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Cat. No.: MPDK-250 (for 250T)

Description

Mycoplasma PCR Detection Kit is a reagent kit that utilizes Nested PCR to detect the presence of Mycoplasma contamination in cell cultures and other biological materials.

Mycoplasma are the smallest and simplest prokaryotes, characterized by the absence of a cell wall. Consequently, common antibiotics targeting cell walls, such as penicillin or β-lactam antibiotics, are ineffective against Mycoplasma. With a size between that of bacteria and viruses (approximately 0.2-0.8 μm), some Mycoplasma can pass through a 0.22 μm filter, rendering standard filtration ineffective. Due to their limited biosynthetic capabilities, many Mycoplasma rely on host cells for nutrition, often adhering to or spreading between cell surfaces. These traits pose a risk of Mycoplasma contamination during cell culture, a widespread global issue.

Mycoplasma contamination can severely impact cell status, leading to altered gene expression and metabolic characteristics, resulting in abnormal growth, differentiation, and cell death, thereby compromising cell function. These effects significantly undermine experimental results' reliability, reproducibility, and consistency, underscoring the vital importance of Mycoplasma contamination detection.

While bacterial, yeast, or fungal contaminations in cell cultures are visible under an optical microscope, Mycoplasma contamination is usually invisible and requires specific detection methods. Common methods for detecting Mycoplasma contamination include Mycoplasma isolation culture, ELISA, luminescence assays, biochemical assays, DNA fluorescent staining, among others. Many of these methods can be cumbersome, lack sensitivity, cannot differentiate Mycoplasma species, require specialized equipment, or have extended processing times. In contrast, the PCR method is relatively simple and convenient, whereby detection of Mycoplasma contamination is achieved through gel electrophoresis analysis of PCR amplification products. The size of the amplified fragment can also provide an approximate prediction of the Mycoplasma species involved. If necessary, conventional sequencing of the PCR product can identify the specific Mycoplasma species.

The Mycoplasma PCR Detection Kit utilizes two pairs of primers to specifically amplify Mycoplasma genomic DNA fragments through Nested PCR, achieving high sensitivity and specificity in detection. The rRNA base sequences of prokaryotes are highly conserved, while the DNA intergenic spacer (space region) encoding rRNA on the rRNA operon differs significantly between various species, such as the intergenic spacer between 16S and 23S rRNA. This spacer region's DNA sequence and length exhibit both highly conserved and significantly divergent sections among different Mycoplasma species. For the first round of Nested PCR (1st PCR), a pair of primers (F1/R1) is designed on the conserved region of the DNA encoding 16S and 23S rRNA to amplify the intergenic spacer. This step is used for the initial identification of Mycoplasma contamination. In the second round of Nested PCR (2nd PCR), an additional F2 primer is designed on the conserved region of the DNA encoding 16S and 23S rRNA, along with an R2 primer on the DNA encoding 23S rRNA. This design enables the amplification of the conservative region of the DNA intergenic spacer between 16S and 23S rRNA. Nested PCR greatly enhances detection specificity and sensitivity.

Components

250T

• 1st PCR Primer Mix (50X)    100μl
• 2nd PCR Primer Mix (50X)    100μl
• Control template    100μl

Features

• This assay kit enables rapid, effective, and high-sensitivity detection of Mycoplasma contamination.
• The kit provides a positive control template, facilitating the verification of proper functionality of the PCR detection and the identification of any substances within the samples that might inhibit PCR reactions.
  
• If used in a 20 μl PCR reaction system, this assay kit can perform 250 tests in total. If used in a 50 μl PCR reaction system, this assay kit can perform 100 tests in total.

Storage

Store at -20°C.

Precaution

• Due to the extreme sensitivity of PCR amplification, it can increase the target gene sequence over 10 million times. When using Taq polymerase, be cautious to avoid contamination of trace amounts of DNA to be amplified. It is advisable to consider setting up negative controls without template DNA to confirm the absence of DNA contamination. It is preferable to conduct these procedures in a standard PCR laboratory. In cases where a standard PCR laboratory is unavailable, strict segregation of a dedicated pre-PCR area and a post-PCR area is recommended. These two areas should be in separate rooms and ideally be well-separated to prevent cross-contamination. All reagents, instruments, pipettors, lab notebooks, etc., used in the post-PCR area should not be brought into the pre-PCR area.
• Using filter-tipped pipette tips for setting up the PCR system is recommended to minimize contamination-induced false positives.
• Preferably, use a dedicated pipetting device for preparing the PCR system. Pipettes previously used in cell culture might carry Mycoplasma contamination.
• During experimental procedures, minimize speaking, wear disposable masks, and prevent saliva contamination to avoid Mycoplasma contamination.
• Utilizing a PCR system containing Heat-Labile Uracil DNA Glycosylase (HL-UDG) can effectively minimize contamination of amplification products, reducing the probability of false positives.
• In general, the 1st PCR can provide preliminary identification of Mycoplasma contamination. However, performing the 2nd PCR is recommended for further confirmation.
• This kit cannot detect Human Mycoplasma pneumoniae.
• This product is intended for scientific research by professionals only. It must not be used for clinical diagnosis or treatment, food or drug purposes, and should not be stored in residential settings.
• For your safety and health, please wear lab coats and disposable gloves during operation.

Related:  Mycoplasma qPCR Detection Kit

Only for research and not intended for treatment of humans or animals

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