Line Probe Assay (LPA) is a molecular assay to identify the mutations in the causative agents in different clinical samples. LPA is a rapid diagnostic procedure, and this procedure has an important role in the diagnosis of different diseases. LPA plays an important role in the field of Tuberculosis. Tuberculosis (TB) is an infectious disease caused by bacteria known as Mycobacterium tuberculosis (MTB). TB is associated with different types of symptoms. Different diagnosis tools are used for the diagnosis of TB. In recent days, a new type of TB known as Drug Resistant TB (DR-TB) emerges which needs timely diagnosis for timely treatment. For timely diagnosis of DR-TB LPA also play an important role. This assay plays a specific and important role in the diagnosis of DR-TB in drug-resistant TB patients. This is a strip-based diagnostic procedure and is used in TB labs in the identification of resistance patterns among DR-TB patients which cause due by mutations in the causative agents. Along the resistance pattern, this assay also helps in the presence of MTB. The test is carried out following the special instructions explained by the manufacturers.
Read more: Symptoms of Tuberculosis
The Process of Line Probe Assay (LPA)
The process of LPA consists of three main steps. These steps are;
- DNA Extraction.
- PCR (Multiplex) Amplification.
- Reverse Hybridization Methods.
DNA Extraction
- For DNA extraction, a GenoLyse® kit is used. This kit is also known as a DNA extraction kit. This is the first step and during this DNA from culture or decontaminated pellets is extracted.
- For this purpose, 1 mL of culture or decontaminated sample is transferred to a sterile screw cap microcentrifuge tube (1.5mL) and centrifuged at ten thousand revolutions per minute (RPM) for 15 minutes.
- The pellet is then suspended in 100 µl of Geno Lysis (A-LYS) using a vortex mixer after the supernatant is removed.
- Each tube is incubated for 5 minutes at 95 °C in a thermocycler before receiving 100 of Neutralization Buffer (A-NB).
- After vertexing, the samples are centrifuged for 5 minutes at 15,000 rpm.
- Due to centrifugation, all cell debris is deposited at the bottom of the tube, and approximately 50-100 µl of DNA supernatant is then transferred to another new tube (1.5 ml tube), and these samples are now stored for further amplification process.
Polymerase Chain Reaction (PCR):
- In this stage, master mix/PCR reagents are used. This is for amplification purposes.
- Primers are present in the amplification reagents named AM-A and AM-B.
- Reagents are placed in line according to the serial number of samples.
- About 10µl AM -A and 35µl AM -B are taken and mixed up thoroughly by pipetting up and down in the free area.
- A final concentration of 45µl of the master mix is used for a single sample.
- After that 5µl of the extracted DNA sample is transferred to prepared PCR tubes containing a 45 µl master mix.
- The denaturation process is completed in different phases. Initial denaturation at 94°C for 5 minutes is followed by 40 cycles of denaturation at 94°C for 1 minute, annealing at 65°C for 1.5 minutes, and in the final phase, its extension takes place at 72°C for 10 minutes in a thermocycler.
- The amplified product is stored at temperatures ranging from +8 to -20 degrees Celsius.
Hybridization:
- Hain Life Sciences procedures are used for this process.
- In this step, TwinCubator is used.
- Initially, 20µl denaturation (DEN) solution and 20µl amplified product are mixed.
- A pipette is used to mix them in the corner of each well of the tray, after which this product is incubated at room temperature for 5 min.
- Then 1 ml of pre-warmed hybridization solution (HYB) is added to each well and stirred to homogenize the mixture. The DNA strip is labeled and laid carefully in each well.
- The tray was set on the TwinCubator and then incubated at a forty-five-degree centigrade temperature for thirty minutes.
- These HYB solutions are aspirated completely and then 1 ml of stringent solution (STR solution) is added to each well and incubated for 15 min at 45 °C in the TwinCubator.
- At the next stage, the stringent solution is thoroughly pipetted out with an individual sterile pasture pipette. Moreover, the strips are rinsed by adding 1 ml solution to each well and incubated for 1 min at 25 °C on the TwinCubator.
- Then, here now, after this step, the incubation period is completed, and the solution is carefully pipetted out.
- Then 1 ml of diluted conjugate (10 ul CON-C and 990 ul CON-D) is enhanced into each well, and here again, these wells incubate for 30 min at 25 °C on the TwinCubator.
- The conjugate is then removed completely and each strip is washed twice for 1 min with 1ml of rinse solution (RIN) and finally washed using 1ml of distilled water.
- Finally, diluted substrate (10 ul SUB-C and 990 ul SUB-D) is added and incubated for 2–5 minutes.
- The substrate is then washed two times with distal water as soon as bands are visible.
- The DNA strips are taken from the tray through tweezers, and at the end, the entire DNA strip is separated from the tray and wiped out on absorbent paper.
Read more: Contacts Screening of Multidrug-Resistant Tuberculosis Patients
Line Probe Assay (LPA)
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