eChapters

7.1 Basic concept

What is DNA sequencing?

The genome of any biological organism is a sequence of nucleotides arranged in a specific manner. The nucleotides are chemically a base which usually exists in 4 different forms; Purine – Adenine (A) and Guanine (G) and Pyramidine – Cytosine (C) and Thymine (T). These four nucleotides make up an entire sequence of Deoxyribose Nucleic Acid (DNA) which is encodes the information of any cell in the form of a gene. The genome, therefore, is the complete set of all genes that is required to build and maintain that organism. DNA sequencing is the process wherein the precise order of these nucleotides along the gene is detected and analysed which includes any method or technology that assists in determining the sequence.

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.

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.

Nucleic acid extraction from clinical samples is the first step to be followed in molecular detection of cancer. Nucleic acids are the starting material for all downstream molecular diagnostic tools. Quality of test results largely depends upon the quality of starting material. Thus utmost importance should be given to the extraction protocols to isolate good quantity and good quality nucleic acids from patient samples.

Basic principle: The basic principle of nucleic acid extraction is to disrupt cell membrane and nuclear membrane to release and precipitate nucleic acid in the solution. Following precipitation,removal of other cellular contaminates such as protein, lipids, polysaccharides etc is performed to obtain a good quantity and quality of nucleic acid.

Molecular diagnostic laboratory set-up has basic requirements of space, laboratory benches and equipments. In terms of laboratory space, minimum four chambers, one each for sample preparation and nucleic acid extraction, PCR reaction mixture preparation, thermal cycling and gel analysis are required. In addition there should be sufficient space for storage of consumables and laboratory wares. In addition, there are some laboratory equipments which are needed in a molecular diagnostic laboratory. In the following section the equipments of a molecular diagnostic laboratory are discussed. The pictures are taken from the instruments present in the laboratory of the author. The purpose of this chapter is to introduce the reader to a molecular diagnostic laboratory. None of the pictures below are meant for promotion of any particular brand or manufacturer.

3.1 UV-VIS spectrophotometer

A spectrophotometer is required for determining the quality and quantity of nucleic acids extracted from a sample. Photometric research applications such as DNA, RNA and protein analysis can be carried out in a UV-Vis Spectrophotometer. There are various units that feature a broad wavelength range (including UV area), path length correction and a fast reading speed. Various instruments also provide an interface that allows quick measurements directly from the instrument or comprehensive easy-to use software for demanding assays.

In any organism and biomolecules (enzyme, proteins, etc.), most biological activities could be seen only within a narrow environmental conditions. Therefore in order to carry out a successful experiment, from cell culturing to DNA sequencing or assessing an enzyme’s activity, one has to pay utmost attention in calculating correct reaction components.

A solution is defined as a homogenous mixture of solute and solvent. In this case the amount solute is less as compared to the solvent. It is not always necessary that solvent is liquid and solute is solid. There are several examples where solute and solvents are found in other physical states such as gas or solids. For example, carbonated water CO₂ is solute and water is solvent; and smoke is solution of different smoke particles (solute) in air (solvent). The concentration of solution is described as amount of solute in a given volume of solvent. Several ways are there to express the concentration of a solution, such as molarity, normality, molality, and ppm. In this chapter we would discuss the general terms used in making solutions and a brief introduction about buffer solutions.

Molecular biology is a branch of science that deals with understanding the biological activities at the molecular level. Molecular biologists are involved in understanding the concepts behind various intra and intercellular interaction of molecules, contributing to the knowledge about interrelationship of DNA, RNA and protein synthesis, i.e., how genes are transcribed into RNAs and how RNA is translated into protein, the phenomenon which is commonly known as “Central Dogma”stated by Francis Crick in 1956.

“The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information. It states that such information cannot be transferred back from protein to either protein or nucleic acid”-Francis Crick.

Once a pest, pathogen or a weed has been detected in the introduced planting material, quarantine officials must make all efforts to disinfect/decontaminate the material and make it available for further exploitation in the country without undue delay. However, it may be kept in mind that treatments, which only reduce the inoculum, may be acceptable for general agricultural practices, but they are not acceptable in plant quarantine. For quarantine purposes, tolerances are zero and, therefore, no residual inoculum of exotic pests/pathogens must remain. Fullproof eradicative treatments are required to be employed before release of the planting material from quarantine.

16.1. Fumigation

Fumigation of the material under atmospheric or under reduced pressure has been found acceptable as a quarantine treatment against insects and mites. Fumigants like methyl bromide, HCN, phosphine and EDCT (ethylene dichloride + carbon tetrachloride mixture) are commonly used.

In general, fumigation with Methyl bromide at 48g/cubic metre for 24 hours at 21ºC or above or 32g/cubic metre for 2 hours at 21ºC or above is used based on the kind of infected material and the pest to be salvaged.

15.1. Cereals

15.1.1. Oat (Avena sativa)

The Export-Import (EXIM) Committee was constituted in the Seeds Division to deal with application for exports/imports of seeds and planting materials in accordance with the New Policy on Seed Development and EXIM regulation.

The Policy reiterated that all imports of seeds and planting material would be regulated under the Plant Quarantine Order 2003 and amendments made their under, Import licences would be granted by DGFT/PPA on the recommendations of DAC.

The composition of the EXIM Committee is as under: (i) Secretary (A&C) - Chairperson (ii) Agriculture Commissioner (iii) DDG (CS), ICAR (iv) DDG (Hort.), ICAR (v) JS (IC) (vi) JS (PP) (vii) Horticulture Commissioner (viii) Animal Husbandry Commissioner, Deptt. of Animal Husbandry & Dairying (proposed to incorporate by the 94th Exim Committee) (ix) Director, National Bureau of Plant Genetic Resources (NBPGR) (x) Plant Protection Adviser (xi) Economics and Statistical Adviser (xii) JS (Seeds) - Member Secretary

A small quantity of seeds sought to be imported would be given to ICAR, or farms accredited by ICAR, for trial and evaluation for one crop season. On receipt of applications for commercial import, DAC would consider the trial/ evaluation report on the performance of the seed and their resistance to seed/ soil borne diseases. DAC is required to either reject or recommend the application to DGFT for grant of import licence within 30 days of receipt.

13.1. Quarantine Inspection and Certification

13.1.1. Scope/Purpose

Provides guidance and describes the standard operating procedures for certification of facilities for growing imported plants/plant material including transgenic plant material and other regulated articles such as biological control agents/beneficial organisms and living modified organisms under post-entry quarantine and inspecting the same to ensure free from quarantine pests and other regulated non-quarantine pests.

The purpose is to facilitate adoption of standard operating procedures by all the inspection authorities notified by the Ministry of Agriculture (Department of Agriculture & Cooperation)/PQOs (with PEQ responsibilities) for undertaking certification of post-entry quarantine inspection facilities as well as conducting postentry quarantine inspection of growing plants and other regulated articles under the supervision of concerned inspection authority/ PQOs (with PEQ responsibilities) in accordance with phytosanitary requirements specified under the Plant Quarantine (Regulation of Import into India) Order, 2003 and amendments issued there under to prevent the introduction and spread of destructive pests that affects plants and other regulated articles such as biological control agents and beneficial organisms.

12.1. Introduction

The protocols provides guidance on the structure and content of the International Plant Protection Convention (IPPC) diagnostic protocols for regulated pests. The protocols describe procedures and methods for the official diagnosis of regulated pests that are relevant for international trade. They provide at least the minimum requirements for reliable diagnosis of regulated pests.

This standard sets the framework for the content of diagnostic protocols, their purpose and use, their publication and their development. Diagnostic protocols for specific regulated pests are included as annexes to this standard. Information relevant for diagnosis is provided in the diagnostic protocol on the specified regulated pest, its taxonomic position, and the methods to detect and identify it. Diagnostic protocols contain the minimum requirements for reliable diagnosis of the specified regulated pests and provide flexibility to ensure that methods are appropriate for use in the full range of circumstances.

The methods included in diagnostic protocols are selected on the basis of their sensitivity, specificity and reproducibility, and information related to these factors is provided for each of these methods. Detailed information and guidance for the detection of pests is provided on, for example, signs and/or symptoms associated with the pest, illustrations (where appropriate), developmental stages of the pest, and methods for detecting the pest in a commodity, as well as methods for extracting, recovering and collecting the pests from plants.

11.1. History of Plant Quarantine in India

The first recorded plant quarantine measure in India dates back to 1906, when perceiving the danger of introducing the Mexican boll weevil, the Government of India directed that all cotton imported from the New World should only be admitted to India after fumigation with carbon disulphide at the port of entry.

In India two categories of regulatory measures are in operation for controlling pests, diseases and weeds. In the first category regulatory measures are aimed to prevent the introduction of exotic pests and diseases into the country or their spread from one State or Union Territory to another (Plant Quarantine). The second pertains to suppression or prevention of spread of pests and diseases in localized areas within a State or Union Territory.

The former derives its authority from the Destructive Insects and Pests (DIP) Act 1914 of the Central Government and the latter from Agricultural Pests and Diseases Acts of the various States. The legislative measures against crop pests and diseases were initiated under the DIP Act of 1914 which was passed by the then Governor General of India in Council on 3 February 1914.

Prior to the establishment of the Directorate of Plant Protection, Quarantine and Storage in 1946, under the Ministry of Food and Agriculture, the various rules and regulations of the DIP Act were enforced by the customs department. The quarantine regulations are operative through The Destructive Insects and Pests Act, 1914 (which has been revised 8 times from 1930 to 1956 and amended in 1967 and 1992).

The IPPC Secretariat provides support for the International Plant Protection Convention (IPPC) and coordinates the annual work programme put in place by the Interim Commission on Phytosanitary Measures. The three key activities of the Secretariat are: standard setting, technical assistance and information exchange. To facilitate the information exchange obligations laid out in the IPPC, the Secretariat adapted an existing Web-based environment to allow users to be able to both deposit and retrieve data from various sources. This Web site was named the “International Phytosanitary Portal” (IPP). As the IPP continues to evolve every effort are being made to ensure that specific issues necessary for the management of invasive alien species are addressed.

Classical biological control is one of the important tools for the management of invasive species. This involves the introduction and release of natural enemies, usually from the invaders’ area of origin. During the past decade, increasing concerns have been raised about the potential negative impact of introduced biological control agents on non-target organisms. This has highlighted the need to examine and guide the process of importation and release of natural enemies. The third international standard for phytosanitary measures, ISPM 3: Code of conduct for the import and release of exotic biological control agents, is specifically concerned with this process. ISPM 3 was endorsed by members of the Food and Agriculture Organization of the United Nations in 1995.

There is a growing demand and need for biological control across the globe. This has been fuelled not only by the increasing spate of invasive species but also by the need to develop integrated pest management approaches as well as the increased interest in organic production. Classical biological control of arthropod pests and weeds has been carried out successfully for more than a century (Greathead and Greathead, 1992; Julien and Griffiths, 1998). However, over the years, practitioners have become increasingly aware that introduced biological control agents may have undesirable side-effects (Thomas and Willis, 1998; Howarth, 2000; Follet and Duan, 2000; Lynch and Thomas, 2000; Wajnberg, Scott and Quimby, 2000; Henneman and Memmott, 2001). Initially, this concern was limited to the possible impact of these introduced agents on economically important plants and insects (notably, honey bees, silk moths and weed biological control agents). More recently, increased environmental awareness has drawn attention to the potential danger to all indigenous fauna and flora, particularly rare and endangered species.

To address these concerns, a code of conduct for the import and release of exotic biological control agents was developed with a view to fostering safe importation and release of biological control agents (Greathead, 1997). It was developed over a period of six years and was finally endorsed by FAO member countries at the end of 1995. The code of conduct, the third in a series of international standards for phytosanitary measures developed by the International Plant Protection Convention, was formally published in 1996 as ISPM 3: Code of conduct for the import and release of exotic biological control agents and is available, with other ISPMs

8.1. Phytosanitary Capacity Evaluation (PCE): The Tool, and Its Relation to Invasive Alien Species

Phytosanitary Capacity Evaluation (PCE) is a diagnostic tool enabling a country to assess the weaknesses and strengths of its phytosanitary system in relation to its ability to fully implement the International Plant Protection Convention and other international phytosanitary obligations. PCE has also been applied as a cross-disciplinary tool among the sanitary, phytosanitary and food safety areas in the Andean subregion. Since “quarantine pests”, as defined by the convention, constitute a subset of invasive alien species, PCE results are already useful in relation to invasive alien species. PCE methodology has the potential to be further developed to cover a country’s needs in implementing Article 8(h) of the Convention on Biological Diversity.

Article XX of the International Plant Protection Convention establishes the agreement by contracting parties to promote the provision of technical assistance to facilitate implementation of the IPPC. Phytosanitary Capacity Evaluation (PCE) is the first tool approved by the Interim Commission of Phytosanitary Measures to address that commitment, assisting contracting parties to perform a needs assessment and identification of their constraints for achieving full implementation of the IPPC at the national level.

7.1. BioNET International

7.1.1. What is BioNet International

BioNET-International, is the Global Network for Taxonomy, and is dedicated to creating sustainable mechanisms to assist developing countries to overcome the Taxonomic Impediment by becoming self-reliant in taxonomy i.e. self-reliant in the skills, infrastructure (collections etc.) and technologies needed to discover, identify, name and classify and to understand the relationships of all organisms on this planet.

It is concerned with helping developing countries to recognise and know the organisms that constitute and threaten their biodiversity, not for taxonomy’s own sake, but rather to support national programmes for sustainable agricultural development, and conservation and sustainable use of the environment.

BioNET-International is particularly focused on helping countries implement the various environmental conventions such as the International Plant Protection Convention (IPPC; www.ippc.int) and the Convention on Biological Diversity (CBD; www.biodiv.org), especially its taxonomic capacity building compnent, the Global Taxonomy Initiative (GTI). The subregional networks of BioNET-International have been identified as appropriate structures through which much of the GTI (www.biodiv.org/programmes/cross-cutting/taxonomy/default.asp) can be effectively implemented.

6.1. Speed Detection of Pests in China for Phytosanitary Programme

Phytosanitary procedures such as pest detection and identification rely on technology. With the rapid development of science, it is necessary for national plant protection organizations to keep informed and to make diagnoses more sensitive and accurate. Previous general pest detection methods frequently have limitations for the diagnosis of quarantine pests. In the past ten years, China has conducted considerable research into how to improve its diagnostic measures and methods.

6.1.1. General Pest Detection Measures and Methods

Traditional determination of morphology by microscope is used mostly to detect fungi and adult insects. The morphological method may be used, for example, in the separate identification of wheat dwarf bunt (Tilletia controversa), wheat karnal bunt (T. indica) and wheat common bunt (T. tritici). On the other hand, it is hard to detect seed-borne pests using the morphological method.

Isolation culture is also used to detect fungi and bacteria in China. It is an effective method for detecting seed-borne disease or systemic infection. However, it takes a long time (more than 1-2 weeks). Inoculating the indicated plant (a healthy plant that is a host for the suspected pest organism) is used mainly to identify viruses. This was the principal and time-consuming detection method used before immunological and molecular biological methods were developed. Because viruses are very important kinds of quarantine pests of young plants, effective and rapid methods for detection were urgently needed.

5.1. Australia’s Experience and Approach to Pest Risk Analysis

Australia applies the principles of pest risk analysis for the identification and management of sanitary and phytosanitary risks associated with new trade proposals and, where the need is identified, to the review of sanitary and phytosanitary risks of existing trade. One such trade activity is the intentional importation of exotic plant species not yet established in Australia.

Of approximately 250 000 plant species in the world, 30 000 have been intentionally introduced to Australia since European colonization, along with an unknown number of unintentional introductions, of which about 2 700 have naturalized (Cunningham et al., 2003). Some become problematic plants of which about 300 have become serious weeds of agriculture and natural ecosystems. Clearly, the majority of plant species have yet to be introduced to Australia and some of these would become weeds if allowed into the country.

The weed risk assessment (WRA) system is a decision support tool that is employed by Australia in applying the principles of pest risk analysis to the review of sanitary and phytosanitary risks associated with the intentional importation of exotic plant species not yet established in the country. The WRA system is used specifically to assess the weed potential of such species. It generates a score for a species based on its history as a weed in other parts of the world, climatic suitability to Australia and biological attributes. The weed risk assessment (WRA) system is one means by which the assessment component of pest risk analysis is addressed. The WRA system is a question-based scoring method that can be operated using a computer or manually using paper-based forms. A Microsoft® Excel spreadsheet version of the system, including documentation, has been made available through the International Phytosanitary Portal

In Article IX of IPPC, it is stated that RPPOs shall

• Function as coordinating bodies in the areas covered by the IPPC (including information sharing)
• Cooperate with the IPPC Secretariat in achieving the objectives of the IPPC (including the development of ISPMs)
• Cooperate with other RPPOs and participate in the regular Technical Consultations for RPPOs that are convened by the IPPC Secretariat

At present, there are 10 RPPOs covering the different regions of the world. Each RPPO has its own independent statutes and conducts its own regional cooperation programme. RPPOs produce regional standards for their members. They cooperate with each other and with the IPPC Secretariat. In particular, they meet in Technical Consultations to promote the development and use of relevant ISPMs and to encourage inter-regional cooperation on phytosanitary measures for controlling quarantine pests and preventing their introduction and spread.