1 
 NATIONAL STANDARDS FOR PHYTO SANITARY MEASURES  
 
 
 
 
 
NSPM: 27 
 
 
 
 
Diagnostic protocols  for regulated pests  
2013  
 
 
 
 
 
 
(This standard is approved by (the NPPO and) the Quarantine Committee of Nepal, chaired by the 
secretary of the Ministry of Agriculture Dev elopment on 1st December 2013 and, is submitted by National 
Plant Quarantine Program to the National Notification Authority to be notified to the WTO member states)  
 
 
 2 
 Table of Contents  
1. Introduct ion ................................ ................................ ................................ ................................ .............  3 
1.1 Scope  ................................ ................................ ................................ ................................ ................  3 
1.2 References  ................................ ................................ ................................ ................................ .......  3 
1.3 Definitions  ................................ ................................ ................................ ................................ .........  4 
1.4 Outlines of requirements  ................................ ................................ ................................ ................  4 
2.  Background  ................................ ................................ ................................ ................................ ............  5 
3. Purpose and use of diagn ostic proposals  ................................ ................................ ..........................  5 
4. Requirement  ................................ ................................ ................................ ................................ ...........  6 
4.1  General Requirements for Diagnostic Protocols1 ................................ ................................ .. 6 
4. 2 Specific requirements for a Diagnostic Protocal  ................................ ................................ ........  7 
4.2.1 Pest information  ................................ ................................ ................................ ........................  7 
4.2.2 Taxonomic informa tion ................................ ................................ ................................ ............  7 
4.2.3 Detection  ................................ ................................ ................................ ................................ .... 7 
4.2.4 Identification  ................................ ................................ ................................ ..............................  8 
4. 2.5 Records  ................................ ................................ ................................ ................................ ..... 9 
4. 2.6 Contact points for further information  ................................ ................................ .................  10 
4.2.7 Acknowledgements  ................................ ................................ ................................ ................  10 
4.2.8 References  ................................ ................................ ................................ ..............................  10 
5.  Publication of Diagnostic Protocols  ................................ ................................ ...........................  10 
Appendix 1: Suggested main elements of procedures for  diagnostic protocols  .............................  11 
1. Development of Diagnostic Protocols  ................................ ................................ .......................  11 
2. Review of Existing Diagnostic Protocols  ................................ ................................ ......................  11 
3. Requests for New Diagnostic Protocols  ................................ ................................ .......................  11 
4. Request for Diagnosis to Laboratory  ................................ ................................ ............................  11 3 
  
1. Introduction  
 
1.1 Sco pe 
This National standard provides guidance on the structure and content of diagnostic protocols 
for regulated pests. The protocols describe procedures and methods for the official diagnosis of 
regulated pests that are relevant for international trade. The  standard provides requirements for 
reliable diagnosis of regulated pests.    NSPM preparation based on guidelines and 
recommendations developed within the framework of the IPPC. This standard also adopted the 
principles, recommendations and format of ISPM to achieve international harmonization of 
phytosanitary measures with the aim to facilitate trade.  
 
1.2 References  
IPPC . 1997. International Plant Protection Convention . Rome, IPPC, FAO.   
ISPM 4 . 1995. Requirements for the establishment of pest free areas . Rome, IPPC, FAO. 
[published 1996]  
ISPM 5 . Glossary of phytosanitary terms . Rome, IPPC, FAO.   
ISPM 6 . 1997. Guidelines for surveillance . Rome, IPPC, FAO.  
ISPM 7 . 1997. Export certification system . Rome, IPPC, FAO. [revised; now ISPM 7:2011]  
ISPM 8 . 1998. Determination of pest status in an area . Rome, IPPC, FAO.  
ISPM 9 . 1998. Guidelines for pest eradication programmes . Rome, IPPC, FAO.  
ISPM 10 . 1999. Requirements for the establishment of pest free places of production and pest 
free production sites . Rome, IPPC, FAO.  
ISPM 13 . 2001. Guidelines for the notification of non -compliance and emergency action . Rome,  
IPPC, FAO.  
ISPM 14 . 2002. The use of integrated measures in a systems approach for pest risk 
management . Rome, IPPC, FAO.  
ISPM 17 . 2002. Pest r eporting . Rome, IPPC, FAO.  
ISPM 20 . 2004. Guidelines for a phytosanitary import regulatory system . Rome, IPPC, FAO.  4 
 ISPM 22 . 2005. Requirements for the establishment of areas of low pest prevalence .  Rome, 
IPPC, FAO.  
ISPM 23 . 2005. Guidelines for inspec tion. Rome, IPPC, FAO.  
ISPM 27 .2006. Diagnostic protocols for regulated pests . 
 
1.3 Definitions  
Definitions of phytosanitary terms used in the present standard can be found in ISPM 5 
(Glossary of phytosanitary terms ).  
 
1.4 Outlines of requirements  
This s tandard sets the framework for the content of diagnostic protocols, their purpose and use, 
their publication and their development. Diagnostic protocols for specific regulated pests will be 
developed base on this standard and will be 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 requirements for reliable diagn osis 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 re producibility, 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 a ppropriate), 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. Information and guidance for the 
identification of pests includes detailed  information on morphological and morphometric 
methods, methods based on biological properties, and methods based on biochemical and 
molecular properties of the pest. Furthermore, detailed guidance is provided on the records that 
should be kept.   
 
 
Diagno stic protocols are intended to be used by laboratories performing pest diagnosis as part 
of phytosanitary measures. They are subject to review and amendment to take into account new  
developments in pest diagnosis. The standard also provides guidance on ho w these protocols 
will be initiated, developed, reviewed and published.  5 
 2.  Background  
Proper diagnosis is crucial for the appropriate application of phytosanitary measures (see for 
example ISPM 4:1995,  ISPM 6:1997,  ISPM 7:1997,  ISPM 9:1998  and NSPM : Guidelines for a 
phytosanitary import regulatory system ).  In international trade, contracting parties should agree on 
diagnostic procedures for determination of pest status and pest reporting (ISPM 8:1998; NSPM : 
Pest reporting ), and the diagnosis of pests in imported consignments (ISPM 13:2001).  
 
The laboratories of National Plant Protection Organization,  Nepal are doing diagnostic works 
since many years. However, NPPOs of Nepal have not produced national standard on 
diagnostic protocols for regulated pest s in order to adequately fulfill responsibilities according to 
Article IV of the IPPC   (1997), in particular regarding surveillance, import inspections and export 
certification. In response to the need for regional harmonization, several regional plant 
protection organizations (RPPOs) have developed a significant number of regional diagnostic 
standards. This underlines the need for national standard for diagnostic protocol. Plant 
Protection Division, Department of Agriculture recognizes  that there is a need  for national 
standard for diagnostic protocol and approves the formation of a Technical Panel on Diagnostic 
Protocol (TPDP).  
3. Purpose and use of diagnostic proposals  
The purpose of harmonized diagnostic protocols is to support efficient phytosanitary me asures 
in a wide range of circumstances and to enhance the mutual recognition of diagnostic results by 
NPPOs, which will also facilitate trade. Furthermore these protocols should aid the development 
of expertise and technical cooperation, and they may also  be relevant to the accreditation 
and/or approval of laboratories. The protocol will be helpful for the different laboratories working 
with the diagnostic work of pest of different crops. This protocol will be basis for developing 
protocols for specific pe sts of different crops important in international trade.  
 
Diagnostic protocols may be used in different circumstances that may require methods with 
different characteristics. Examples of such circumstances grouped according to an increased 
need for high se nsitivity, specificity and reliability are:   
 routine diagnosis of a pest widely established in a country  
 general surveillance for pest status  
 testing of material for compliance with certification schemes  
 surveillance for latent infection by pests   
 surveillance as part of an official control or eradication programme  
 pest diagnostic associated with phytosanitary certification  
 routine diagnosis for pests found in imported consignments  
 detection of a pest in an area where it is not known to occur  
 cases  where a pest is identified by a laboratory for the first time  
 detection of a pest in a consignment originating in a country where the pest is declared 
to be absent.  
 
For example, in the case of routine diagnosis, the speed and cost of a test method may be more  
relevant than sensitivity or specificity. However, the identification of a pest by a laboratory or in 6 
 an area for the first time may require methods with a high level of specificity and reproducibility. 
The significance of the outcome of a diagnos is is often dependent on proper sampling 
procedures (NSPM -Sampling).  
 
Diagnostic protocols provide the requirements for reliable diagnosis of regulated pests. This 
may be achieved by a single method or a combination of methods. Diagnostic protocols also 
provide additional methods to cover the full range of circumstances for which a diagnostic 
protocol may be used. The level of sensitivity, specificity and reproducibility of each method is 
indicated where possible. NPPOs may use these criteria to determine t he method or 
combination of methods that are appropriate for the relevant circumstances.  
 
Diagnostic protocols are intended to be used by laboratories performing pest diagnosis. Such 
laboratories may be established under or may be authorized by the NPPO t o perform these 
activities in such manner that the results of the pest diagnosis may be considered as part of a 
phytosanitary measure of the NPPO.  
 
The main elements of the procedure for the development of diagnostic protocols are presented 
in Appendix 1.   
4. Requirement  
4.1  General Requirements for Diagnostic Protocols  
Each protocol contains the methods and guidance necessary for the regulated pest(s) to be 
detected and positively identified by an expert (i.e. an entomologist, mycologist, virologist, 
bacteriologist,  nematologist, weed scientist, molecular biologist) or competent staff who are 
specifically trained.  
 
The methods included in diagnostic protocols are selected on the basis of their sensitivity, 
specificity and reproducibility. In addition, th e availability of equipment, the expertise required for 
these methods and their practicability (for example ease of use, speed and cost) are taken into 
account when selecting methods for inclusion in the diagnostic protocol. These methods and 
their associa ted information should also be published. It may be necessary that some methods 
are validated before  inclusion in the protocols. Such validation may include, for example, the 
use of a set of known  samples, including checks, prepared to verify sensitivity, specificity and 
reproducibility.   
 
Each diagnostic protocol usually describes more than one method to take into account the 
capabilities of laboratories and the situations for which the methods are applied. Such situations 
include finding of different dev elopmental stages of organisms, which require different 
methodologies, the need for an alternative diagnostic technique because of uncertainties of the 
initial diagnosis, as well as varying requirements for the level of sensitivity, specificity and 
reliabi lity. For some purposes a single method may be sufficient, for other purposes a 
combination of methods may be necessary. Each protocol contains introductory information, 
information on the taxonomic position of the pest, methods for detection and identific ation of the 
pest, records to be kept, and references to appropriate scientific publications. In  many cases a 
wide range of supplementary information is available which may support diagnosis, for example 
geographical distribution of the pest and host list s, but diagnostic protocols focus on the critical 
methods and procedures for pest diagnosis.  
 
The aspects of quality assurance and in particular the reference materials that are required by  
diagnostic protocols (such as inclusion of positive and negative controls or collection of 7 
 specimens) are specifically indicated in the corresponding section of the protocol.   
 
The following general provisions apply to all diagnostic protocols:   
 Laboratory tests may involve the use of chemicals or equipment which pres ent a certain 
hazard. In all cases, national safety procedures should be strictly followed.  
 Use of names of chemicals or equipment in these diagnostic protocols implies no approval  
of them to the exclusion of others that may also be suitable.  
 Laboratory procedures presented in the protocols may be adjusted to the standards of 
individual laboratories, provided that they are adequately validated.  
 
4. 2 Specific requirements for a Diagnostic Protocal  
Diagnostic protocols are arranged according to the follo wing sections:  
 Pest information  
 Taxonomic information  
 Detection  
 Identification  
 Records  
 Contact points for further information  
 Acknowledgements  
 References.   
 
4.2.1 Pest information  
Information is provided on the pest, including, where appropriate, its life cycle, morphology, 
variation (morphological and/or biological), relationship with other organisms, host range (in 
general), effects on hosts, present and past geographical distribution (in general), mode of 
transmission, dissemination (vectors and  pathways), economic importance, and  past history. 
When available, reference to a pest data sheet should also be provided.  
4.2.2 Taxonomic information  
This section provides information on the taxonomy of the pest involved and includes:  
 name (current sci entific name, author and year (for fungi, teleomorph name if known ))  
 synonyms (including former names)  
 accepted common names, anamorph name of fungi (including synonyms)  
 acronym of viruses and viroids  
 taxonomic position (including information on subsp ecies classifications where relevant).  
4.2.3 Detection  
This section of the diagnostic protocol provides information and guidance on:  
 the plants, plant products or other articles capable of harbouring the pest  
 the signs and/or symptoms associated with the pest (characteristic features, differences 
or similarities with signs and/or symptoms from other causes), including illustrations, 
where appropriate  
 the part(s) of the plant, plant products or other articles on/in which the pest may be found  
 the developm ental stages of the pest that may be detected, together with their likely 
abundance and distribution on/in the plants/plant products or other articles  
 the likely occurrence of the pest associated with developmental stages of the host(s), 
climatic conditio ns and seasonality  
 methods for detecting the pest in the commodity (e.g. visual, hand lens)  8 
  methods for extracting, recovering and collecting the pest from the plants, plant products 
or other articles, or for demonstrating the presence of the pest in the plants, plant 
products or other articles   
 methods for indicating the presence of the pest in asymptomatic plant material or other  
 materials (e.g. soil or water), such as ELISA  (Enzyme linked immunosorbent assay)  
tests or culturing on selective media viab ility of the pest.  
 
For all methods included in this section, information is provided on their sensitivity, especially  
and reproductively, where relevant.  Where appropriate, guidance is provided on positive and 
negative controls and reference material to b e included in tests. Guidance is also provided on 
resolving possible  confusion with similar signs and/or symptoms due to other causes.  
4.2.4 Identification  
This section provides information and guidance on methods that either used alone or in 
combination l ead to the identification of the pest. When several methods are mentioned, their 
advantages/disadvantages are given as well as the extent to which the methods or 
combinations of methods are equivalent. A flow diagram may be presented if several methods 
are needed to identify the pest or many alternative methods are included.  
 
Main types of methodologies used in diagnostic protocols include those based on morphological 
and morphometric characteristics, biological properties such as virulence or host range o f a 
pest, and those based on biochemical and molecular properties. Morphological characteristics 
may be investigated directly or after culturing or isolation of the pest. Culturing and/or isolation 
may also be required for biochemical and/or molecular assa ys. Details are provided when 
culturing or isolation procedures are necessary components of methods.  
For morphological and morphometric identifications, details are provided, as appropriate, on:  
 methods to prepare, mount and examine the pest (such as for light microscopy, electron  
microscopy and measurement techniques)  
 identification keys (to family, genus, species)  
 descriptions of the morphology of the pest or of its colonies, including illustrations of 
morphological diagnostic characteristics, and an indication of any difficulties in seeing  
particular structures  
 comparison with similar or related species  
 relevant reference specimens or cultures.  
 
For biochemical or molecular identifications, each method (e.g. serological methods, 
electrophoresis, PC R (Polymerase chain reaction) , DNA barcoding, RFLP  (Restriction fragment 
length polymorphism) , DNA sequencing) is described separately in sufficient detail (including 
equipment, reagents and consumables) to perform the test. Where approp riate, reference may 
be made to methodology described in other diagnostic protocols annexed to this standard. In 
cases where more than one method can be used reliably, other appropriate methods may be  
presented as alternative or supplementary methods, e.g.  where morphological methods can be 
used reliably and appropriate molecular methods are also available.  
 
Where appropriate, methods for isolation of pests from asymptomatic plants or plant products 
(such as tests for latent infection) are given, as well a s methods for extraction, recovery and 
collection of pests from plant or other material. In these cases, methods may also be provided 
for direct identification of pests using biochemical or molecular tests on asymptomatic material.  
 9 
 For all the methods in cluded in this section, information is provided on their sensitivity, 
specificity and reproducibility, where relevant. Where appropriate, guidance is provided on 
positive and negative controls and reference material to be included in tests. Guidance is als o 
provided on removing possible confusion with similar and related species or taxa.  
Diagnostic protocols provide guidance on the criteria for the determination of a positive or 
negative result for each method or information necessary to determine if an al ternative method 
be applied.   
Those cases where the use of appropriate controls for a specific technique, including where 
relevant  reference material, is essential are clearly indicated in the protocol. When appropriate 
controls are not available, other te sts, preferably based on different identification principles, may 
increase the certainty of the identification. Alternatively, a sample, specimen or, where 
appropriate, an image should be sent to another laboratory with experience in diagnosis of the 
suspe cted pest and possessing the required control or reference materials. Specimen(s) or 
material for reference purposes should be properly preserved.  
Methods for quick, preliminary indications of identity (which will later need to be confirmed) may 
also be i ncluded in diagnostic protocols.  
4. 2.5 Records  
This section provides information on the records that should be kept:  
 Scientific name of pest identified  
 Strain, biotype and other difference, if appropriate  
 Code or reference number of the sample (for tr aceability)  
 Nature of the infested material including scientific name of host, variety, stages of plant part             
affected, where applicable   
 Origin (including the geographic location if known) of the infested material, and location of 
interceptio n or detection  
 Description of signs or symptoms (including photographs where relevant), or their absence 
Methods, including controls, used in the diagnosis and the results obtained with each  
method   
 For morphological or morphometric methods, measurements , drawings or photographs of 
the diagnostic features (where relevant) and, if applicable, an indication of the 
developmental stage(s)  
 For biochemical and molecular methods, documentation of test results such as photographs 
of diagnostic gels or ELISA prin touts of results on which the diagnosis was based  
 Where appropriate, the magnitude of any infestation (how many individual pests found, how 
much damaged tissue)  
 The name and address of the laboratory and, where appropriate, the name of the person(s) 
responsible for and/or who performed the diagnosis  
 Dates of collection of the sample, and of detection and identification of the pest  
 Where appropriate, state of the pest, alive or dead, or viability of its development stages.  
 Sample preserved condition  
 
 
Evidence such as culture(s) of the pest, nucleic acid of the pest, preserved/mounted specimens 
or test materials (e.g. photograph of gels, ELISA plate printout results) should be retained, in 
particular in cases of non -compliance (ISPM 13:2001) and where pest s are found for the first 
time ( NSPM : Pest reporting ). Additional items may be required under other ISPMs such as ISPM 
8:1998.   10 
  
The period for which records should be kept depends on the purpose for which a diagnosis is 
made. In cases where other contrac ting parties may be adversely affected by the results of the 
diagnosis, records and evidence of the results of the diagnosis should be retained for at least 
one year.  
 
4. 2.6 Contact points for further information  
Contact details of organizations or indivi duals with particular expertise on the pest(s) should be 
provided; they may be consulted regarding details on the diagnostic protocol.   
4.2.7 Acknowledgements   
The name and address of the experts who wrote the first draft of the diagnostic protocol shoul d 
be given, together with those of any others who made major contributions.  
4.2.8 References   
References to accessible scientific publications and/or published laboratory manuals should be 
given that may provide further guidance on the methods and proced ures contained in the 
diagnostic protocol.  
5.  Publication of Diagnostic Protocols  
Diagnostic protocols are published as annexes to this NSPM and thus are individual 
publications under the IPPC framework with a specific publication and/or revision date. If 
appropriate, they may also form part of other ISPMs. The process of their adoption includes 
stringent review by internationally acknowledged scientists/experts for the relevant discipline.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 11 
  
 
 
 
 
 
 
 
6.  Main elements of procedures for diagnostic protocols  
 
6.1.Development of Diagnostic Protocols  
Plant Protection Directorate, with the initiation of National Plant Quarantine Program (NPQP)  
should create a Technical Panel on Diagnostic Protocols (TPDP) for Nepal. The panel should  
commi ssion  an expert to lead the  development of a diagnostic protocol by adopting as 
appropriate, protocols that have already  been approved by RPPOs, or other international or 
national organization s orby developing a new prot ocol. Research institutes like Plant  Pathology , 
Entomology of NARC  and other institutes of Nepal have longtime experience  on diagnostic work. 
The institutes  should be involved to develop diagnostic protocol. Official  mechanism that will share  
both work and opportunities  among NPQP and other institutes  should be developed. The 
diagnostic  protocol  should  be developed further by a small  group of experts selected by the 
TPDP, Nepal  and will then be submitted to TPDP, Nepal.  
 
6.2. Review of Existing Diagnostic Protocols  
 
TPDP members will review the diagnostic protocols in their discipline on an annual basis or as 
determined by the TPDP. A request for a revision to a diagnostic protocol may also be 
submitted   by NPPOs, or subsidiary bodies through the NPQP, which will in turn forward it to the 
TPDP. The TPDP will evaluate the request, identify those diagnostic protocols that require 
revision and  oversee their revision. New methods should be at least equivalent to existing 
methods or provide a significant advantage for their national application suc h as costs, 
sensitivity or specificity. Appropriate evidence should be provided to support any claims.   
 
6.3. Requests for New Diagnostic Protocols  
 
Requests for new diagnostic protocols, in addition to those identified in the work programme of the 
TPDP, s hould be sent by NPPOs, or subsidiary bodies through NPQP using a form for the topics and 
priorities for standards.  
 
7. Request for Diagnosis to Laboratory  
When diagnosis can not be done at the inspection site or at field, the samples or specimens of pests , 
diseases or weeds should be sent  to accridated laboratory for diagnosis. Following considerations 
should  be made while sending samples/specimens to the laboratory : 12 
  Preserve specimens in proper condition so that specimens reach in the laboratory in good 
condition without spoilage  
 Use ice -box, if necessary in the summer when temperature is too high for transportation.  
 Use sterilized hand -gloves and tools to collect specimens  
 Collect specimens (plant parts with sign or symptoms) with least disturbance  
 Collec t good amount of specimens and send to the laboratory so that it can be used in 
duplicate  
 Collect specimens use envelops, preferably, “Nepali khams” to keep plant parts like leaves, 
stems and roots  
 Do not use plastic envelops to keep specimens  
 If soil -borne diseases like nematodes or soil insects are suspected, collect soil from 
rhizosphere and keep in plastic bags. Do not seal plastic bags. Just staple the bag two or 
three places  
 Provide detail information about crops and environments that will help for id entification 
(narrowing down the process) examples; crop, variety, season, time of seeding/planting, time 
when disease(symptom) appeared, pattern of disease(uniform or patchy), critical cultural 
practices done before disease or insect observed like irrigat ion (flooding), spraying fungicide 
or insecticide or other chemicals, fertilizer application etc.  
 If insects are observed, kill or demobilize insect properly and send to laboratory  
 Provide, if available all stages of insects.