Instruction Manual
Research Use Only

Myco-Visible
Mycoplasma LAMP Detection Kit
Sensitive and Rapid Detection of Mycoplasma contamination incell cultures

Size: 40 preps
Storage: -20 °C
Cat. No.: 093050601
Content Version: August 2022

Table of Contents
1. Introduction to Myco-Visible Mycoplasma LAMP Detection Kit .......... 3
2. Kit Components and User Supplied Materials .............................. 4
3. Storage and Kit Stability ...................................................... 5
4. Important Consideration Before Use ........................................ 5
5. Protocol .......................................................................... 56. Flow Chart ....................................................................... 7
7. Data .............................................................................. 8
8. Troubleshooting ................................................................ 9
9. Product Use Limitation & Warranty ....................................... 10

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2Myco-Visible Mycoplasma LAMP
Detection Kit
1. Introduction to Myco-Visible Mycoplasma LAMP Detection Kit
Mycoplasmas are commonly found in research laboratories and unaffected by manyof the antibiotics that have been used to control bacterial contamination in cellcultures, especially those that affect cell wall formation, such as penicillin. The lack ofa cell wall also means that some cells may be able to pass through 0.2 μm filters. There
have been several studies to determine the prevalence of mycoplasma contaminationin cell cultures. Some have estimated that it could be as high as 80%, while studies in
the USA suggest a lower contamination rate of 10-15%. An average figure between 25%
and 50% worldwide seems quite plausible. This is a very significant problem for theindustry and could lead to serious economic losses. Most cases of cell culture
contamination are caused by a group of about six to eight species with human, porcine,or bovine hosts, of which Mycoplasma orale and Mycoplasma hyorrhinis are the mostcommon.
Myco-Visible Mycoplasma LAMP detection kit is highly sensitive, specific, and rapid assay
for the detection of mycoplasma contaminations in cell cultures. Myco-Visible
Mycoplasma LAMP detection kit achieves this by targeting and amplifying the 16S rRNA
coding region of the mycoplasma genome at loci that are well conserved in all
Mollicutes, including Acholeplasma laidlawii, Mycoplasma arginini, Mycoplasma
fermentans, Mycoplasma hominis, Mycoplasma hyorhinis, and Mycoplasma orale. The
detection procedure can be completed within 45 mins and requires as little as 10 16S
rRNA copies or 10 fg of Mycoplasma genomic DNA per reaction. No cross-reactivity withother bacterial, fungal and mammalian DNA by Primer-BLAST. The test assay is easy toperform with no thermal cycler required. Positive results can be visualized by color
changes from pink to yellow.

3

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2. Kit Components and User Supplied Materials
2.1 Myco-Visible Mycoplasma LAMP Detection Kit Component
Components

PackageCat. No.

Master Mix

1 vial (510 µL)

093050602

Primer Mix

1 vial (220 µL)

093050603

Nuclease-free water

1 vial (510 µL)

093050604

Positive Control

1 vial (50 µL)

093050605

Quick-start Protocol

1 ea-Instruction Manual

Available www.mpbio.com-MSDS & CoA

Available www.mpbio.com-2.2 User Supplied Materials•Microcentrifuge capable to work at speed of ≥14,000 x g•

Heat block or dry bath at 65 °C and 95 °C•

Nuclease-free 0.5 mL and 1.5 mL sterile microcentrifuge tubes•Sterile PBS•Pipettes with corresponding filter tips

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4Myco-Visible Mycoplasma LAMP
Detection Kit
3. Storage and Kit Stability
All Myco-Visible Mycoplasma LAMP detection kit components are guaranteed for at least
12 months from the date of manufacture when stored appropriately.
Upon receipt, store at -20 °C.

4. Important Consideration Before Use❑ Thaw the kit at 4 °C or room temperature. Do not leave it at room temperature formore than 1 hour.
❑ Use clean, disposable gloves when performing the assay and make sure that the work
area is cleaned with 70 % alcohol or 10 % household bleach before the assay setup.
❑ Incubation of LAMP reaction mix (at 65 °C) should be performed in a separate locationfrom sample preparation and LAMP reaction setup.
❑ Perform the experiment using sterile, DNases, and RNases-free filter pipette tips ishighly advisable.
❑ Do not open the tubes containing amplified LAMP product to prevent carryover
contamination (air contamination of previous PCR product). Samples and amplified
products are biohazards that must be sterilized before discarding.

5

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5. Protocol
5.1 Sample Preparation
1. Confluency of test cell culture should be >80 %. Detach the cells using scraper (avoidtrypsin and EDTA).
2. Count cell numbers using standard counting methods. Transfer 1 mL of cell culture
samples with a minimum of 5x104 cells to a 1.5 mL microcentrifuge tube.
3. Centrifuge at 14,000 x g for 2 mins and carefully decant the supernatant.
4. Resuspend cell pellets in 100 µL of sterile PBS.
5. Incubate the sample at 95 °C for 5 mins.
6. Centrifuge at 14,000 x g for 2 mins and transfer the supernatant to a new 0.5 mL tube.
This supernatant will be used as template for LAMP reaction in Section 5.2.

5.2 LAMP Protocol
1. Setup LAMP-PCR reactions (including 1X positive control (PC) and negative control (NC))
with 0.5 mL tubes and incubate at 65°C for 30 mins (incubation should be performed ina separate location from assay setup)
LAMP reaction (25 µL)

Volume (µL)

Master Mix

12.5

Primer Mix

5.0

Template*

1.0 – 5.0

Nuclease-free waterTop up to 25* For PC and NC, use 1 uL of positive control and nuclease-free water respectively(supplied in the kit) as template.
2. Result color interpretation: Negative results are indicated in pink and positive results
are indicated by a change to yellow.

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6Myco-Visible Mycoplasma LAMPDetection Kit
6. Flow Chart

7

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7. Data
Myco-Visible Mycoplasma LAMP detection kit demonstrates high sensitivity andspecificity on detection of Mycoplasma spp. contamination in cell cultures. Analyticalsensitivity tests were carried out using the known-copy number genomic DNA from 6Mycoplasma spp. commonly found in the laboratory cell cultures.
NC101

102103A. laidlawii

10NC

10NC

101102103M. hyorhinis

NC101102103M. arginini

1102103M. orale

NCM. fermentans

NC

1011021031102103M. pneumoniae

Figure 1: Analytical sensitivity test of Myco-Visible Mycoplasma LAMP detect Kit. 10-fold serial dilution from 10 to 103
copies/µl have been performed using six strains of mycoplasma spp. Results shown that the kit is ablet to detect as low as
10 16S rRNA copies or 10 fg of Mycoplasma genomic DNA.

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8Myco-Visible Mycoplasma LAMP
Detection Kit
8. Troubleshooting
ProblemPossible Cause

Recommendation

NC color changes
intermittently

Long reaction time

Check the color again when tubes equilibrated
to room temperature.

Trace contamination of thereagent•

Do not exceed incubation time (65 °C @ 30mins) as this significantly impacts thereaction•

PC color does notchange to yellow

Degradation of reagent

Faint positive
result for samples

Weak Mycoplasma infection ofthe sample

Discard current reagent and use new one.
All procedures should be performed in a
dedicated environment that is free ofcontamination. Use filter tips will reducethe contamination.
Avoid frequent freeze-thaw cycles of the LAMP
reagent•••

9Increase the volume of template (up to 5µL)
Grow the cell culture for additional 48
hours and repeat the test
Use first-time LAMP product as template torepeat the test.

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9. Product Use Limitation & Warranty
The products presented in this instruction manual are for research or manufacturing use
only. They are not to be used as drugs or medical devices in order to diagnose, cure,mitigate, treat or prevent diseases in humans or animals, either as part of an accepted
course of therapy or in experimental clinical investigation. These products are not to be
used as food, food additives or general household items. Purchase of MP Biomedicals
products does not grant rights to reproduce, modify, or repackage the products or any
derivative thereof to third parties. MP Biomedicals makes no warranty of any kind,
expressed or implied, including merchantability or fitness for any particular purpose, exceptthat the products sold will meet our specifications at the time of delivery.

Buyer’s exclusive remedy and the sole liability of MP Biomedicals hereunder shall be limited
to, at our discretion, no replacement or compensation, product credits, refund of thepurchase price of, or the replacement of materials that do not meet our specification. By
acceptance of the product, Buyer indemnifies and holds MP Biomedicals harmless against,and assumes all liability for, the consequence of its use or misuse by the Buyer, its
employees, or others, including, but not limited to, the cost of handling. Said refund orreplacement is conditioned on Buyer notifying within thirty (30) days of receipt of product.
Failure of Buyer to give said notice within thirty (30) days shall constitute a waiver by theBuyer of all claims hereunder with respect to said material(s).

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