Nucleic acid based tests

The most widely used methods for detecting microbial DNA fall into three categories:

1) Direct hybridization (non-amplified assay)

2) Amplification methods using the polymerase chain reaction (PCR)

3) DNA microarrays

Although not likely to completely replace culture techniques in the near future, nucleic acid–based

tests for the diagnosis of infectious diseases are gaining wider acceptance.

Direct detection of pathogens without target amplification

This highly specific method of pathogen detection involves identification of the DNA of the pathogen

in a patient sample or, more commonly, organisms isolated in culture. The basic strategy is to detect a

relatively short sequence of nucleotide bases of DNA sequence) that is unique to the pathogen. This is

done by hybridization with a probe, a single-stranded piece of DNA (usually labeled with a fluorescent

molecule) containing a complementary sequence of bases. In bacteria, DNA sequences coding for 16S

ribosomal RNA sequences (rRNA) are commonly used targets because each microorganism contains

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multiple copies of its specific 16S rRNA gene, thereby increasing the sensitivity of the assay. When

the probe is bound to the target, the label will give off a signal after the free probe is washed away. A

limitation of standard direct probe hybridization is the requirement for a 104

or greater number of

copies of target nucleic acid for detection.

Nucleic acid amplification for diagnosis

Nucleic acid amplification overcomes the principal limitation of direct detection with nucleic acid

probes by selectively amplifying specific DNA targets present in low concentrations. The bacterial

16S rRNA gene has emerged as the most useful marker for microbial detection and identification.

Ribosomal DNA genes contain highly conserved areas (that are used as targets for primers) separated

by internal transcribed sequences containing variable, species-specific regions. These sequences are

like fingerprints. Comparing certain locations on a 16s rRNA gene with a database of known

organisms allows the identification of organisms. For virus detection, primers are constructed to target

highly conserved DNA or RNA sequences unique to the pathogen. Amplification and detection of the

viral genomes are highly sensitive and are especially valuable when the viral load is too low to be

detected by culture or when results are needed rapidly.

Conventional polymerase chain reaction

DNA polymerase repetitively amplifies targeted portions of DNA (ideally sequences that are highly

conserved and unique to the pathogen). Each cycle of amplification doubles the amount of DNA in the

sample, leading to an exponential increase in DNA with repeated cycles of amplification. The

amplified DNA sequence can then be analyzed by gel electrophoresis, Southern blotting, or direct

sequence determination.

Real-time polymerase chain reaction (RT-PCR)

This variant of PCR combines nucleic acid amplification and fluorescent detection of the amplified

product in the same closed automated system. Real-time PCR limits the risk of contamination and

provides a rapid (30-40 minutes) diagnosis. Real-time PCR is a quantitative method and allows the

determination of the concentration of pathogens in various samples.

Advantages of polymerase chain reaction:

1- Methods employing nucleic acid amplification techniques have a major advantage over direct

detection with nucleic acid probes because amplification methods allow specific DNA or RNA

target sequences of the pathogen to be amplified millions of times without having to culture the

microorganism itself for extended periods

2- PCR also permits identification of non-cultivatable or slow-growing organisms, such as

Mycobacteria, anaerobic bacteria, and viruses

3- Nucleic acid amplification methods are sensitive, specific for the target organism, and are

unaffected by the prior administration of antibiotics

4- Nucleic acid amplification techniques are generally quick, easy, and accurate

5- They are useful in the detection of organisms that require complex media or cell cultures or

prolonged incubation times

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PCR amplification is limited by the occurrence of spurious false-positives due to cross-contamination

with other microorganisms’ nucleic acid. PCR tests are often costly and require skilled personnel

DNA microarrays

Although microarrays are now routinely used to measure gene expression, the technique is an

emerging technology in the diagnostic microbiology laboratory. Microarrays have the unprecedented

potential to simultaneously detect and identify many pathogens from the same specimen. For example,

an oligonucleotide microarray targeting the 16S rRNA gene has been developed for the detection of a

panel of forty predominant human intestinal bacterial pathogens in human fecal samples. DNA

microarray consists of microscopic spots of immobilized DNA oligonucleotide, each containing

specific DNA sequences, known as probes. The probes are constructed to be complementary to

specific gene sequences of interest in suspected pathogens. DNA of the microorganism obtained from

a clinical specimen, known as the target, is extracted and amplified using PCR and fluorescent

labeling techniques. The target DNA is exposed to the probe microarray. If the labeled DNA from the

microorganism and the immobilized probe has a complementary base sequence, they will hybridize,

thereby increasing fluorescence intensity. After washing off of nonspecific bonding sequences only

strongly paired strands will remain hybridized and fluoresce. The intensity of fluorescence at each spot

is a measure of the amount of that particular microbial DNA in the sample. Correlating fluorescence

with the identity of the probe allows for the detection and quantitation of specific pathogens.

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Lecture one

Sterilization and Disinfection

Sterilization is the process of killing or removing all viable organisms achieved by: physical and chemical

Disinfection is the process of removing or killing most but not all, viable organisms

Methods:

1. Chemical the substance is called a disinfectant it kills pathogens but may not kill viruses or spores

2. Physical process boiling or low pressure steam it reduces only the bioburden

Antiseptics: A particular group of disinfectants which used to reduce the number of viable organisms in the skin;

that act differentially on organism and host tissue

1. Germicide is a chemical agent capable of killing microbes

2. Sporicide is a germicide capable of killing bacterial spores

Pasteurization: it reduces the total number of viable microbes in bulk fluids such as milk and fruit juices without

destroying flavor and palatability.

Decision whether to use Sterilization or Disinfection it depended upon:

Cost

Damage involved

Low bioburden is a prerequisite for cost-effective sterilization.

Uses of sterilization and disinfection

1. Prevention of hospital infection:

a. sterile equipments, instruments and dressings

b. isolation facilities

c. safe disposal of infected materials

Figure shows the Antiseptics effect on normal skin flora

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2. Microbiologists: production of sterile media and the laboratory activities

3. Central to almost all areas of medical practice like surgery (from hand washing to needles and prosthesis)

Process: The rate of killing of microorganisms depends upon the concentration of the killing agent and time of

exposure.

N = 1/CT

N – Number of survivors

C – Concentration of agent

T – Time of exposure to the agent

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Mechanisms of action of antimicrobial agents:

1. Damage cell membrane

2. Denature proteins

3. Modify functional groups of proteins and nucleic acids

Activity of a particular disinfectant may result from one or combination of pathways.

Factors affecting efficacy:

1. Physical environment

2. Presence of moisture

3. Temperature and pH

4. Concentration of the agent

5. Hardness of water

6. Bioburden and the object

7. Mature and state of microbes in bioburden

8. Ability of microbes to inactivate the chemical agent

Techniques for Sterilization

I. Heat the preferred choice because of:

a. ease of use

b. controllability

c. low cost

d. efficiency

Types:

1. Dry heat

 Incineration

 use of Bunsen burner

 Hot air oven (one hour) 160-180 °C / 1 hour.

2. Moist heat under pressure

a- Autoclave the most effective for sterilization (121 °C / 15 minutes) in which the pressure aids in penetration;

in sterilization of surgical instruments, dressing and heat resistant pharmaceuticals or culture media

b- Boiling water for a few minutes can be used as a rapid emergency measure to disinfect instruments; it kills

vegetative bacteria but not all spores

c- Pasteurization: done at 62.8 – 65.6°C for 30 minutes, which used for fluids as it reduce the number of bacteria

and eliminate pathogen present in small numbers

Flash pasteurization (71.7 °C for 15 seconds). After the process, the fluid should be kept at a temperature below

10°C to minimize, subsequent bacterial growth.

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II. Irradiation: it include gamma and X-rays

Gamma irradiation which used for sterilizing large batches of small volume items such as:

1. Needles, syringes, catheters, gloves

2. Vaccines

3. To prevent food spoilage

4. Capital cost is high but process is 100% efficient

5. Killing mechanism involves production of free radicals that break the bonds in DNA

6. Sporocidal at higher doses (4.5 megarads)

III- Filtration

1-Used to produce particles and pyrogen-free fluid.

2-Composed of nitrocellulose

3-Work by electrostatic attraction and physical pore size

4-Purify drinking water

5-To recover very small number of organism from large volumes of fluid

6-Can be used for quantitating bacteria in fluids

Disinfection by chemical agents

1- Alkylating agents which include gases as

a- ethylene oxide (toxic and explosive)

b-Formaldehyde (extremely unpleasant odor; and irritant to mucous membrane decontaminate rooms and exhaust

protective cabinets)

2- Liquid

I- Glutaraldehyde - disinfect heat–sensitive articles (endoscopes surfaces)

II- Oxidizing agents

1-Hydrogen peroxide (H2O2)

3 – 6% kills most bacteria

10-25% kills all including spores

(Disinfects plastic implants, contact lenses and surgical prosthesis)

III-Halogens it includes:

a-Iodine compounds

1-Most effective, virtually effective among all organisms including spore- formers and Mycobacterium

2-Effective in acid pH because more free iodine is liberated

3-Acts more rapidly than other halogens and quaternary NH4 (ammonium) compounds

4- Effectivity reduced by serum, feces, body fluids.

Povidone iodine used to disinfect metal surfaces and tissues.

b-Chlorine compounds

It available in 3 forms:

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Cl2

HOCl (hypochlorous acid)

OCl (hypochlorite)

It has a good germicide activity, though spores are resistant to it.

IV. Phenolic compounds

a. rarely used as disinfectant nowadays

b. historical interest it was used as comparative standard for assessing the activity of germicidal compounds

V- Quaternary Ammonium compounds which are organic groups linked to nitrogen (Benzalkonium chloride and

Cetyl pyridinium chloride. Bacteriostatic at low concentrations, bactericidal at high. Not effective against

Pseudomonas, Mycobacterium and Trichophyton)

VI-Alcohols