These tests were carried out on sputum samples for which the Xpert MTB/RIF test had detected rifampicin resistance and on some culture samples. These tests were performed in three steps in dedicated facilities, i.e. DNA extraction, resistance gene amplification and hybridization. DNA was extracted using the alkaline lysis method in the GenoLyse kit provided with the reagent. PCR amplification was run in a GTQ-Cycler 96 (Hain Lifescience) thermocycler and hybridization was carried out in a TwinCubator (Hain Lifescience) with nitrocellulose DNA strips on which the complementary probes of the main wild-type (non-mutant) and mutant sequences involved in MDR-TB have been bound.
Twincubator
Manufactured by Hain Lifescience
Sourced in Germany
The TwinCubator is a dual-chamber incubator designed for cell culture applications. It maintains a controlled temperature and environment for the simultaneous cultivation of two separate cell samples or experiments.
Automatically generated - may contain errors
5 protocols using twincubator
1
Multiplex PCR for MDR-TB Detection
2
Comprehensive Microbial Identification and HPV Typing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
An ISOLATE II DNA Mini kit with a silica membrane was used to isolate the DNA (Bioline, Meridian Bioscience, Memphis, TN, USA). The isolation protocol was according to the manufacturer’s instructions.
The pathogenic bacteria associated with periodontitis were detected by micro-IDent®plus11 kit (Hain Lifescience GmbH, Nehren, Germany). Reactions were performed according to the manufacturer’s instructions using a FluoroCycler® 12 PCR apparatus (Hain Lifescience GmbH, Nehren, Germany), and a TwinCubator (Hain Lifescience GmbH, Nehren, Germany) was used to perform the hybridization.
An Amplisens kit (Ecoli Dx, s.r.o., Prague, Czech Republic) was used to detect HPV types 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, and 66. The protocols were according to the manufacturer’s instructions.
The pathogenic bacteria associated with periodontitis were detected by micro-IDent®plus11 kit (Hain Lifescience GmbH, Nehren, Germany). Reactions were performed according to the manufacturer’s instructions using a FluoroCycler® 12 PCR apparatus (Hain Lifescience GmbH, Nehren, Germany), and a TwinCubator (Hain Lifescience GmbH, Nehren, Germany) was used to perform the hybridization.
An Amplisens kit (Ecoli Dx, s.r.o., Prague, Czech Republic) was used to detect HPV types 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, and 66. The protocols were according to the manufacturer’s instructions.
3
Molecular Detection of TB Drug Resistance
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The identification of genotypic drug resistance to RIF (based on mutations the rpoB gene) and INH (based on mutations in katG and inhA genes) was carried out using the manufacturer’s instruction27 in the TB Laboratory at ALIPB-AAU. Accordingly, 50 µL of PCR mixture consisting of 10 µL AM-A GT MTBDRplus ver 2.0, 35 µL AM-B GT MTBDRplus ver 2.0 and 5µL DNA template from an isolate was used to perform this assay. M. tuberculosis H37Rv DNA template was used as a positive control. Water (Qiagen NV, Venlo, the Netherlands, product) served as a negative control. A thermal cycler (VWR, Leicestershire, UK) was programmed as follows: 15 min for enzyme activation at 95°C followed by 10 cycles of 30 sec denaturation at 95°C, 2 min annealing at 58°C, 20 cycles of 25 sec denaturation at 95°C, 40 sec annealing at 53°C, 40 sec elongation at 70°C, and finally elongation at 70°C for 8 min. Biotin-labeled amplicons were hybridized to DNA probes attached to a DNA strip®. Hybridization was done using the TwinCubator (Hain Lifescience, Nehren, Germany). Results were interpreted based on the presence and absence of wild-type (WT) and mutation (MUT) probes.
4
GenoType MTBDR plus Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Heat-killed bacterial colonies grown on LJ media were used for the GenoType MTBDRplus assay (Hain Lifescience, Nehren, GmbH, Germany). The bacterial colonies were collected with an inoculation loop, suspended in 100 μL of lysis buffer (A-LYS) and heated at 95°C for 5 minutes to inactivate the vegetative bacilli. After a brief spinning down, an additional 100 μL of neutralization (A-NB) buffer was added. Following 5 minutes of spinning the supernatant, DNA was used for PCR amplification. The master mix was prepared according to the manufacturer's instructions. The amplification profiles included 15 min of denaturation at 95°C, followed by 1 cycle of 30 sec at 95°C and 2 min at 65°C, followed by an additional 10 cycles of 25 sec at 95°C, 40 sec at 50°C, and 40 sec at 70°C, for a total of 20 cycles, and then an 8-minute extension at 70°C in 1 cycle. A TwinCubator (Hain Lifescience, Nehren, GmbH, Germany) was used for hybridization and detection [20 ].
5
Mycobacterial Species Identification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PCR was carried out in the GTQ Cycler 96 using the protocol in the user guide (Hain Lifescience, Nehren, Germany). Speciation was carried out using GenoType®CM/AS (Hain Lifescience, Nehren, Germany) protocol on TwinCubator (Hain Lifescience, Nehren, Germany). Species-specific probes are mounted to the DNA strips to determine complementary strands from amplified DNA samples [28 (link), 29 (link)]. Table 1
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!