Overview

• Detection kits for H5N1
• Available in TaqMan format for analysis

Influenza virus infection of birds, humans and other animals is a major public health problem worldwide. Influenza viruses are classified as either type A, B or C based on differences in their nucleoproteins and matrix proteins. The type A viruses are the most virulent human pathogens among the three influenza types and cause the most severe disease and epidemics. The different types can be further classified into subtypes based on antigenic differences in two surface glycoproteins; hemagglutinin and neuroamidase. All known subtypes of influenza A can be found in birds (H1-H16, N1-N9), while a limited number of the subtypes have been found in humans (H1-H3, N1 and N2). However, over the past few years, various subtypes of Influenza A viruses, including H5N1, have been reported to infect humans (WHO, 2006). In addition, the coexistence of human influenza viruses and avian influenza viruses may provide an opportunity for genetic material to be exchanged between these viruses. This could potentially create a new virulent influenza strain that is easily transmissible and lethal to humans (Food Safety Research Information Office, 2006). Thus, there is the need for sensitive diagnostic tests to allow for the rapid and early detection of these H5 influenza virus infections, to help reduce the risk of epidemics or pandemics in both animals and humans.

H5N1 TaqMan RT-PCR Kit, 100 reactions

• Ready to use format, including Master Mix for the target and PCR control to monitor for PCR inhibition and validate the quality
• Specific Primer and Probe mix for the pathogen/virus/viroid of interest
• Primer and Probe mix
• Positive and negative control to confirm the integrity of the kit reagents

H5N1 TaqMan RT-PCR Probe/Primer Set and Controls, 100 reactions

• Specific Primer/Probe mix and Positive Control for the pathogen/virus/viroid of interest
• Nuclease-free water
• Can be used together with Norgen’s RT-PCR Master Mix (#28113) or customer supplied master mix

For research use only and NOT intended for in vitro diagnostics.

Details

Supporting Data

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Storage Conditions and Product Stability
All kit components can be stored for 1 year after the date of production without showing any reduction in performance.

All kit components should be stored at -20°C upon arrival. Repeated thawing and freezing (> 2 x) of the Master Mix and Positive Control should be avoided, as this may affect the performance of the assay. If the reagents are to be used only intermittently, they should be frozen in aliquots.

Component	Cat. TM35450 (100 preps)	Cat. TM35410 (100 preps)
MDx TaqMan 2X PCR Master Mix	2 x 700 μL	–
H5N1 Primer & Probe Mix	280 μL	280 μL
H5N1 Positive Control	150 μL	150 μL
Nuclease-Free Water (Negative Control)	1.25 mL	1.25 mL
Product Insert	1	1

Introduction

HiPure PCR Pure Mini Kit uses proprietary chemistry and HiPure technology to recover DNA Fragments between 70bp-30kbp with yields exceeding 85%. DNA is suitable for ligations, PCR, sequencing, restriction digestion, or various labeling reactions. In addition, this kit can be also used to recover DNA directly from enzymatic reactions such as PCR and enzyme digestion reactions.

Compared with the gel purification kit, this product uses guanidine hydrochloride mediated adsorption, which have better A260/A230 ratio.

Details

Specifications

Features	Specifications
Main Functions	Recover DNA fragments >100bp from various pcr products, enzymatic reaction solutions, labeling reaction solutions, crude DNA, etc.
Applications	PCR, sequencing, labeling reactions, ligations and restriction digestion, etc.
Purification method	Mini spin column
Purification technology	Silica technology
Process method	Manual (centrifugation or vacuum)
Sample type	PCR products, enzymatic reaction solution
Sample amount	10-200μl
Recovery	≥85%
Elution volume	≥15μl
Time per run	≤10 minutes
Liquid carrying volume per column	800µl
Binding yield of column	35µg

Principle

The HiPure system uses a simple bind-wash-elute procedure. Gel slices are dissolved in a buffer containing a pH indicator, allowing easy determination of the optimal pH for DNA binding, and the mixture is applied to the column. Nucleic acids adsorb to the silica-gel membrane in the high-salt conditions provided by the buffer. Impurities are washed away and pure DNA is eluted with a small volume of low-salt buffer provided or water, ready to use in subsequent applications.

Advantages

• High recovery efficiency – ≥85% DNA recovery
• Fast – isolation can be completed in 10 minutes by column gel method

Kit Contents

Contents	D212102	D212103
Purification Times	100 Preps	250 Preps
Buffer DP	60 ml	120 ml
Buffer DW2	20 ml	50 ml
Elution Buffer	15 ml	30 ml
HiPure DNA Mini Columns II	100	250
2 ml Collection Tubes	100	250

Storage and Stability

The Kit should be stored dry at room temperature (15-25°C) and are stable for at least 18 months under these conditions. If any precipitates form in the buffers, warm at 37℃ to dissolve.

Experiment Data

HiPure PCR Pure Mini Kit uses proprietary chemistry and HiPure technology to recover DNA Fragments between 70bp-30kbp with yields exceeding 85%. DNA is suitable for ligations, PCR, sequencing, restriction digestion, or various labeling reactions. In addition, this kit can be also used to recover DNA directly from enzymatic reactions such as PCR and enzyme digestion reactions.

Compared with the gel purification kit, this product uses guanidine hydrochloride mediated adsorption, which have better A260/A230 ratio.

Description

Brucella abortus is an intracellular, blood-borne parasite. It is a Gram-negative coccobacillus that causes an infectious and contagious disease called Brucellosis. The disease primarily affects cattle but it can also be transmitted to humans from infected animals and consuming their products. The disease can lead to great economic loss especially in the dairy and agricultural industry. The Brucella abortus genome contains two DNA chromosomes in a circular confirmation; the first chromosome is approximately 2.1 Mb and the second chromosome is approximately 1.2Mb. Unusually it does not contain any plasmids or genomic islands that relate to pathogenicity and lacks many other genes that code for common virulence factors including capsules, fimbriae, exotoxins, cytolysins, resistance forms, or antigenic variation. The most common mode of transmission to humans is through the ingestion of unpasteurized milk and cheese products as the bacteria are present in the milk glands of infected female cows. In cattle transmission can also be through ingestion but in addition, the bacteria can persist in the reproductive tracts of males, namely seminal vesicles, ampullae, testicles, and epididymides, allowing sexual transmission. In humans the bacteria enter macrophages by phagocytosis and then live in compartments of vacuolar space along the endoplasmic reticulum. They persist by inhibiting host apoptosis and go onto form chronic disease causing lesions in the liver, spleen, bone marrow and kidneys. In cattle the bacteria additionally infect the trophoblast epithelial cells, which provide nutrition to the embryo. The trophoblast cells eventually lyse, releasing further bacteria into the blood stream of the embryo. The B. abortus cells in the blood stream go on to colonize the placenta and fetus in pregnant female cows, resulting in abortion of the fetus. Abortion can also result from insufficient anti-Brucella activity in the amniotic fluid. In humans, the disease can be either acute or chronic and some of the symptoms include fluctuating fever, chills, sweating, headache, muscle pain and weight loss. Once a person becomes infected they are prescribed a combination of tetracycline and streptomycin for 3-6 weeks. In cattle, additional symptoms include arthritic joints and retained after-birth.

Exceptional value for money
Rapid detection of all clinically relevant subtypes
Positive copy number standard curve for quantification
Highly specific detection profile
High priming efficiency
Broad dynamic detection range (>6 logs)
Sensitive to < 100 copies of target Accurate controls to confirm findings