||-1.7-1.90||-1.8-1.88|
|DURATION||-2 to 4 hours||-2 to 2.5 hours||-30 to 45 mins|
|COST||Low cost||High cost||Medium cost|
|SAMPLE REQUIREMENT||500- 1.5mL||500-1.5mL||100- 200|
|CsCl Gradient Centrifugation With EtBr||1950s||High purity and yield of nucleic acid.||Laborious, costly and time consuming.|
|Oligo(Dt) Cellulose Chromatography||1972||Fast protocol, good yield.||Purification bias for mRNAs.|
|Alkaline Extraction||1979||Fastest, reliable, and relatively easy procedure.||Medium purity and fragmentation of genomic DNA.|
|FTA Paper||1980s||Consistent results may be obtained without quantification.||Due to static electricity, dry paper punches do not remain stable.|
|Phenol- Chloroform||1987||High purity and yield of DNA.||Hazardous chemicals.|
|Chelex Extraction||1991||Quick and simple protocol; no use of hazardous chemicals.||Low purity of nucleic acids.|
|CTAB Extraction||1993||Efficient method for plant and other “hard to lyse” samples.||Laborious, time-consuming; use of hazardous chemicals.|
|High-purity DNA, easy to perform, and reproducible.||Unable to recover small DNA fragments; one-time use.|
|Simple, sensitive, and reproducible.||High cost; requirement of equipment.|
|Easy to use and storage||Extraction protocols being complex and prone to error.|
|Reduced pipetting error, shorter protocol||High cost|
Anion exchange material
|Reusable resins; delivers high quality DNA for optimal results in sensitive applications||Presence of high salt concentrations|
|No centrifugation, best choice for automation, virtually equipment-free|
Interference in PCR amplification
- Nucleic Acid Amplification and Detection: Presently various automated platforms are available commercially, however, PCR, the gold standard platform and its various chemistries are being excessively employed to amplify purified genetic material due to its convenience and cost effective nature without compromising other parameters, which being apt for commercial setups. The Real-Time chemistry of PCR enabled combined amplification and detection for various applications of viral load monitoring, pathogen detection and quantification and target gene amplification thus being selected as the suitable platform for amplification and detection test. Nevertheless it is not the only amplification platform; various other automated devices that are capable of simultaneous extraction, amplification and detection are also utilized. Platforms such as Cobas Ampliprep, Cobas Taqman, GeneXpert.
PCR: Polymerase chain reaction, LAMP: Loop-mediated isothermal amplification, SR: Sequence replication, SDA: Strand displacement amplification, LCR: Ligase chain reaction, NASBA: Nucleic acid sequence based amplification, RCA: Rolling circle amplification, DNA: Deoxyribonucleic acid, ELISA: Enzyme-linked immunosorbent assay, ECL: Electrochemiluminescent.
- Amplicon Detection and Analyzing: Conventional detection method like agarose gel electrophoresis, SDS-PAGE are extremely limited in present day clinical laboratories due to their practicality, long turnaround time and complex procedures. Instead today, mostly detection is carried over alongside amplification using RT-PCR, or using specific high resolution end product detectors like Mr.SPOT, or Lumix Xmap. While there are other fully automated platforms such as GeneXpert, Hologic Panther, Aries and veregene by Luminex, which uses various probe binding chemistrie for detection prior to physical extraction and amplification. However for high resolution post amplicon detection, analyzers are used; commonly PCR amplicon sequencing is the go to step, this enables high degree of analysis of the gene of interest. Others methods like Sanger Sequencing and Next Generation Sequencing are also employed for ultra-high precision analysis and are presently been successfully absorbed for high clinically relevant applications.
PCR: Polymerase Chain Reaction, LAMP: Loop mediated isothermal Amplification, TMA: Transcription Mediated Amplification, NASBA: Nucleic Acid Sequence Based Amplification, RPA: Recombinase Polymerase Amplification, RTF: Real Time Fluorescence, RTB: Real Time Bioluminescence, BDS: Bi-directional Synthesis
- Indian Molecular Diagnostic
The field of molecular biology came into recognition in the 1980s with the convergence of various disciplines, until then clinical diagnosis of infectious and non-infectious diseases remained stagnant. Previous diagnostic microbiological techniques such as microbial culture followed by microscopic examination, microbial identification using biochemical test, serodiagnosis, and antimicrobial and antibiotic susceptibility tests were the go-to methods for Indian clinical diagnostics. With basic technologies that remained unchanged for decades, clinical microbiologists could detect only a limited number of organisms and as a result, a multitude of infections were left undiagnosed until the ‘Molecular Revolution‘ which took place in the late twentieth century. This led to the emergence of a distinct field of molecular and genomic laboratories which pivoted the Indian clinical diagnostic sector. One of the earliest adopters of this field in India was SRL Ltd, which transitioned in as early 2000s, followed by other large private laboratories in India. A decade later, PCR and its advanced systems were prevalent in diagnostic and research laboratories thought the nation. One big driver for this change was the pandemic 2009 influenza A H1N1, this incident persuaded the adoption of rapid test to result technologies. Past another decade, molecular testing sector grew rapidly within the country due to the rising demand. The private sector was able to tap this potential by serving majority of the nation’s population, at times in collaboration with government which led to adoption of many high-end international diagnostic equipment and technology to cater the needs of the enormous Indian population.
Currently, advanced NAAT techniques and technologies such as PCR, ddPCR, Real Time PCR, PCR Sequencing, Multiplex Ligation-dependent Probe Amplification, Amplified Fragment Length Polymorphism (MLPA), GeneXpert, Pyrosequencing and NGS have been successfully employed for clinically relevant applications such as for screening of infectious and non-infectious diseases, genetic and familial disorders, cardiovascular diseases as well as oncological marker identification, in India. The State-of-the-art private laboratories like SRL Ltd, Dr.Lal PathLabs, Dr.Dangs Lab, Oncquest, Core Diagnostics etc. which operate in the Northern parts of India utilize high end high-throughput fully automated technologies such as Cobas Ampliprep, Cobas Taqman, Abbott ms 2000sp and ms2000rt systems etc.
Nucleic acid amplification based methods have proven their clinical relevance inmedical diagnostics due to their edge over traditional methods (Appendix 1). The low limit of detection, rapid turnaround time, ability to identify culture independent microorganisms, and specific genetic disorder in a compact and versatile form factor empowered its implementation into diverse fields such as oncology, pathology, virology, hereditary genetic disorder and syndromes testing, pharmacogenomics, transplantation, and gene expression studies. In conclusion, NAAT is a robust tool in the present day clinical diagnostic laboratory. Although numerous cutting-edge NAAT technologies exist throughout the globe, accommodation of these into the Indian diagnostic market took enormous resources and time. The influence of multinational diagnostic centers played a crucial role in the adoption of these novel technologies. However, these were confined to major metro cities. On the other hand, NAAT dependent point of care technologies witnessed increasing demand in high disease burden developing countries like India. Enormous growth of the diagnostic sector in India contributed by multiple factors such as demand over healthcare facilities, increasing disease burden along with rapidly growing infection rate, rising awareness for prevention and testing combined with accessible and affordable diagnostic networks. Overall NAAT based technologies proved to be sustainable, and heads its path to replace conventional gold standard tests through rigorous evaluation, demonstration and assessments leading to better test outcomes and positively impacting overall healthcare sector. The future of medical diagnostics lies in the genomics and proteomics, thereby reassuring the significance and demand for NAAT in the clinical sector.
-Able to accommodate large sample volumes
|-Sensitivity limited by use of antibiotics and antifungals|
-Sensitivity limited for fastidious organisms
-Limited use in viral testing
-Long time to result, especially in acid-fast and fungal cultures
|Microscopy and staining||-Rapid|
|-Low sensitivity unless there is a high burden of disease|
|Serology||-May be negative during early infection|
-False-negatives in humoral immune deficiencies
-Potential for quantitative PCR
|-Depends on hypothesis|
-Requires primers that may not always work
-Limited to a very small portion of genome
|Multiplex PCR||-Low specificity and false positives for many organisms due to difficulty in quantitation|
-Often requires more than one amplification
-Limited to a small portion of genome
-Requires primers that may not always work
|Targeted universal multiplex PCR for Sanger sequencing||-Can differentiate multiple species within one pathogen type||-Requires primers that may not always work|
-Limited to a very small portion of genome
|Targeted universal multiplex PCR for NGS|
-Can differentiate multiple species within one pathogen type
-Potential for quantitation
-Requires primers that may not always work
-Expensive and time consuming
-Often requires more than one amplification
-Limited to a very small portion of genome
|Targeted NGS||-Sensitive detection for selected organism types|
-Potential for quantitation
-Potential to be combined with multiplex NGS
-Sequencing library preparation
-More complex, typically with more than one amplification
-Hypothesis-free, or unbiased, testing
-Discovery of new or unexpected organisms
-Potential for quantitation
-Ability to detect any portion of genome
-Must also sequence human host background
-Not all genomes are available
-Prone to contamination with environmental species
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