Polymerase Chain Reaction (PCR) Assay for Mycobacterium Tuberculosis
Tuberculosis is a worldwide, chronic and communicable disease caused by the "Koch bacillus" Mycobacterium tuberculosis, which usually affects the lungs, but may cause lesions in any organ or tissue of the human body. The Human Immunodeficiency Virus (HIV) epidemic is having a profound impact on the Tuberculosis problem in both industrialized and developing countries. India has one third of the world's TB patients. Sadly, one of the main contributors to the above situation is the fact that this disease ever so often remains undiagnosed and worse, untreated.
Conventional Diagnostic Tools
TB is conventionally diagnosed based on X-ray findings, elevated Erythrocyte Sedimentation Rate (ESR), Mantoux test and on various clinical symptoms like weight loss, low-grade fever and persistent cough.
Advanced Diagnostic Tools
Mycobacterium tuberculosis is diagnosed using the Ziehl-Neelsen smear and culture on Lowenstein-Jensen (LJ) medium. Since M. tuberculosis takes six to eight weeks (sometimes even 12 weeks) to grow on Lowenstein-Jensen (LJ) medium, the patients often start receiving anti-tuberculosis treatment (ATT) on the basis of (a) presumptive clinical diagnosis (b) an acid fast bacilli (AFB) positive smear. Identification of acid-fast bacilli in specimens by smear examination has been shown to be insensitive (10,000 bacilli/ml of sample are required for a ZN smear examination to reveal a positive result) and nonspecific (MOTT species can be picked up), potentially leading to inappropriate patient management. Thus, there is a need for a TB diagnostic test of increased sensitivity, specificity and rapidity. Rapid culture methods have a sensitivity of 60-70% only, especially in patients on antibiotics, which is often the case in India. In this context, detection of Mycobacterial DNA in clinical samples appears to hold promise in early diagnosis of tuberculosis. DNA amplification by Polymerase Chain Reaction (PCR) is a technique, which fulfills the above requisites, with a sensitivity and specificity of 97-100%, with it being possible to obtain results within 24 hours. Currently, many different sets of primers have been used to amplify different target regions of the M. tuberculosis genome, of which the most commonly used primers are Pt-8, and Pt-9 based on the insertion sequence 6110. (Eisenach et al., 1991)
Problems with already existing PCR Assays
Currently, the PCR assays used in India are based upon protocols and kits developed in Europe and USA. The PCR assays innovated in the developed countries are based on the genetic make-up of strains of Mycobacterial species circulating in those regions. For example IS 6110 sequence is often used as a target sequence for amplification in PCR for tuberculosis protocols developed in western countries. There have been recent reports that the Mycobacterial isolates from some geographical areas like the Indian subcontinent contain less copies of insertion sequence - IS 6110 (many PCR assays are based on this gene sequence) as compared with the 8 to 15 copies usually found in strains from most developed countries (Das et al., 1995).
As the number of copies of the target gene sequence is an important determinant of PCR sensitivity, it would be lower for strains such as those circulating in India having only a few copies of IS 6110 (Rush et al., 1996).
It is well known that India accounts for a large proportion of tuberculosis cases. Thus, it has become necessary to evaluate PCR protocols based on other genes of M.tuberculosis in developing countries like India and others in sub continent. In this context, Auroprobe Laboratories has introduced a rapid, specific and sensitive PCR assay based on the clinical isolates of Mycobacterium tuberculosis of Indian origin- AURO-TB-PCR-1.
AURO-TB-PCR-1
The basic principle behind the AURO-TB-PCR-1 is a rapid, highly specific Polymerase Chain Reaction (PCR). This assay amplifies a portion of DNA which codes for a specific protein, MPB64 (Yamaguchi et al., 1989) and is present only in the members of M. tuberculosis complex i.e. M. tuberculosis, M. bovis and some strains of BCG of Indian subcontinent.
Auro-TB-PCR-1 besides being highly specific is so rapid that the result can be known within one working day and it is highly sensitive i.e. the test can detect as low as 10 copy numbers. It’s most important advantage over other PCR assays for Tuberculosis currently available in India, is that it is especially standardized for the Indian strains of Mycobacterium tuberculosis.
Information For Collection of Samples
| Test Name |
PCR for Mycobacterium tuberculosis |
| Test Code |
AURO-TB-PCR-1 |
Specimen |
Collection |
Minimum Volume* |
Storage |
Transportation |
Fresh tissue biopsies (including endometrial biopsies and menstrual blood) |
In sterile normal saline |
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Refrigerator
(40C) |
On ice or in Gel Pack Box*** |
Paraffin Embedded Tissue |
In box or envelope |
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At room temp. |
In box or envelope at room temp. |
Blood** |
Fasting whole blood in ACD vacutainer (yellow top tube) |
One 5 ml Tube |
Refrigerator
(40C) |
In Gel Pack Box*** |
Bone Marrow Aspirate |
In sterile container(2 drops of 20% sodium citrate should be added per 10 ml of fluid) |
2 ml |
Refrigerator
(40C) |
In Gel Pack Box*** |
Exudative fluids/ CSF |
In sterile container (2 drops of 20% sodium citrate should be added per 10 ml of fluid) |
2 ml |
Refrigerator
(40C) or Freezer
(-20C) |
On ice or in Gel Pack Box*** |
Pleural/peritoneal fluid |
In sterile container |
30 ml |
Refrigerator
(40C) or Freezer
(-20C) |
On ice or in Gel Pack Box*** |
Urine |
First morning urine on 3-5 consecutive days*** |
10 ml |
Refrigerator
(40C) or Freezer
(-20C) |
On ice or in Gel Pack Box*** |
Sputum |
In sterile container, first morning sputum on 3-5 consecutive days*** |
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Refrigerator
(40C) or Freezer
(-20C) |
On ice or in Gel Pack Box*** |
|
This is the minimum recommended volume. Although volumes lower than this will be processed, a negative report may not be reliable |
|
Blood should only be sent in cases of miliary or disseminated tuberculosis. |
|
Gel pack box with instructions for transportation can be obtained from Auroprobe Laboratories on request |
|
The samples can be stored at 40C and pooled before sending to Auroprobe Laboratories |
