eChapters

The history of the pregnancy test is a meaningful history embedded in magic, myth, politics, and science. From ancient Egyptian to modern times, the pregnancy test holds a cultural omnipresence that forces women to confront their fertility, femininity, and their future. Today we know that a pregnancy test that detects the human hormone known as human chorionic gonadatropin (hCG) offers the quickest and most accurate result for determining pregnancy. HCG is secreted by the placenta after the fertilized egg implants in the uterus and can be detected in both urine and blood. Urine pregnancy tests are convenient for home use, but a doctor may also opt to test blood as it provides a quantitative hCG number. Both methods are highly accurate and common. Pregnancy tests, however, were not always so accurate or so culturally accepted.
 
The diagnosis of pregnancy requires a multifaceted approach using three main diagnostic tools. These are (a) history and physical examination, (b)laboratory evaluation, and (c) ultrasonography. Currently, physicians may use all of these tools to diagnose pregnancy at early gestation and to help rule out other pathologies.

The increasing use of antibiotics in livestock and poultr y led to development of antibiotic resistant micro organisms and antibiotic residues in livestock products besides other side effects. These caused alarm bells ringing all over the world and by the end of June, 1999 majority of antibiotics as growth promoters in monogastric diets had been banned within the EU. The ban on use of antibiotic at sub therapeutic level in Europe and the potential for a ban in the United States have made scientists to seriously think of alternatives to antibiotics. In fact, the use of probiotics for farm animals was already stimulated by the findings of the Schwann Committee in 1969 which recommended that antibiotics in animal feeds should be restricted. However, the concept of probiotics gained momentum in later part of the last century. In recent years, with the public disapproval of using antibiotics in livestock and poultry production due to their residual effects in their products, there has been a greater emphasis on the use of probiotics in poultry.

The genera Proteus and Providencia belong to the tribe Proteae of the family Enterobacteriaeceae. The members of both these genera are gram negative, motile bacilli, aerobes and facultative anaerobes and can grow on basic media. A characteristic feature which distinguishes tribe Proteae from other members of Enterobactereaceae is the presence of the enzyme phenylalanine-deaminase which converts phenylalanine to phenylpyruvic acid (PPA reaction).They also produces a powerful urease enzyme which rapidly hydrolyses urea to ammonia.
Genus Proteus
They are gram-negative bacilli, 1-3 μm long and 0.6 μm wide. They are noncapsulated and are actively motile by peritrichous flagella. The name ‘Proteus’ refers to their pleomorphism, after the Greek God Proteus who could assume any shape. Four species: Proteus mirabilis, P.vulgaris, P.penneri and P.myxofaciens are recognized. Proteus mirabilis, P.vulgaris are widely recognised as human pathogens.
Culture Characteristics
These can grow on ordinary media like nutrient agar with a characteristic fishy or seminal odour. On MacConkey and Teepol lactose agar, lactose nonfermenting pale colonies, around 2-3 mm in size are formed. On non-inhibitory solid media such as blood and nutrient agar Proteus mirabilis and P vulgaris show characteristic swarming growth in the form of a uniform film, which spreads over the whole surface of the plate. In young swarming cultures, many of the bacteria are long, curved and filamentous, sometimes reaching upto 80 μm in length. When two different strains of swarming proteus mirabilis encounter one another on an agar plate, swarming ceases and a visibile line of demarcation forms. This is known as the Dienes phenomenon. In liquid medium (peptone water, nutrient broth), Proteus produces uniform
turbidity with a slight powdery deposit and an ammoniacal odour.

6.1 Designing primers

Designing correct primers are very important to get the expected result in PCR experiment. There are two primers (Forward & Reverse) required for the amplification of the desired DNA fragment. One primer should anneal to the sense strand (oriented to the 5'→3' direction) and another primer should complement the antisense strand (oriented in the 5'→3' direction).

There are various characteristics to be considered to design a good primer including Tm, length, GC content, secondary structure and specificity.

There are commercially available softwares besides free online tools at NCBI (primer blast) for designing PCR primers. In addition, primer sequences published in peer reviewed literature can also be used.

For RT-PCR, the extracted total RNA is first reverse transcribed to cDNA using reverse transcriptase enzyme. Following are the protocols for cDNA sysnthesis and RT-PCR.

Note: cDNA synthesis and RT-PCR can be performed according to the manufacturer protocol from where the specific reagents for the above methodologies are purchased.

8.1 Purpose

To lay down a uniform protocol for the synthesis of c-DNA from total RNA

8.2 Scope

The scope of this Standard Operating Procedure is to lay down a standard procedure for the synthesis of c-DNA from total RNA

5.1 Purpose

To employ a standard protocol to perform agarose gel electrophoresis.

5.2 Principle

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb. Migration rate of DNA molecule depends on various factors like 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under UV light.

11.1 Principle

In situ hybridization techniques involve detection of specific nucleic acid sequences to in morphologically preserved chromosomes, cells or tissue sections. Along with immunocytochemistry, in situ hybridization can correlate microscopic topological information to gene activity at the level of DNA, mRNA or protein. The FISH protocol involves 1) denaturation and hybridisation performed on Day 1 and 2) washing and detection performed on Day 2. DNA of the chromosomes and the probes are denatured and allowed to hybridize overnight on the first day. On day 2, slides are washed to remove unbound DNA sequences followed by detection, counterstaining and mounting of the slides.

10.1 Purpose

To lay down a standard protocol to perform qPCR for quantitative analysis of target genomic sequence.

10.2 Principle

qPCR or quantitative polymerase chain reaction or real-time polymerase chain reaction, is a molecular biology technique to amplify and at a time quantify a targeted DNA molecule and based on the polymerase chain reaction (PCR). It is called real time as the amplified DNA is detected as the reaction progresses and holds an advantage over standard PCR where the reaction is detected at its end. There are two common methods in quantitative PCR to detect the products i) non-specific fluorescent dyes which intercalates with any double stranded DNA ii) sequence-specific DNA probes labeled with a fluorescent reporter consisting oligonucleotides. This reporters helps in detection of the complementary hybridization of the probe and quantifies amount of DNA in cells or tissues.

9.1 Purpose

To lay down a standard protocol to perform RT-PCR for quantitative analysis of target RNA.

9.2 Principle

RT-PCR technology is used to amplify a specific RNA sequence (target sequence) to measure its expression inside the cell. RT-PCR follows the same principle as of normal PCR, but instead of DNA here cDNA is used as template for amplification. Extracted RNA is reverse transcribed to cDNA with the help of reverse transcription enzyme. The cDNA is then subjected for PCR amplification with the help of DNA polymerase, target specific primers, nucleotides and reaction buffer. In molecular diagnosis, RT-PCR is used to detect the expression of oncogenes at the time of diagnosis. It is also used to detect minimal residual disease during treatment

Proto – first; Zoon – animal

Protozoa are single celled eukaryotic organisms, which lack cell wall and ability to move at some stage of their life cycle.

They are referred as the first animals.

There are about 65000 described species grouped into 7 phyla. Among them, 50 per cent are not available as living forms (as fossils) and out of remaining 50%, 22,000 species are free-living and 10,000 species are parasitic forms.

The protozoa are distinguished from other organisms by following characters

History
Pseudomonas is comprised of two Greek words-Pseudo meaning false and Monas meaning unit, so literally the term means “false unit”. However Pseudomonas is a real bacteria so we really do not know the basis of the nomenclature by Walter Migula. A scientist by the name of Migula gave the Genus name of Pseudomonas in 1894 to these bacteria. Classification
The classification of Pseudomonas is given below:
• Class : Gamma Proteobacteria
• Order : Pseudomonadales
• Family : Pseudomonadaceae
• Genus : Pseudomonas
• Eight groups : P. aeruginosa ; P. chlororaphis; P. fluorescens;
P.pertucinogena; P.putida; P. stutzeri; P. syringae; P.
incertae sedis.
The family has 191 valid species. These include bacteria which are saprophytic, free living, and human, animal and plant pathogens. The type species is Pseudomonas aeruginosa.
Usual Habitat
Pseudomonas species are environmental organisms which occur worldwide in water and soil, and on plants. Pseudomonas aeruginasa is also found on the skin, on mucous membranes and in faeces. Burkholderia pseudomallei, which is found in soils, occasionally infectsanimals and man. Wild rodents can act as reservoirs of this organism. It is widely distributed in some tropical and subtropical regions of Southeast Asia and Australia.Although B.mallei can survive in the environment for upto 6 weeks its reservoir is infected Equidae.

Introduction
Antimicrobial resistance (AMR) has emerged as one of the most pressing global health threats, with implications that extend beyond human medicine to animal health, food security, and the environment. In livestock production systems, the misuse and overuse of antibiotics contribute significantly to the development of resistant pathogens. These resistant organisms can spread from animals to humans through direct contact, contaminated food products, and environmental pathways. The containment of AMR in livestock is not solely a technical or regulatory challenge—it is also a social and behavioral issue. Public awareness, education, and behavioural change are key pillars of any effective AMR mitigation strategy. Schools, community networks, and media platforms play vital roles in shaping knowledge, attitudes, and practices related to antibiotic use in livestock.
Causes of AMR in Livestock
Over-prescription and misuse of antibiotics in veterinary care Use of antimicrobials as growth promoters rather than for therapeutic purposes Poor farm bio-security and hygiene leading to frequent infections
Inadequate regulation and over-the-counter sale of antibiotics without veterinary oversight
Public Health Impact
Transmission of resistant pathogens via the food chain Contamination of soil and water with resistant genes through manure
Emergence of zoonotic diseases with limited treatment options

What so ever work you have done, it will not be recognized in the scientific world unless you publish it hence publishing the search work is of prime importance for a scientist. As it provides you recognition, you must pay greatest attention on quality of your research work which depends on the keenness of your observations. It is must to confirm and re-confirm your observations prior publishing them as once they are published, it will come in public domain with nothing you can do (except of publishing another paper to refute your earlier finding). As publishing the observations will form opinion about your scientific integrity and knowledge among the peers, the paper, if not cared and contained silly technical mistakes, will reflect ignorance of the workers on the subject.

Molecular diagnostics has witnessed groundbreaking technological advances in recent times, starting from automated sample preparation to real time amplification technology. Possibility to develop and run assays for ever increasing number of clinical queries has become effortless due to the technological advances. However, the state of the art in quality assurance and quality control practices for molecular diagnostics has seldom been up to the expectations if one considers accuracy for once in a lifetime genetic tests or new genetic test targets appearing almost every single day. In the face of such issues, clinical laboratories are struggling to develop appropriate quality assurance programs for the molecular diagnostic tests they conduct. In addition, legislation and government policies threaten to increase oversight of genetic tests to a level not seen before in laboratory medicine, for example, FDA’s guidance on in vitro diagnostic multivariate index assays (IVDMIA).

To relate patient results to previous test results or to absolute values in clinical practice guidelines, those results need to be comparable across time and methods. This may either be achieved by designing the identical value across methods and test versions or by using reliable and stable conversions. Therefore, the establishment of international standards and reference materials is of paramount importance. Monitoring molecular test system outputs and statistical analysis might provide a good way to obtain such standards; however, such traditional QC (Quality Control) strategies can be slow to take root in molecular diagnostics.This chapter highlights general and specific issues relevant for quality assurance and quality control in the routine molecular diagnostics laboratory and guidelines for designing quality manuals for a molecular diagnostic laboratory.

Introduction
The safety of meat and its derivatives is a paramount concern in the dynamic realm of food trade. Consumers demand food products that are not only affordable and possess an extended shelf life but are also of high safety standards. Consequently, meat inspections require strict adherence to good manufacturing practices and safety labelling that aligns with regulatory standards. Food-borne pathogens, including Listeria monocytogenes, Staphylococcus aureus, pathogenic Escherichia coli, Clostridium perfringens, Campylobacter spp., and Vibrio spp., are responsible for numerous illnesses, thereby exerting a significant impact on public health and the economy. According to the World Health Organization (WHO), food contaminated with pathogens, chemicals, and allergens results in 600 million cases of food-borne illnesses and approximately 400,000 deaths globally each year. Furthermore, 56 million individuals die annually, with about 7.7% of the global population affected by foodborne diseases. Meat and meat products are essential sources of nutrients for humans, providing high-quality protein, essential amino acids, B vitamins, and minerals. However, due to their high-water activity and nutrient content, they also create a conducive environment for spoilage microorganisms and food-borne pathogens. Until a few decades ago, the meat industry was largely dominated by the unorganized sector, with limited access to knowledge that could enhance productivity. However, in the post-World Trade Organization (WTO) era, meat entrepreneurs have succeeded in increasing productivity, although they have faced challenges in ensuring product quality that aligns with the legislative standards of numerous developed and developing countries. The use of antibiotics as antimicrobial growth promoters in animal food production has been a contentious issue, as their unauthorized application has inadvertently contributed to the emergence of antibiotic-resistant bacteria within the food chain. Recently, the United States Trade Representative’s office estimated that international trade has expanded significantly since the General Agreement on Tariffs and Trade (GATT) was signed in 1947. The establishment of the WTO in 1995 further facilitated trade in animal-origin foods and live animals between nations. However, the emergence and re-emergence of diseases caused by pathogenic bacteria have become a significant concern in this new pattern of food trade. Some of these pathogens are so virulent that they were not previously reported. According to a 1998 report by the Centers for Disease Control and Prevention (CDC), the annual cost of foodborne illnesses in the United States is nearly 10 billion US dollars. The bacterial pathogens most commonly associated with illnesses from beef products include Salmonella spp., Clostridium perfringens, and Staphylococcus aureus. Interest in Escherichia coli O157: H7 increased following a highly publicized food poisoning outbreak linked to undercooked beef patties in the United States in 1993, although it was largely confined to North America until the mid-1990s. Similarly, multidrug-resistant Salmonella typhimurium DT-104 has spread widely since it was first identified in the United Kingdom. In India, the incidence of Salmonella in red meat was recorded at up to 9%. These potentially harmful bacterial pathogens can reside on the hides or in the intestinal tracts of foodproducing animals or may originate indirectly through cross-contamination or the processing environment. Other emerging foodborne diseases include Listeriosis, which has spread throughout France and Canada, where meat and meat products have been implicated as sources of Listeria monocytogenes. Similarly, Staphylococcal food poisoning or food intoxication syndrome, first reported in 1894, has become a global issue in the meat industry.

Restriction enzymes cut only at specific sequences of 4 to 8 bases

A long restriction site is rare. Cutting rare sites will produce a few large pieces of DNA.

Restriction enzymes with long sites → small number of large fragments

Restriction enzymes with short sites → large number of small fragments