|Kit content||25 Preps||100 Preps|
|Pro MTB Mix||250µl||1000µl|
|MTB Positive Control||40µl||150µl|
Viga MTB molecular diagnostic kit is an in vitro nucleic acid amplification test for the detection of all members of the M. tuberculosis complex (M. tuberculosis, M. africanum, M. bovis, M. bovis BCG, M. microti, M. pinnipedii) in human sputum, BAL, bronchial secretion, CSF, stomach fluid or peritoneal punction samples. This diagnostic test kit utilizes the polymerase chain reaction (PCR) and is configured with Real Time-PCR instruments.
- Highly sensitive detection of as few as 25 Copies/ml.
- Non-competitive internal control to monitor extraction and PCR efficiency.
- Specificity, no cross reaction with other respiratory pathogens
Sample Storage and Preparation
Human sputum, BAL, bronchial secretion, CSF, stomach fluid, or peritoneal punction samples
Fresh specimen must either be processed immediately as per sample procedure outlined in the section on Sample processing protocol or stored frozen at -20°C. Frozen samples must be brought to room temperature before starting sample processing. Sample Pre-treatment decontaminates the specimen and makes it ready for extraction.
Take out each component from the kit and place them on the bench top. Allow the reagents to equilibrate to room temperature, then briefly vortex each tube for later use.
For Mycobacterium tuberculosis nucleic acid isolation use, DNall Plus kit (DN983049 or DN983048), DNall Pro (DN983064 or DN983063) or other kits with confirmation of ministry health.
Take out each component from the kit and place them on the bench top. Allow the reagents to equilibrate to room temperature, then briefly vortex each tube for later use. The volume of isolated sample in this test should be 5μl. Prepare PCR reaction refer to Table 1 and then perform Realtime PCR refer to Table 2.
Table 1: PCR reaction preparation
|Pro MTB Mix||10µl|
Table 2: Thermal profile for PCR reaction
|Stage||Temperature||Incubation Time||Cycle Numbers|
|Pre-Denaturation||95°C||3 min||1 |
|Denaturation||95°C||10 sec|| |
|Annealing and acquisition on channel Green and Yellow||60°C||40 sec|
Interpretation of results
- Data analysis for each gene should be performed separately by using a manual threshold.
- The threshold for each sample should be in the exponential phase of the fluorescence curves and above any background signal.
- FAM Fluorophore (green) for the IS6110 gene of M. tuberculosis and HEX Fluorophore (Yellow) is for the IC gene (internal control).
- A negative control is used as contamination control. The magnitude increase of the Fluorescence curve in the negative control does not cross the threshold. If Ct is less than 35 (Ct<35), it is considered as possible contamination. Strong signals above 35 in the NTC can be PCR artifacts, which in these cases, the shape of the curve can be considered (the S-shaped curve is typical for a positive result).
- Internal control should be positive for all clinical specimens at Ct 35 or less than 35, indicating sufficient nucleic acid from the human gene and the sample has acceptable quality.
- Internal control curve Ct>33 or without Ct indicates low sample concentration or inhibitors in the reaction (the isolated sample is recommended to dilute at least ½). If the test result is not acceptable again during the retest, another new sample should be taken from the patient, and the test must be repeated.
- A positive clinical specimen should have Ct≤40 for the target gene (IS6110).
- If the expected positive reaction is not achieved (typical S-shaped curve), the performed test is not acceptable, and the test must be repeated by following the kit instructions accurately.
- Determine the reason for the failure of positive control, take the corrective action, and document corrective action results.
The acceptable situation for positive and negative control
Table 3: Control conditions for a valid PCR Run
|Results||IC (HEX)||MTB (FAM)|
|Positive control*||Not considered||Ct≤40 (+)|
|Negative control||Ct>35 (+)||–|
|Invalid and not accepted||–||–|
- A false-negative result is obtained in case of low titration of MTB in the patient sample, improper transportation, and lack of quality of sample isolation.
- All controls must be checked before the interpretation of results. If the controls are not valid, the patient’s results cannot be interpreted. The diagnostic limit of this kit equals Ct≤40, and also, the user must review the fluorescence curve before final interpretation. All positive curves must have an amplification peak.
- Non-observance of proper storage conditions of the kit can lead to false-negative results.
- Handling this kit needs experienced and trained personnel. Any personnel error may lead to invalid results.
- The results of this diagnostic kit are only acceptable if they are along with clinical evidence for diagnosing MTB. Definitive diagnosis and treatment of patients must be based on a combination of this test with other tests results, medical records, and how to respond to treatment.