A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)

We describe a tuberculosis molecular bacterial load assay test performed after heat inactivation of sputum. Heat inactivation renders sputum samples noninfectious and obviates the need for containment level 3 laboratories for tuberculosis molecular tests.

Transcript

This Tuberculosis Molecular Bacterial Assay, answers three key questions. One:What is the magnitude of the patient's disease burden? Two:How does the patient disease burden respond to the anti-tuberculosis medication?

And three:What is the relationship between disease burden and treatment response? This assay produces a quantitative result of the patient tuberculosis burden, and measures how this burden changes with treatment in a short time. With modification, this methodology can be applied to other bacterial pathogens.

We have already developed similar technology for diagnosis of nontuberculous mycobacteria, and bacteria associated with chronic obstructive pulmonary disease. When performing this technique for the first time, watch the video and practice the steps. It's very important to practice with samples that are not needed for patient diagnostic results.

Visual demonstration is critical because it simplifies learning and mastering of steps, especially those parts of the method that are difficult to explain in text. Start by preparing the cell cultures. On a clean lab bench or class one cabin, harvest one milliliter aliquots of exponential phase Bacille Calmette-Guerin or BCG culture into 15 milliliter plastic tubes and tightly close them.

When preparing patient's sputum samples, work in a well ventilated space and follow the guidelines in GL One Stop TB handbook. Carefully open the specimen cup and pipette one milliliter aliquots into 15 milliliter plastic centrifuge tubes. Then, tightly close the tubes.

Transfer the sample tubes to a holding rack immersed in a water bath that is preheated to 95 degrees Celsius, making sure that 3/4 of each tube is immersed. Boil the samples for 20 minutes. Then, transfer the tubes to the bench to cool at room temperature for five minutes.

Perform RNA extraction in a fume hood if using a kit with phenol chloroform or tumor capital ethanol. The RNA procedure illustrated here is the phenol chloroform extraction procedure. However, alternative methods for RNA extraction may also be used.

Transfer one milliliter aliquots of the heat inactivated sample to 1.5 milliliter tubes and spike 100 microliters of extraction control into each sample. Close the tubes and mix them by inverting three times. Centrifuge the tubes at 20, 000 times g for 10 minutes.

Then, aspirate the supernatant, leaving 50 microliters of sediment. We suspend the sediment in 950 microliters of lysis buffer by pipetting and transfer the whole suspension into the lysing matrix tubes supplied with the RNA extraction kit. Tightly close the tubes and ensure that they are labeled on both the lid and the side.

Then, homogenize the samples for 40 seconds at 6, 000 rpm. Centrifuge the lysate at 12, 000 times g for five minutes at room temperature. Meanwhile, prepare fresh one milliliter tubes, and add 300 microliters of chloroform.

Use a one milliliter pipette to carefully aspirate the supernatant without touching the lysing matrix and transfer it to the tube with the chloroform. Vortex the tubes for five seconds and leave them to settle for five minutes or until three phases are clearly visible. Centrifuge the tubes at 12, 000 times g for five minutes.

Then, carefully pipette the upper phase and transfer it to a 1.5 milliliter tube. Add 500 microliters of ice cold 100%ethanol to the sample, close the tube, and invert it three times to mix. Incubate the tubes at minus 80 degrees Celsius for 15 minutes or at minus 20 degrees Celsius for 30 minutes.

Then, load the tubes into a pre-chilled micro centrifuge and centrifuge for 20 minutes at 13, 000 times g and four degrees Celsius. Discard the supernatant, add 500 microliters of 70%ice cold ethanol and centrifuge for another 10 minutes at 13, 000 times g. Discard all the supernatant.

And incubate the tubes at 50 degrees Celsius for 20 minutes to dry the nucleic acid pellet, making sure to keep the tubes partially open to enable evaporation of ethanol. Next, add 100 microliters of nucleus free water to the pellet and incubate at room temperature for five minutes. Vortex the sample for three seconds and proceed with the DNA removal or store it at minus 80 degrees Celsius until ready to use.

Prepared DNA's one mix according to the manuscript directions and pipette 11 microliters into each tube containing RNA extract. Vortex for three seconds and spin down briefly to remove any droplets from the wall of the tube. Then, incubate the tube at 37 degrees Celsius for 30 minutes in the hot block or incubator.

After the incubation, add an additional one microliter of DNA's one enzyme directly to the tube, and mix well by vortexing. Then, incubate the tubes for another 30 minutes at 37 degrees Celsius. Thaw and vortex the DNA's inactivation reagent.

Then, add 10 microliters to each RNA extract. And incubate the tubes at room temperature for five minutes. Vortex the tubes three times during the incubation step.

Then, centrifuge the mixture at 13, 000 times g for two minutes and carefully transfer the supernatant to a 1.5 milliliter RNA's free tube, making sure to not touch any of the inactivation matrix. Dilute all unknown RNA samples one to 10 in RNA's free water then, mix them by vortexing for five seconds and briefly spinning them down. Thaw MTB and EC-RNA samples and to make seven and six tenfold dilutions respectively for a standard curve.

Prepare RT plus and RT minus PCR master mixes according to the manuscript directions. Vortex the RT plus mix and transfer 16 microliters into each RT plus PCR tube. Then, vortex the RT minus mix and add 16 microliters into each RT minus PCR tube.

Add four microliters of RNA extract or water and duplicate to the RT plus tubes. The water is the non-template control or NTC. Add four microliters of RNA extract or water to the RT minus tubes.

Load the reaction tubes into the real time PCR machine, program the reaction as described in the manuscript and run the reaction. To interpret the treatment response, use the standard curve to convert CQ values into bacterial load and calculate the response as the change in bacterial load over the treatment follow up period. The fall in bacterial load following treatment signifies a positive response while no change or rise in bacterial load implies a negative response.

To verify that all M.tuberculosis bacilli were heat inactivated, optical density of the cells was measured and compared to that of live cells. No change in OD over time was observed for the heat inactivated samples indicating no growth. Heat inactivated samples were incubated at 37 degrees Celsius to determine whether RNA degrades following heat inactivation of cells.

No difference was found between the RNA harvested immediately after heat inactivation, and the RNA isolated one, two, three, and four days later in both BCG cultures and TB positive sputum. When RNA's A enzyme was exogenously added to the samples, before and after heat inactivation, RNA loss occurred in all heat inactivated samples across four days of incubation. The effect of heat inactivation on ribosomal RNA was measured in BCG cultures and sputum from TB positive patients.

The measured bacterial load of control culture was higher than the combined bacterial load of heat inactivated culture at 80 degrees Celsius, 85 degrees Celsius, and 95 degrees Celsius for both BCG and sputum samples. Heat inactivation of the samples causes minimal loss of RNA, leaving adequate RNA for downstream TB-MBLA and other downstream molecular tests. Transmission Electron Microscopy was used to investigate whether cells lysed by heat inactivation.

Inspection of the cells at lower and higher magnification revealed the intact cell walls and the visible intracellular lipid bodies. The cells appeared elongated, but not lysed. When attempting this procedure, it's critical to remember to add the extraction control, which controls for any false negative results due to poor RNA extraction.

This approach can be applied to bacteria associated with chronic obstructive pulmonary disease, nontuberculous mycobacteria infection, and other respiratory pathogens. The quantitative results from TB-MBLA have enabled mathematical and pharmaco-modeling of how patients respond to an tuberculosis therapy. We look forward to applying the technique in routine care where tuberculosis patients are being managed.

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Journal (Immunology and Infection)

Journal (Immunology and Infection)

A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)

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