1. Soil borne pathogens have significantly impacted human health over centuries by potentially enabling the development of Europe and America through wiping out half the native populations. 2. Real-time PCR allows for the detection and quantification of soil borne pathogens during each cycle of the reaction through the use of sequence specific DNA probes, eliminating the need for gel electrophoresis. 3. Screening samples for soil borne pathogens using real-time PCR enables early disease diagnosis and prophylactic measures while minimizing laboratory hazards compared to culture-based techniques, and can inform spatial epidemiology through mapping of positive areas.

1. REAL-TIME PCR FOR THE DETECTION OF SOIL BORNE PATHOGENS
Haroon Rashid Chaudhry, Tariq Jamil, Umair Ahsan and Shehzad Ashraf Khan Baloch
Haroon.rashid@iub.edu.pk
College of Veterinary & Animal Sciences, The Islamia University of Bahawalpur
Department of Microbiology, UVAS Lahore
Across the realm of time in units of centuries soil borne pathogens have had an incredible impact on human
health. Sometimes scientists believe that the advent of Europe and America was based on the infections
caused by these soil borne pathogens which paved the way for the development caused by wiping out
half the population of these countries leading to the survival of the fittest theory to input its design.
These pathogens inhabit the soil mostly in minority with respect to other soil community, which is
found in abundance in a biological commune. These include a number of viral, bacterial, parasitic
and/or protozoan agents in nature. Many of them are of zoonotic and of public health importance and
are placed for Category A bioterrorism diseases due to their severity and peracute nature of the disease
they cause. A few of the biological threat diseases are e.g. Anthrax, Botulism, Plague, Tularemia,
Glanders to name a few. In animals, these soil borne bacteria gain entry through the contaminated feed,
water and inhalation which may be through grazing or through the ingestion of contaminated, and or
soiled hay, whereas, in humans, it is through ingestion of dust polluted food and contaminated water
sources. As they reside in soil in a commune, soil may act as a possible reservoir for transmission of
such pathogens. In most of the developed countries we have not experienced an outbreak by the causal
agents but with the problem caused by Anthrax letters in America and the potential socioeconomic
losses incurred to USA to control these diseases it has been established that the organisms survive in
unique niches in the third world countries where they may be procured and be used as a biological agent
or may cause a spontaneous outbreak in the area leading to devastation in the world. Thus it has been
considered by considerate countries to study and make screening of these public health significant
organisms from soil. As there is only a small quantity of the agent in the soil commune its detection is
difficult and has become crucially important in order to determine local threat and prevent any future
occurrences. This is an area where conventional ELISA has proved of little significance due to poor
results. Routine culturing and identification practices may be of use but since these are category ‘A’
bioterrorism agents growing them may be dangerous not only to the community but may become an
international threat. To grow them the legislations are strict and require very expensive techniques and
require expensive equipment.
PCR is a molecular technique used to amplify DNA sequences. This technique enables us to early
diagnose genetic disorders and to detect non-culturable, slow growing and infectious microorganisms. This
technique is also used to differentiate between pathogenic and non-pathogenic strains of microorganisms.
Sometimes inhibitors in the reaction mixture block PCR due to self annealing of the products that make the
band results unreliable.
Real-Time PCR is a technique that solves this problem. This technique allows detection and
quantification of the PCR products during each cycle of the reaction. It is possible by introducing sequence
specific DNA probes attached with a dye (FAM or TAMRA) that binds to template DNA and gives
florescence when cleaved by the polymerase enzyme by its 5’-nuclease activity. Thus Real-Time PCR saves
time by eliminating the need of Gel Electrophoresis by the end. This makes Real-Time PCR technique to be
used for quantification and screening of the samples on the basis of nucleic acids of diagnostic importance
(infectious diseases) both.
Screening of samples by using Real-Time PCR is helpful in early diagnosis of the disease and also
for prophylactic measures where positive samples are found. This technique also does not require

2. sophisticated laboratory facilities that are required to culture such pathogens hence minimizing the potential
laboratory hazards. The positive areas may be mapped further to observe spatial ecology and epidemiology
using GIS/GPS techniques. Information on natural prevalence of these public health significant pathogens
can help understanding of potential source of the disease in future and design policies and prevention plans.