Sunday, July 7, 2019

Introduction to Real time PCR

Real-time PCR is a sensitive method for RNA analysis based on fluorescence measurement. It is used in basic research, applied molecular medicine, and biotechnology. Real-time PCR assays are easy to perform and combine high sensitivity with reliability. The technology is evolving rapidly with the introduction of new reagents and instrumentation. It can be used for the confirmation of data acquired by microarray analysis of gene expression. We review basic principles of real-time PCR and describe its application in hematology and especially in multiple myeloma research.


Ref: www.researchgate.net/publication/


An Overview Of Molecular Biology

DNA, the chromosomal material in the cell nucleus, is transcribed by polymerases to form RNA species with different functions. These include messenger RNA (mRNA) produced from each of the ∼20,000 protein coding genes, microRNAs (mIRs) transcribed from the ∼500 regulatory mIR genes, and ribosomal and transfer RNAs that are components of the ribosome and the protein biosynthesis machinery. mRNAs are then translated into proteins by the ribosome and then typically degraded quickly because of the actions of mIRs and cellular nucleases. The set of mRNAs and mIR genes that get transcribed in any particular cell is regulated by growth factor–responsive transcription factors, cell type–specific enhancer complexes, and the epigenetic state of the DNA surrounding genes as well as their scaffold histone proteins. Epigenetic modulation of DNA and histones occurs commonly through methylation and acetylation and is dynamically regulated during hematopoietic cell development and during the development of leukemias and lymphomas.1,2 Acquired (somatic) defects in one or more of these processes underlie the development of hematologic conditions . In addition, inherited gene defects or normal population variations in these cellular functions lead to predisposition to subsequent development of hematologic conditions.11,12 With improved understanding of the basic mechanisms underlying disease, therapies which target the type of molecular aberrations in hematologic conditions have increasingly been developed.


Figure 4.1
 Polymerase chain reaction (PCR). A. A three-stage conventional PCR, with denaturation, annealing, and extension steps. Components of the typical PCR are illustrated including a DNA template (e.g., target gene), unlabeled nucleotides (dNTPs), a DNA polymerase to copy the templates and forward (F) and reverse (R) DNA primers, one of which is fluorescently labeled (*). B. Fluorescent products from the above PCR are then detected by capillary electrophoresis. Shown is a trace with a normally sized 167 base pair NPM1 gene product and an abnormal copy with a 4 base pair insertion (171b) characteristic of acute myeloid leukemia. C. Quantitative PCR using the TaqMan method with four samples showing differing amounts of the target gene as indicated by Cts ranging from 23 to 39 cycles (arrows). A graph showing 10-fold dilutions of a reference sample is plotted below, which are used to convert Ct in patient sample into copy number. D. Design of a TaqMan qPCR assay for detection of the JAK2 V617F mutation, with identical F and R primers but two different fluorescent probes; the red one detecting the normal JAK2 sequence (“G” at that position), and a green probe recognizing the mutated “T” sequence. The black 3′ moiety on the probes represents the quencher dye.

Polymerase Chain Reaction: The Indispensable Molecular Technique

From its first application to bacterial genetics in the early 1980s, PCR has been the central technique for amplifying genes so they can be sized to look for pathogenic insertions or deletions; sequenced to look for base pair mutations; and labeled with radioactivity, fluorochromes, or chromogenic moieties to use as probes in blots and reverse microarrays. The PCR technique involves the sequential amplification by repeated cycles of DNA denaturation, reannealing, and polymerase extension of DNA targets using flanking oligonucleotides (Fig. 4.1A). In the initial cycles of the PCR, the target is exponentially amplified before gradually plateauing when the large amount of product present tends to favor reannealing of double-stranded templates rather than primer binding/extension.
To detect the products that have been amplified by PCR, the reaction is typically run out on a solid agarose or polyacrylamide substrate or gel. These PCR amplicons can be detected by a laser using capillary electrophoresis if one of the primers has been labeled with a fluorochrome (Fig. 4.1B), or by slab gel electrophoresis followed by post-staining with a DNA-binding dye (e.g., ethidium bromide) that can be visualized with ultraviolet light (see Fig. 4.2, Step 1). As described above, if RNA is to be analyzed by PCR, it is first converted into cDNA in a technique known as RT-PCR. If fluorescent probes are added into the reaction, real-time or quantitative PCR (qPCR) can be performed to calculate the amount of an RNA or DNA target present in the initial sample. A common qPCR design is the TaqMan short, gene-specific probe that has a reporter fluorophore at its 5′ end and a quencher molecule at the 3′ end. The probe hybridizes to its target amplicon during the annealing step of each PCR cycle and is then hydrolyzed by the 5′ exonuclease activity of Taq polymerase during DNA extension. When the TaqMan probe is hydrolyzed, the reporter fluorophore is detached from the adjacent quencher molecule and fluoresces in an amount proportional to the degree of PCR product amplification. Thus, as probe is bound to template and its reporter released by the polymerase extension, the detected fluorescence rises exponentially. In qPCR, the amount of initial target present in a PCR is backcalculated by observing the PCR cycle in which the fluorescence signal first becomes detectable. This threshold cycle (Ct) can then be used for absolute or relative quantitation. For absolute quantitation, the observed Ct is converted to a target copy number by plotting it on a standard curve (log Ct vs. starting copy number) constructed from samples with a known target copy number (Fig. 4.1C). For relative quantitation, target quantities are expressed relative to a co-amplified normalizer control (e.g., a highly expressed housekeeping gene such as ACTB [b-ACTIN] or ABL1). The quantity is then represented as a relative ratio most commonly the delta-Ct calculation: [relative quantity] = 2−(Ct of gene target – Ct of reference gene). A specialized form of qPCR used to detect single base pair changes in DNA is allele-specific (AS)-PCR. This method compares the amplification levels of a PCR probe or primer that recognizes one allele versus the signal from a probe that recognizes only the other allele. This same protocol can also be used to sensitively detect the level of mutated sequences in neoplasms.19 This method can routinely detect the presence of a mutation down to 0.1% of the template in the sample (Fig. 4.1D).

DNA Sequencing: The Technique Driving the Genomic Revolution

The DNA sequence of genes is built up from combinations of four nucleotides, adenine (A), cytosine (C), guanine (G), and thymine (T), and their epigenetically modified variants, particularly 5-methylcytosine. DNA sequencing to determine the base composition of the genome was first routinely applied in the late 1970s but has remained a difficult and expensive technique until the last several years. The accurate but costly gold-standard technique for determining DNA base composition, developed by Frederic Sanger, is called the dideoxy chain termination method.20 After an initial PCR step to amplify the gene of interest, this method relies on a second asymmetric PCR step in which stops in the PCR extension are randomly introduced at each position in the product by adding fluorescently labeled chain terminating variants of the A, C, G, and T nucleotides, each terminating nucleotide being labeled with a different color (green, blue, black, and red). This range of DNA molecules each terminated at a different position are then separated by size using electrophoresis and the sequence read by laser detection of the terminally labeled nucleotide (Fig. 4.2).

                                                              Figure 4.2
 DNA Sequencing.

 Steps in the dideoxy chain termination (Sanger) method include: Step 1: Standard polymerase chain reaction (PCR) to produce large amounts of a genespecific template, detected by slab electrophoresis followed by ethidium bromide staining of the gel. Step 2: Unidirectional (or asymmetric) PCR using the template from the first PCR along with either a forward or reverse primer in a reaction containing normal nucleotides mixed with chain terminating A, C, G, and T bases. Step 3: The range of products from the asymmetric PCR which are terminated at every possible base in the PCR amplicon are then separated by capillary electrophoresis and detected by a laser recognizing the fluorochrome/nucleotide present at the end of products. Base-calling is performed using software which normalizes the peak heights to produce the depicted electropherogram.

Molecular Diagnostic Applications In Hematology 

The diagnosis of specific types of lymphoid and myeloid malignancies is discussed elsewhere in this volume, but here we summarize generally how molecular techniques are used to assist in their diagnosis. The current schema for diagnosis of hematologic neoplasms is the World Health Organization (WHO) Classification of Hematologic and Lymphoid Neoplasms.23 This classification incorporates morphology and immunophenotypic features but also increasingly relies on molecular and cytogenetic testing for definitive diagnosis.


Saturday, February 5, 2011

Typhoid Fever

What Is Typhoid Fever?
Typhoid fever is a life-threatening illness caused by the bacterium Salmonella typhi. In the United States, about 400 cases occur each year, and 75 percent of these are acquired while traveling internationally. Typhoid fever is still common in the developing world, where it affects about 21.5 million people each year.

Common Symptoms

The most common typhoid fever symptoms include:


  • Fever as high as 103° to 104° F (39° to 40° C)
  • Weakness
  • Stomach pains
  • Headache
  • Loss of appetite
  • Decreased heart rate
  • Constipation
  • Bloody nose
  • Rash of flat, rose-colored spots.


If the Symptoms Go Untreated


  • Severe loss of appetite
  • Intestinal bleeding
  • Weight loss
  • Hepatitis
  • Meningitis
  • Bronchitis
  • Pneumonia
  • Arthritis
  • Osteomyelitis
  • Orchitis
  • Parotitis
  • Death.

How does the bacteria cause disease, and how is it diagnosed?

After the ingestion of contaminated food or water, the Salmonella bacteria invade the small intestine and enter the bloodstream temporarily. The bacteria are carried by white blood cells in the liver, spleen, and bone marrow. The bacteria then multiply in the cells of these organs and reenter the bloodstream. Patients develop symptoms, including fever, when the organism reenters the bloodstream. Bacteria invade the gallbladder, biliary system, and the lymphatic tissue of the bowel. Here, they multiply in high numbers. The bacteria pass into the intestinal tract and can be identified for diagnosis in cultures from the stool tested in the laboratory. Stool cultures are sensitive in the early and late stages of the disease but often must be supplemented with blood cultures to make the definite diagnosis.


How is typhoid fever treated, and what is the prognosis?

Typhoid fever is treated with antibiotics that kill the Salmonella bacteria. Prior to the use of antibiotics, the fatality rate was 20%. Death occurred from overwhelming infection, pneumonia, intestinal bleeding, or intestinal perforation. With antibiotics and supportive care, mortality has been reduced to 1%-2%. With appropriate antibiotic therapy, there is usually improvement within one to two days and recovery within seven to 10 days.
Several antibiotics are effective for the treatment of typhoid fever. Chloramphenicol was the original drug of choice for many years. Because of rare serious side effects, chloramphenicol has been replaced by other effective antibiotics. The choice of antibiotics needs to be guided by identifying the geographic region where the organism was acquired and the results of cultures once available. (Certain strains from South America show a significant resistance to some antibiotics.) Ciprofloxacin (Cipro), ampicillin (Omnipen, Polycillin, Principen), and trimethoprim-sulfamethoxazole (Bactrim, Septra) are frequently prescribed antibiotics. If relapses occur, patients are retreated with antibiotics.
The carrier state, which occurs in 3%-5% of those infected, can be treated with prolonged antibiotics. Often, removal of the gallbladder, the site of chronic infection, will cure the carrier state.
For those traveling to high-risk areas, vaccines are now available.


Preventions
  1. Avoid risky foods and drinks.
  2. 2. Get vaccinated against typhoid fever.
It may surprise you, but watching what you eat and drink when you travel is as important as being vaccinated. This is because the vaccines are not completely effective. Avoiding risky foods will also help protect you from other illnesses, including travelers' diarrhea, cholera, dysentery, and hepatitis A.

"Boil it, cook it, peel it, or forget it"
  • If you drink water, buy it bottled or bring it to a rolling boil for 1 minute before you drink it. Bottled carbonated water is safer than uncarbonated water.
  • Ask for drinks without ice unless the ice is made from bottled or boiled water. Avoid popsicles and flavored ices that may have been made with contaminated water.
  • Eat foods that have been thoroughly cooked and that are still hot and steaming.
  • Avoid raw vegetables and fruits that cannot be peeled. Vegetables like lettuce are easily contaminated and are very hard to wash well.
  • When you eat raw fruit or vegetables that can be peeled, peel them yourself. (Wash your hands with soap first.) Do not eat the peelings.
  • Avoid foods and beverages from street vendors. It is difficult for food to be kept clean on the street, and many travelers get sick from food bought from street vendors.
Getting vaccinated

If you are traveling to a country where typhoid is common, you should consider being vaccinated against typhoid. Visit a doctor or travel clinic to discuss your vaccination options.

Remember that you will need to complete your vaccination at least 1 week before you travel so that the vaccine has time to take effect. Typhoid vaccines lose effectiveness after several years; if you were vaccinated in the past, check with your doctor to see if it is time for a booster vaccination. Taking antibiotics will not prevent typhoid fever; they only help treat it.


Leptospirosis



What is Leptospirosis?
Leptospirosis, also known as canicola fever, hemorrhagic jaundice, infectious jaundice, mud fever, spirochetal jaundice, swamp fever, swineherd's disease, caver's flu or sewerman's flu, is a bacterial infection resulting from exposure to the Leptospira interrogans bacterium. There is an acute form of human infection known as Weil's disease, where the patient suffers from jaundice, though this term is often (incorrectly) used to describe any case of infection. Weil's disease is comparatively rare, though 'mild' cases of leptospirosis happen everywhere there are carriers, and it is believed that leptospirosis is one of the most common zoonotic infections in the world. Millions of people are infected each year, but information and treatment can be limited, especially in the developed world where cases are considered 'rare' by the medical community. The goal of the LIC and this website is to increase awareness of the infection and how to manage the risks, with clear advice and guidance for all aspects of work and leisure.

Symptoms
Symptoms can take 2 - 26 days (average 10 days) to develop, and may include:
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  • Dry cough
  • Fever
  • Headache
  • Muscle pain
  • Nausea, vomiting, and diarrhea
  • Shaking chills
Less common symptoms include:

  • Abdominal pain
  • Abnormal lung sounds
  • Bone pain
  • Conjunctivitis
  • Enlarged lymph glands
  • Enlarged spleen or liver
  • Joint aches
  • Muscle rigidity
  • Muscle tenderness
  • Skin rash
  • Leptospirosis in kidney
  • Sore throat

Diagnostic Test for Leptospirosis:

The list of medical tests mentioned in various sources as used in the diagnosis of Leptospirosis includes:
  • Blood culture
  • Urine culture
  • CSF culture
  • Blood antibody tests

Home Diagnostic Testing

These home medical diagnostic tests may be relevant to Leptospirosis:
  • Cold & Flu: Home Testing:
    • Home Fever Tests
    • Home Ear Infection Test Kits
    • Home Flu Tests
    • Home Strep A Tests 
Treatment for leptospirosis
The treatment of leptospirosis involves high doses of antibiotics. Antibiotic treatment (doxycycline [Vibramycin, Oracea, Adoxa, Atridox], penicillin) is most effective when initiated early in the course of the illness. Severely ill patients may need hospitalization for IV fluid and antibiotic treatment. Severe liver and kidney manifestations of the infection may require intensive medical care and sometimes dialysis treatment. However, even in severe cases, liver and kidney function often does return after recovery from the illness.
Mortality rates for severe illness with leptospirosis can range from 5%-40%, depending on the severity of organ dysfunction and the patient's general health prior to infection. Most previously healthy patients have a good prognosis and will make a full recovery.

Swine Flu

About Swine flu
Swine flu (swine influenza) is a respiratory disease caused by viruses (influenza viruses) that infect the respiratory tract of pigs and result in nasal secretions, a barking-like cough, decreased appetite, and listless behavior. Swine flu produces most of the same symptoms in pigs as human flu produces in people. Swine flu can last about one to two weeks in pigs that survive. Swine influenza virus was first isolated from pigs in 1930 in the U.S. and has been recognized by pork producers and veterinarians to cause infections in pigs worldwide. In a number of instances, people have developed the swine flu infection when they are closely associated with pigs (for example, farmers, pork processors), and likewise, pig populations have occasionally been infected with the human flu infection. In most instances, the cross-species infections (swine virus to man; human flu virus to pigs) have remained in local areas and have not caused national or worldwide infections in either pigs or humans. Unfortunately, this cross-species situation with influenza viruses has had the potential to change. Investigators think the 2009 swine flu strain, first seen in Mexico, should be termed novel H1N1 flu since it is mainly found infecting people and exhibits two main surface antigens, H1 (hemagglutinin type 1) and N1 (neuraminidase type1). Recent investigations show the eight RNA strands from novel H1N1 flu have one strand derived from human flu strains, two from avian (bird) strains, and five from swine strains.

symptoms of swine flu (H1N1)?
Symptoms of swine flu are similar to most influenza infections: fever (100F or greater), cough, nasal secretions, fatigue, and headache, with fatigue being reported in most infected individuals. Some patients also get nausea, vomiting, and diarrhea.


Flu Symptoms at a Glance


Symptoms

Cold
Flu
Fever
Fever is pretty rare with a cold.
Fever is usually present with the flu. A temperature of 101°F or higher for 3 to 4 days is associated with the flu.
Chills
Chills are uncommon with a cold.
Chills are fairly common in most flu cases. Chills and shivering are a normal reaction to a cold environment, but unexplained chills can also be a sign of the flu.
Tiredness
Tiredness is fairly mild with a cold.
Tiredness is moderate to severe with the flu. It’s normal to feel tired at the end of a long day or when yo don’t get adequate sleep, but unexplained tiredness can be a sign of the flu.
Sudden Symptoms
Cold symptoms are not sudden and develop over a few days.
The flu has a rapid onset with 3-6 hours. The flu hits hard and includes sudden symptoms like high fever, aches and pains.
Coughing
A hacking, productive (mucus producing) cough is often present with a cold.
A dry, nonproductive cough that does not produce mucus is usually present with the flu.
Sneezing
Sneezing is commonly present with a cold.
Sneezing is not as common, but can accompany the flu.
Stuffy or Runny Nose
A stuffy or runny nose usually accompanies a cold and typically resolves spontaneously within a week.
Stuffy or runny nose can be present with the flu.
Sore Throat
Sore throat is commonly present with a cold. A sore throat is pain and inflammation is in the throat that usually comes with a cold.
Sore throat is not as common, but can be present with the flu.
Chest Discomfort
Chest discomfort is mild to moderate with a cold.
Chest discomfort is often severe with the flu. Chest discomfort is pain or abnormal sensations that you feel anywhere along the front of your body between your neck and upper abdomen.
Headache
A headache is fairly uncommon with a cold.
A headache is very common with the flu, present in 80% of flu cases.



Diagnostic
new molecular probe improves on the existing PCR assay used to diagnose seasonal influenza and enables detection of both the seasonal and H1N1 influenza A viruses in the same patient sample using a simple test protocol.

In positive samples this is followed by the addition of two probes that are able to discriminate between the seasonal and swine H1N1 viruses to yield a definitive diagnosis.

Early, accurate identification of infected individuals will expedite appropriate antiviral therapy and enhance control and containment efforts.

The new molecular test specifically amplifies and characterizes the viral genetic material, enabling rapid detection of new viral strains as they evolve.

The researchers say that using these genetic sequence data and making minor alterations to the PCR 



Treatment is available for swine flu (H1N1)



The best treatment for influenza infections in humans is prevention by vaccination. Work by several laboratories has recently produced vaccines. The first vaccine released in early October 2009 was a nasal spray vaccine. It is approved for use in healthy individuals ages 2 through 49. This vaccine consists of a live attenuated H1N1 virus and should not be used in anyone who is pregnant or immunocompromised. The injectable vaccine, made from killed H1N1, became available in the second week of October. This vaccine is approved for use in ages 6 months to the elderly, including pregnant females. Both of these vaccines have been approved by the CDC only after they had conducted clinical trials to prove that the vaccines were safe and effective. However, caregivers should be aware of the vaccine guidelines that come with the vaccines, as occasionally, the guidelines change. Please see the sections below titled "Can novel H1N1 swine flu be prevented with a vaccine?" and the timeline update for the current information on the vaccines.
Two antiviral agents have been reported to help prevent or reduce the effects of swine flu. They are zanamivir (Relenza) and oseltamivir (Tamiflu), both of which are also used to prevent or reduce influenza A and B symptoms. These drugs should not be used indiscriminately, because viral resistance to them can and has occurred. Also, they are not recommended if the flu symptoms already have been present for 48 hours or more, although hospitalized patients may still be treated past the 48-hour guideline. Severe infections in some patients may require additional supportive measures such as ventilation support and treatment of other infections like pneumonia that can occur in patients with a severe flu infection. The CDC has suggested in their interim guidelines that pregnant females can be treated with the two antiviral agents.


Prevention
            The three best ways to protect yourself from this current threat and any that may follow are: 1) maintain a potent immune system; 2) use common sense measures to protect yourself; and 3) encourage the same in those around you (see the second image below).
There are many natural substances that help support immunity. Below, I’ve put together a list of several inexpensive options that are widely available and that also promote other aspects of good health. All of these measures are very safe and have what I call “side benefits” (as opposed to “side effects”).
  • Don’t Panic: Stress suppresses your ability to fight infections (1,2). You can literally worry yourself sick or find a way to constructively process these very real feelings. Go for a walk with a friend. Practice deep breathing or guided imagery. Meditate. Get a massage. Watch funny movies – laugh! (1,2) You may not be able to control the viral realm of the world, but you can help your body and mind be strong in the face of any viral challenge.
  • Don’t Comfort Yourself with Sugar: As tempting as it may be, try to avoid reaching for starchy and sugary snacks to soothe negative feelings. A famous study from 1973 indicates that consuming large amounts of carbohydrates and sugar can drastically lower the activity of neutrophils. Neutrophils account for about 50-70% of white blood cells and are the first responders when an infection is detected by the body. The effects of these unhealthy carbohydrates can last for over 5 hours.
  • Sleep It Off: When in the midst of an illness the body often demands more sleep, simply because sleep helps strengthen the body’s defenses. The same is true when trying to stay well. The key is to get adequate sleep (usually between 7-9 hours) in a completely dark environment. Darkness is necessary in order for the brain to produce adequate/optimal levels of melatonin, which may be responsible for some of the immune-boosting effects of sleep. (1,2)
  • Let the Sunshine In: There’s a growing body of evidence that vitamin D is imperative for optimal immune function. You can prime your body’s immune system by getting regular sun exposure. The key is to spend time in the sun during the morning and evening hours so as not to get a sunburn. Some experts recommend 15 minutes or more of “smart sun exposure” several times a week. Nutritional supplements can also be used to ensure a consistent and adequate supply of vitamin D.
  • Know Your Berries: Black currant and black elderberry extracts  may help combat influenza. Dr. Madeleine Mumcuoglu, an Israeli virologist, proposes that black elderberries may interfere with the flu virus’ ability to attach to healthy cells.
  • Drink and Gargle with Tea: A study from September 2006 found that gargling with a green tea extract helped to prevent the incidence of influenza in a nursing home setting. Subjects gargled 3 times a day for 3 months with a green tea concentrate that was standardized for catechin and ECGC content. A 1997 study also found anti-influenza activity in patients who gargled with a black tea extract. Many other laboratory experiments suggest a role for tea in the prevention of flu-related illnesses and in supporting the immune system (1,2,3)
  • Take Your Multi: There are many nutrients that are absolutely vital to ensure healthy immunity. The simplest way to nutritionally “cover your bases” is to take a high potency multivitamin/mineral. Ideally, this means taking a supplement that needs to be consumed 2-3 times a day with meals or snacks.