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Cat. No.: MPFLA-25 (for 25T)

Cat. No.: MPFLA-100 (for 100T)

Description

Mycoplasma Pneumoniae Fluorescence LAMP Assay Kit is a diagnostic kit that amplifies Mycoplasma pneumoniae (MP) DNA using Loop‑Mediated Isothermal Amplification (LAMP) and determines MP infection by monitoring fluorescence curves generated on a real‑time PCR instrument. The kit incorporates dU and UDG enzyme to effectively prevent carryover contamination from previous LAMP reactions, thereby reducing false‑positive results. It enables rapid, efficient, and highly sensitive detection of Mycoplasma pneumoniae.

Mycoplasma pneumoniae (MP) is a prokaryotic microorganism positioned between bacteria and viruses. It belongs to the class Mollicutes, order Mycoplasmatales, family Mycoplasmataceae, and genus Mycoplasma. Lacking a cell wall and exhibiting marked pleomorphism, MP is primarily transmitted via respiratory droplets and can cause pharyngitis, tracheobronchitis, and pneumonia. It is one of the major pathogens responsible for respiratory infections in both children and adults, accounting for approximately 10–40% of community‑acquired pneumonia (CAP) cases. Clinically and radiologically, MP pneumonia is often indistinguishable from pneumonia caused by other pathogens. Moreover, MP is intrinsically resistant to commonly used antibiotics for pneumonia or upper respiratory infections (e.g., penicillins and cephalosporins). Therefore, timely and accurate laboratory diagnosis is essential for effective management of MP pneumonia.

Conventional detection methods for MP include culture and serological assays. Culture is considered the gold standard for clinical diagnosis, as isolation of MP from sputum, nasal secretions, or throat swabs provides definitive evidence of infection. However, MP is fastidious, requires stringent culture conditions, grows slowly, and yields low positive rates, limiting its routine clinical use and making it unsuitable for early or emergency diagnosis. Serological assays diagnose infection by measuring changes in MP‑specific antibody titers and include enzyme‑linked immunosorbent assay (ELISA), cold agglutinin test (CAT), and indirect immunofluorescence test (IFT). However, IgM and IgG antibodies typically require about one week to develop, leading to potential false‑negative results during early infection. In contrast, conventional PCR, real‑time PCR, and isothermal nucleic acid amplification technologies are widely used due to their speed, sensitivity, and specificity. Compared with colloidal gold antigen tests, nucleic acid–based assays offer significantly higher sensitivity and are more suitable for early detection.

Isothermal amplification technology is an in‑vitro nucleic acid amplification method that operates at a constant temperature. By combining enzymes with specific activities and corresponding primers, rapid nucleic acid amplification can be achieved without thermal cycling. This kit employs loop‑mediated isothermal amplification (LAMP), which uses 4–6 primers targeting six distinct regions of the MP genome. DNA synthesis is initiated by a strand‑displacing DNA polymerase (Bst DNA Polymerase), forming a dumbbell‑shaped structure that subsequently undergoes continuous strand displacement to enter the exponential amplification phase. The final LAMP products consist of a mixture of DNA molecules with various stem‑loop structures and cauliflower‑like concatemers. LAMP reactions can be completed within 30–60 minutes under isothermal conditions (e.g., 60–65°C). Compared with conventional PCR, LAMP does not require template denaturation, thermal cycling, electrophoresis, or UV visualization, offering advantages such as simplicity, speed, high sensitivity, and strong specificity. This kit targets the most conserved region of the MP P1 gene, using specially designed primers together with fluorescent dyes and a qPCR instrument to monitor fluorescence signals in real time, enabling objective and accurate interpretation of results.

The kit offers high sensitivity and a short reaction time. Its sensitivity is 2–5 times higher than that of traditional PCR methods, with a detection limit of approximately 20–500 copies/μL based on quantified positive plasmid standards. The entire detection process can be completed in about 45 minutes.

Components

• LAMP Fluorescence Mix with UDG (2X)
• LAMP Primer Mix (10X)
• Bst DNA Polymerase
• Positive Control
• Ultrapure Water

Features

• This kit demonstrates excellent specificity. Six highly specific primers were designed based on conserved regions of Mycoplasma pneumoniae, ensuring that no amplification occurs with DNA from other Mycoplasma species or from other respiratory pathogens.
• The kit incorporates an effective anti‑contamination system. By using dU and a heat‑labile UDG enzyme, dU is incorporated into the amplification products, and a 5–15 minute incubation at 37°C prior to isothermal amplification efficiently eliminates carryover contamination from previous reactions. During the subsequent isothermal amplification step, the heat‑labile UDG enzyme becomes inactivated and therefore does not interfere with downstream amplification.
• A Positive Control is included in the kit to verify that the assay system is functioning properly. It is recommended to include the Positive Control in each run.

Storage 

Store at −20°C for up to one year. The LAMP Fluorescence Mix with UDG (2X) must be protected from light. Avoid repeated freeze–thaw cycles whenever possible.

Precautions

• Ensure all reagents are completely thawed before use. Mix gently and avoid generating bubbles during mixing.
• Validation studies show that up to 10 freeze–thaw cycles do not significantly affect product performance; however, repeated freeze–thawing should still be minimized, as it may lead to performance degradation.
• Isothermal amplification is an ultra‑sensitive detection method. Perform all assays in a standard PCR laboratory. The reaction setup area should be kept free from any potential sources of amplification product contamination.
• Although this kit incorporates UDG‑based anti‑contamination technology, do not open reaction tubes after amplification. Amplification products should remain sealed and be disposed of according to post‑amplification waste‑handling guidelines to prevent aerosol contamination from high‑concentration amplicons.
• This isothermal amplification system requires a high concentration of divalent ions. Samples must not contain high concentrations of EDTA or other metal‑ion chelators.
• Use filter tips when preparing the reaction system to minimize contamination and reduce the risk of false‑positive results.
• It is recommended to treat pipettes with DNA‑removal reagents before and after use to avoid potential false positives.
• This product is intended for scientific research use only by trained professionals. It is not for clinical diagnosis or treatment, not for food or drug applications, and should not be stored in residential environments.
• For your safety, wear a lab coat and disposable gloves during operation.

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

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SBS Genetech is a long-term sponsor of Cold Spring Harbor Laboratory