Overview

• Detection kits for Cryptosporidium
• CE-IVDD marked in accordance with the European Commission Directive 98/79/EC.
• Research version available
• Available in Taqman Probe format for analysis

Cryptosporidium is a parasite found in water that causes an infection in mammals termed cryptosporidiosis. It is one of the most common water-borne diseases and is found world-wide. It affects the intestines of mammals and typically causes an acute short-term infection. The most common symptom is self-limiting diarrhea in healthy individuals, however in immunocompromised individuals the symptoms are particularly severe and often fatal. There is no specific treatment for cryptosporidiosis other than fluid rehydration and management of any pain. Therefore early detection of Cryptosporidium in water is the foremost action to prevent the infection.

Cryptosporidium TaqMan RT-PCR Kit, 24 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

Details

Supporting Data

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Storage Conditions

The Cryptosporidium TaqMan PCR Kit Dx is shipped on dry ice. The components of the kit should be frozen upon arrival. If one or more of the components is not frozen when the kit is received, or if any of the components have been compromised during shipment, please contact Norgen Biotek for assistance. All kit components should be stored at -20°C upon arrival. Repeated thawing and freezing (> 3 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. DxTM39100 (24 rxns)
MDx TaqMan 2X RT-PCR Master Mix Dx	550 µL
Cryptosporidium Primer & Probe Mix Dx	2 x 70 µL
Cryptosporidium Positive Control Dx – 200,000 μL	50 µL
Nuclease-Free Water	2 x 1.25 mL
Product Insert	1

Introduction

Blood samples contain rich DNA, including mitochondrial DNA, genomic DNA, circulating DNA (mostly released into blood after tumor cell apoptosis) in white blood cells, as well as parasitic viral or microbial DNA. These DNA are important parameters in clinical testing or diagnosis, which are also valuable materials for medical research. There are three main issues with extracting DNA from blood samples:

1. The sample is highly infectious, posing great harm to operators and the environment.

2. The source of DNA is complex and aportion of the nucleic acid, such as viral DNA or free DNA, may be lost during the operation, leading to downstream detection failure;

3. Blood sample contains a large amount of impurities and inhibitory factors.

Currently there are many methods available for extracting DNA from whole blood samples, such as phenol chloroform extraction, salting out method, etc. However, these methods require pre-treatment of blood sample, which removes red blood cells and isolate white blood cells in the first step. Due to the requirement that it cannot inactivate or kill pathogens during the process of removing red blood cells, the waste liquid (red blood cell lysate) and consumables may be contaminated by pathogens and become infectious, posing a danger to the entire laboratory environment and operators. In addition, during the process of removing red blood cells, useful nucleic acid information such as viruses, microorganisms, or circulating DNA is also lost, leading to experiment or detection failures.

The HiPure Blood DNA Kits series provided by Magen Company uses silica gel column purification technology, which can directly lyse whole blood samples without the need for white blood cell separation. Whole blood samples are directly mixed with lysates and proteases, resulting in the inactivation of pathogens, greatly reducing the infectivity, environmental pollution, and the chance of operators being infected. Due to the direct lysis and digestion of samples, except lymphocyte DNA, other circulating DNA as well as DNA from viruses and microorganisms, can also be recovered.

This product provides fast and easy methods for purification of total DNA for reliable PCR and Southern blotting. Total DNA (e.g., genomic, viral, mitochondrial) can be purified from tissue, whole blood, plasma, serum, buffy coat, bone marrow, other body fluids, lymphocytes, cultured cells.

Details

Specifications

Features	Specifications
Main Functions	Isolation total DNA from blood, tissue, culture cells, swab, blood spots using 96 plate
Applications	PCR, southern bolt and virus detection, etc
Purification method	96 well plate
Purification technology	Silica technology
Process method	Manual (centrifugation or vacuum)
Sample type	Blood, serum, plasma, milk, saliva, and other liquid samples and cultured cells
Sample amount
Elution volume
Time per run
Liquid carrying volume per column
Binding yield of column

Principle

This product is based on silica column purification. The sample is lysed and digested with lysate and protease, DNA is released into the lysate. Transfer to an adsorption column. Nucleic acid is adsorbed on the membrane, while protein is not adsorbed and is removed with filtration. After washing proteins and other impurities, Nucleic acid was finally eluted with low-salt buffer (10mm Tris, pH9.0, 0.5mm EDTA).

Advantages

• High quality DNA – meet a variety of downstream applications, including PCR, qPCR, enzyme digestion, hybridization, etc.
• High throughput – 96 samples can be processed simultaneously

Kit Contents

Contents	D311701	D311702
Purification Times	1 x 96	4 x 96
HiPure gDNA Plate	1	4
96 well Plate (2.2ml)	1	4
1.6ml Collection Plate	1	4
0.5ml Collection Plate	1	4
Silicon Seal Tape	1	4
Seal Film	5	25
Buffer ATL	30 ml	100 ml
Buffer AL	30 ml	100 ml
Buffer DW1	60 ml	250 ml
Buffer GW2	50 ml	2 x 100 ml
Proteinase K	50 ml	200 ml
Protease Dissolve Buffer	5 ml	15 ml
Buffer AE	30 ml	120 ml

Storage and Stability

Proteinase K should be stored at 2-8°C upon arrival. However, short-term storage (up to 12 weeks) at room temperature (15-25°C) does not affect their performance. The remaining kit components can be stored at room temperature (15-25°C) and are stable for at least 18 months under these conditions.

Purchase Guide

Name	CAT NO	Sample amount	Leukocyte protocol*	Colum type	Elutio volume	Average yield	Time per run
HiPure Blood DNA Mini Kit	D3111	10-200μl	1ml	2ml column	≥20μl	5-9μg/200μl	≤30 minutes
HiPure Tissue&Blood DNA Midi Kit	D3113	0.2-2ml	10ml	1.5ml column	≥300μl	20-40μg/1m	≤80 minutes
HiPure Tissue&Blood DNA Maxi Kit	D3115	3 -10ml	10ml	15ml column	≥700μl	20-40μg/1m	≤90 minutes
HiPure Tissue&Blood DNA 96 Kit	D3117	1-200μl	1ml	96 well plate		3-8μg/200μl

Blood samples contain rich DNA, including mitochondrial DNA, genomic DNA, circulating DNA (mostly released into blood after tumor cell apoptosis) in white blood cells, as well as parasitic viral or microbial DNA. These DNA are important parameters in clinical testing or diagnosis, which are also valuable materials for medical research. There are three main issues with extracting DNA from blood samples:

The kit was developed for construction of high quality libraries for next generation sequencing (illumina and MGI Platforms). The kit needs double strand DNA fragments (blunt and/or sticky) as input DNA for NGS library preparation, and is compatible with DNA fragments generated from both enzymatic methods and physical methods (sonication, nebulization etc.). Library multiplexing is possible with different types of indexes.

The kit was optimized for next generation sequencing (NGS) library preparation with different types of samples. Most of DNA library preparation requires the ligation of sheared DNA fragments to library adaptors and the DNA library preparation is closely related to the quality of NGS data. With BioDynami’s unique DNA library preparation technologies, the fast and simple kit allows high quality NGS library preparation to be completed in 1.5 hours with only 10 minutes of hands-on time.

<img decoding="async" src="https://biodynami.com/wp-content/uploads/2020/07/NGS-DNA-Library-Prep-Kit-illumina-1.png" alt=""

Some genomic regions are very difficult to be covered evenly and usually result in very low coverage rate or gap in these regions.

Typical difficult regions are:
• with high GC contents
• have secondary structures: mainly due to repeat sequences
• the worst cases: have both high GC contents and repeated sequences.

Example: human TERT gene is one of the most difficult regions as shown above. NGS data showed that BioDynami kit has the best performance to cover the extremely difficult human TERT gene region.

Limited GC bias across whole genome

<img decoding="async" src="https://biodynami.com/wp-content/uploads/2020/07/BioDynami-NGS-DNA-Library-Prep-Kit-GC-bias.png" alt="

Three index types are available for the illumina platform kits:

Non-index (illumina Cat.# 30009): Libraries do not have index.

Index (illumina Cat.# 30021): A unique barcode sequence with 6 bases has been included in each of the index primers. RNA Sequencing library multiplexing is possible with up to 48 samples. Index information can be downloaded here.

Unique dual index (illumina Cat.# 30023): RNA Sequencing library multiplexing up to 96 samples is possible with the unique dual indexes. We have developed a 4-Base Difference Index System. The system can generate indexes with at least 4 bases different from others in the 8-base indexing region. the unique dual indexing primers identify sequencing errors such as index hopping, mis-assignment, and de-multiplexing errors. Index information can be downloaded here.

Indexes are available for the MGI platform kits (Cat.# 34021).

The kit was developed for construction of high quality libraries for next generation sequencing (illumina and MGI Platforms). The kit needs double strand DNA fragments (blunt and/or sticky) as input DNA for NGS library preparation, and is compatible with DNA fragments generated from both enzymatic methods and physical methods (sonication, nebulization etc.). Library multiplexing is possible with different types of indexes.