This presentation covers a brief introduction to Diagnostic kit with its different types and examples. Also, this presentation covers examples of some common diseases with their diagnostic test.
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Developing diagnostic test for plant, human and animal diseases
1. Developing Diagnostic
Tests for Plant, Animal
and Human Diseases
Presented By-
Sushant Balasaheb Jadhav (18PBT206)
M. Tech. Pharmaceutical Biotechnology
Institute of Chemical Technology
2. • Diagnostic
• Screening
• Monitoring
Purpose
• Patient Observations
• In Vivo Diagnostics
• In Vitro Diagnostics
Method
• Blood
• Urine
• Stool
Sample
Location
Medical Test
2
3. Diagnostic Test
A diagnostic test is a procedure performed to
confirm or determine the presence of disease in
an individual suspected of having a disease
Accuracy and Precision
Detection – Positive or Negative
Quantification – Discrete or Continuous Values
Interpretation
Risks related to test procedure
3
4. Categories of Diagnostic Tests
4
Test Category Test Purpose Examples
General Chemistry Measurements of base
compounds in the body
Urinalysis test strips
Calcium level test
HbA1c tests
Immunochemistry Match antibody-antigen
response to indicate the
presence or level of a
protein
Antibiotic
susceptibility tests
HIV antibody tests
Tumor marker tests
Hematology /
Cytology
Study of the blood,
blood producing organs,
and cells of the body
Complete blood
count Coagulation
tests (e.g., INR)
5. Categories of Diagnostic Tests
5
Test Category Test Purpose Examples
Microbiology /
Infectious Disease
Detection of disease-
causing agents
Streptococcal testing
Bacterial urine testing /
urine culture
Molecular Study of DNA and
to detect genetic
sequences that may
indicate presence or
susceptibility to
disease
HER2/neu over-
expression testing to
identify patients who are
more likely to respond to
the breast cancer drug
Herceptin HIV viral
testing and other HIV
assays
6. Uses of Diagnostic Tests
6
Test Use Purpose
Screening, Early Disease
Detection
To detect asymptomatic disease or a
predisposition to disease in order to take action
to prevent it by modifying a risk factor or to
it earlier
Diagnosis To make a diagnosis when symptoms,
abnormalities on physical examination, or other
evidence suggests, but does not prove, that a
disease may be present
Disease Staging, Prognosis To determine the extent of disease progression
severity and the likelihood of recovery or risk of
future adverse health outcomes (e.g., cancer
relapse)
7. Uses of Diagnostic Tests
7
Test Use Purpose
Drug Selection, Treatment
Monitoring
To allow accurate and targeted
treatment selection tailored to individual
needs
Disease or Condition
Monitoring and
Management
To understand the course of the disease
or the effect of a therapy in order to
evaluate the success of treatment and
the need for additional testing or
treatment
8. Health Conditions and
Associated Tests
8
Health Condition Test Examples
Heart Disease Lipid panel (cholesterol,
Triglycerides) Troponin
Respiratory Disease Blood gas test Bacterial
culture Viral culture
Diabetes Glucose HbA1c
HIV/AIDS Antibody testing CD4
RNA
Influenza Viral culture Serology
Rapid antigen testing
9. Top 10 Diagnostic Tests
Mammography
Echocardiography
Complete Blood Count (CBC)
Colonoscopy
Prothrombin Time (PT)
Bone Density Study
Magnetic Resonance Imaging (MRI)
Computer Axial Tomography (CT or CAT Scan)
Electrocardiogram (EKG)
Prostate Specific Antigen (PSA Test)
9
10. Diabetes mellitus
The most common endocrine disorder of carbohydrate metabolism
The blood glucose concentration is the major diagnostic criterion
for diabetes with HbA1c level and is a useful tool for patient
monitoring
The majority of the current glucose biosensors are of the
electrochemical type
Enzymatic amperometric glucose biosensors are the most common
devices commercially available, and have been widely studied over
the last few decades. Amperometric sensors monitor currents
generated when electrons are exchanged either directly or
indirectly between a biological system and an electrode
10
11. Diabetes mellitus
Glucose measurements are based on interactions with one of three
enzymes: hexokinase, glucose oxidase (GOx) or glucose-1-
dehydrogenase (GDH)
GOx is the standard enzyme for biosensors; it has a relatively higher
selectivity for glucose
The basic concept of the glucose biosensor is based on the fact that
the immobilized GOx catalyzes the oxidation of β-D-glucose by
molecular oxygen producing gluconic acid and hydrogen peroxide
Glucose + GOx-FAD+ Glucolactone + Gox-FADH2
Gox-FADH2 + O2 Gox-FAD + H2O2
H2O2 2H+ + O2 + 2e- 11
12. Myocardial infarction
Myocardial infarction (MI), commonly known as a heart attack, occurs
when blood flow decreases or stops to a part of the heart, causing
damage to the heart muscle
The most common symptom is chest pain or discomfort which may
travel into the shoulder, arm, back, neck, or jaw
There are a number of different biomarkers used to determine the
presence of cardiac muscle damage
Troponins, measured through a blood test, are considered to be the
best, and are preferred because they have greater sensitivity and
specificity for measuring injury to the heart muscle than other tests. A
rise in troponin occurs within 2–3 hours of injury to the heart muscle,
and peaks within 1–2 days. The level of the troponin, as well as a
change over time, are useful in measuring and diagnosing or excluding
myocardial infarctions, and the diagnostic accuracy of troponin testing
is improving over time. One high-sensitivity cardiac troponin is able to
rule out a heart attack as long as the ECG is normal.
12
13. Myocardial infarction
Mihaela Savin et al prepared and developed lateral flow immunoassay
test for the detection of human heart fatty acid binding protein (hFABP)
Water-soluble CdTe (Cadmium telluride) quantum dots (QDs) were
selected as the fluorescent label and were linked covalently to anti-
hFABP antibodies
Upon conjugation, the secondary structure of the anti-hFABP was
preserved and the fluorescence quantum yield of the CdTe QDs
increased
The labelled antibodies were transferred to the immunoassay test strip
and the antigen–antibody reaction was successfully performed
This evidenced the preserved antibody activity of QD-labelled anti-
hFABP towards hFABP, and provided a rapid means for the quantitation
of hFABP in human serum within the range of 0 to 160 ng ml-1, with a
much lower detection limit of 221 pg. ml-1 compared with other rapid
tests based on lateral flow immunoassays 13
14. Rabies
Viral disease that causes inflammation of brain in humans and other
mammals
Symptoms include neurological problems and a fear of light and
water
The current gold standard for post-mortem diagnosis of human
and animal rabies is the direct fluorescent antibody (DFA) test
An immunohistochemistry procedure, which is recommended by
the World Health Organization (WHO)
The FAT relies on the ability of a detector molecule (usually
fluorescein isothiocyanate) coupled with a rabies-specific antibody,
forming a conjugate, to bind to and allow the visualization of rabies
antigen using fluorescent microscopy techniques 14
15. Rabies
The LN34 pan-lyssavirus real-time RT-PCR assay represents a
strong candidate for rabies post-mortem diagnostics due to
its ability to detect RNA across the diverse Lyssavirus genus,
its high sensitivity, its potential for use with deteriorated
tissues, and its simple, easy to implement design
Beta-Actin Assay
Differential Diagnosis
Following the vaccination requirements for pets helps
prevent and control rabies
15
16. Q Fever
Q fever is a widespread disease caused by the bacteria Coxiella
burnetii, which is able to infect mammals, birds, reptiles and
arthropods. It causes a mild disease in ruminants, but can cause
abortions and still births in cattle, sheep and goats
C. burnetii is shed in milk, urine and feces. But most importantly,
during parturition, huge concentrations of bacteria, up to a billion
per cubic centimetre, are found in the amniotic fluids and the
placenta.
Outside the animal the bacteria becomes a small, dense, long
lasting spore-like form which is able to resist heat and drying. It can
then contaminate dust and be spread by wind for long distances. It
so highly infectious that a single inhaled organism can cause clinical
illness in an animal or person.
Outbreaks typically occur following a birth or abortion where the
environment becomes contaminated with birthing fluids.
16
17. Q Fever
In samples from aborted or affected animals the diagnosis is
confirmed by identifying the bacteria or more commonly
using serological test to identify antibodies according to the
standards found in the OIE
Q fever is diagnosed with a blood antibody test. According
to the Centers for Disease Control (CDC), an antibody test
frequently appears negative in the first seven to 10 days of
sickness
Chest X-ray and echocardiogram as a cough and chest pain
while breathing are symptoms of this disease
17
18. Rice Bakanae
Rice bakanae is an important disease that causes serious rice
production loss worldwide
Zhenyang Rong et al, described a new method for rapid
diagnosis of rice bakanae caused by Fusarium fujikuroi and F.
proliferatum, based on loop-mediated isothermal
amplification (LAMP) assays
After screening, primers were selected to target Fusarium
DNA sequences, that is, the intergenic spacer (IGS) region of
the nuclear ribosomal operon and reductase-coding region
(RED1) in F. fujikuroi and F. proliferatum, respectively
18
19. Rice Bakanae
Both LAMP assays efficiently amplified target genes in 70 min
at 62°C.
A colour change from purple to sky blue (visible to the
unaided eye) was observed in the presence of the DNA of
the targeted pathogens only, by adding hydroxynaphthol
blue to the reaction system prior to amplification.
The minimum of genomic DNA needed in the assays was 67
and 346 pg/μl for F. fujikuroi and F. proliferatum, respectively
19
20. Angular leaf spot of Strawberry
M. Getaz et al, applied comparative genomics approach using
genomes of different Xanthomonas species and pathovars to
identify highly specific targets in the genome of Xanthomonas
fragariae, the causal agent of angular leaf spot of strawberry, listed
under quarantine regulations in Europe
A reliable and sensitive loop-mediated isothermal amplification
(LAMP) assay was designed using a unique marker providing a
highly specific and rapid detection, making this technique
convenient for on-site detection
Specificity of the designed assay was tested on 37 culture collection
strains of X. fragariae, 82 strains of other Xanthomonas species and
pathovars and 11 strains of other bacterial genera isolated from
strawberry leaves 20
21. Angular leaf spot of Strawberry
Detection limit of 102 fg was achieved, approximating 20 genome
copies per reaction.
When performing analyses with crude plant material, a consistent
lower detection efficiency of 102 CFU ml-1 was achieved.
The LAMP assay designed in this study was adapted to work on
crude plant material without any prior extensive extraction steps or
incubation period.
Moreover, it does not require advanced analytical knowledge or a
fully equipped laboratory.
Results were produced within 7 to 20 min depending on the
pathogen concentration, thus providing a high throughput and
user-friendly method for detection and screening of plant material
in support of quarantine regulations.
21
22. References
Lewin, Laboratory Medicine: A National Status Report, pp.
67-69.
https://en.wikipedia.org/wiki/Medical_test
https://www.littletonhealthcare.org/top_procedures.php
https://en.wikipedia.org/wiki/Rabies
Gigante et al, Multi-site evaluation of the LN34 panlyssavirus
real-time RT-PCR assay for postmortem rabies diagnostics
http://www.oie.int/animal-health-in-the-world/animal-
diseases/q-fever/
22
23. References
https://en.wikipedia.org/wiki/Myocardial_infarction
Mihaela Savin et al, A quantum dot-based lateral flow
immunoassay for the sensitive detection of human heart fatty
acid binding protein (hFABP) in human serum
Zhenyang Rong et al, Rapid diagnosis of rice bakanae
caused by Fusarium fujikuroi and F. proliferatum using loop-
mediated Isothermal amplification assays
M. Gentaz et al, A diagnostic tool for improved detection of
Xanthomonas fragariae using a rapid and highly specific
LAMP assay designed with comparative genomics 23
A medical test is a medical procedure performed to detect, diagnose, or monitor diseases, disease processes, susceptibility, or to determine a course of treatment. Medical tests relate to clinical chemistry and molecular diagnostics, and are typically performed in a medical laboratory
A blood calcium test is ordered to screen for, diagnose, and monitor a range of conditions relating to the bones, heart, nerves, kidneys, and teeth. The test may also be ordered if a person has symptoms of a parathyroid disorder, malabsorption, or an overactive thyroid.
he hemoglobin A1c test tells you your average level of blood sugar over the past 2 to 3 months. It's also called HbA1c, glycated hemoglobin test, and glycol hemoglobin. People who have diabetes need this test regularly to see if their levels are staying within range
A urine test strip or dipstick test is a basic diagnostic tool used to determine pathological changes in a patient's urine in standard urinalysis
An arterial blood gas (ABG) test measures the acidity (pH) and the levels of oxygen and carbon dioxide in the blood from an artery. This test is used to check how well your lungs are able to move oxygen into the blood and remove carbon dioxide from the blood.
Biomarker - a naturally occurring molecule, gene, or characteristic by which a particular pathological or physiological process, disease, etc. can be identified
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from the Japanese for "foolish seedling", is a disease that infects the rice plant.
LAMP is an isothermal nucleic acid amplification technique