A Rapid Nucleic Acid Probe Assay for Detection of  Mycobacterium Tuberculosis in Human Sample

For investigational use only  

Intended Use:

SLAP^TM TB kit is intended for detection of mycobacterium tuberculosis, the etiologic agent for tuberculosis in a human sample.

 Tuberculosis background:

Tuberculosis (TB) is a chronic bacterial infection that kills approximately 3 million people each year. The global epidemic is growing at an alarming rate, and becoming increasingly more dangerous.  The sudden increase of TB infections is attributed to spread of HIV infection, resulting in acquired immunodeficiency syndrome (AIDS).  It is estimated that between 2002 and 2020, approximately 1,000 million people will be newly infected with TB, over 150 million people will get sick, and 36 million will die of TB, if control measures are not further strengthened (1).

 Mycobacterium tuberculosis is the etiological agent of TB. The aim of all diagnostic methods available today is the detection of mycobacterium tuberculosis complex, which include M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. microti and M. canetti.

 TB is a contagious disease.  Like the common cold, the organism spreads through the air.  It can infect several organs of the human body, including the brain, the kidneys, and the bones, but most commonly it infects the lungs with manifestation of pulmonary tuberculosis.  Only people who are sick with pulmonary TB are infectious. When infectious people cough, sneeze, talk or spit, they propel TB bacilli, M. tuberculosis, into the air.

 TB is a leading cause of death among people who are HIV-positive. It accounts for about 11% of AIDS deaths worldwide.  In Africa, HIV is the single most important factor determining the increased incidence of TB in years. Global trade and the increased number of people traveling by airplanes have contributed to the dissemination and spread of tuberculosis dramatically. In many industrialized countries, at least one-half of TB cases are among foreign-born people. Another factor in the global dissemination of TB is the increase in the number of refugees and displaced people worldwide. Untreated TB spreads quickly in crowded refugee camps and homeless shelters. In addition it is difficult to treat mobile populations, as treatment takes at least six months. As many as 50% of the world's refugees could be infected with TB. As these displaced people move, they constitute a major source of dissemination and spread of TB.

 The number of people dying of tuberculosis every year is on the rise. Each year, more people are dying of TB, and the number of deaths is increasing globally after almost 40 years of decline. According to WHO (1), the biggest burdens of TB are in southeast Asia, Eastern Europe and Africa.

• TB kills about 2 million people each year (including persons infected with HIV).

• More than 8 million people become sick with TB each year.

• About 2 million TB cases per year occur in sub-Saharan Africa. This number is rising rapidly as a result of the HIV/AIDS epidemic.

• Around 3 million TB cases per year occur in Southeast Asia.

•  Over a quarter of a million TB cases per year occur in Eastern Europe.

Reference: World Health Organization. (2002).”Tuberculosis.” WHO Fact Sheet #104.

 Current assays:

 Several diagnostic tests are used in the diagnosis of tuberculosis, including chest x-ray, TB skin tests (also called Mantoux test or purified protein derivative (PPD) test), sputum test for tuberculosis, lung fluid tests for tuberculosis, biopsy, and lymph node biopsy.  These traditional methods, though helpful, require 1 to 8 weeks to yield results.  The introduction of rapid and molecular diagnostic tests into the diagnosis of TB has made it possible to detect M. tuberculosis complex within 3 to 5 hours.  These tests can be used to rule out tuberculosis (TB) and to allow for appropriate follow up when a patient has a sputum smear that is positive for acid fast bacilli (AFB).   

 TB can be diagnosed by a nucleic acid probe hybridization assay (Laszio, A. (1999) Tuberculosis: Laboratory aspects of diagnosis, CMAJ 160:1725-1729).  The disease can also be detected by immunoassay methods (Daniel, T.M. (1988) Antibody and antigen detection for the immunodiagnosis of tuberculosis, J Infect. Dis. 158: 678-680).

 Nucleic acid assays are more sensitive than immunoassays. Nucleic acid based assays, however, they are very expensive and require well-trained personnel and special laboratory set up. Immunoassays are easy to perform but they are not sensitive enough and do not have the specificity to distinguish between infected patients and those who are BCG vaccinated. Currently commercialized probe assays are complex, slow and prone to false positive results due to amplicon contamination.

 The SLAP^TM TB assay is a sensitive, easy to perform non-target amplification-nucleic acid probe based assay for the detection of mycobacterium tuberculosis complex in a sample. In the assay the target DNA is labeled in a crude cell lysate, hybridized to an immobilized probe and the hybrid is detected by chemiluminescence.  The relative light units from a luminometer are used to interpret the results.

 SLAP^TM TB Kit Components:

Each Kit contains materials to perform 50 tests:

1.                   SLAP^TM TB Sample Labeling Reagent *

2.                   SLAP^TM TB Mag-Probe reagent

3.                   10X Hybridization Solution

4.                   10X Hybrid Wash Solution 

* Highly sensitive to light, to be kept in a brown bottle or wrapped in aluminum foil.

 Materials required but not supplied:

Water baths or Heat Block Heater

Magnetic Separator (available from Caldon Biotech Magnetor Catalogue# AGT-007)

UV lamp 320-365 nm transmission

Luminometer with injectors

5 ml test tubes for hybridization, washing and detection

1.5 ml Eppendorf type transparent tubes for the photo-labeling reaction

Pipette tips and pipettors

Sodium Hydroxide Solution

Hydrogen Peroxide

Hydrochloric Acid

Storage and Stability:

The SLAP^TM TB test kits should be stored at room temperature.  Do not  refrigerate.

Labeling Reagent is highly sensitive to light.  Pipette under diffuse attenuated or red light. 

After uncapping for dispensing, close the vial quickly to protect the reagent from exposure to

 light.

,Precautions:

 1-  The test is for investigational use only

 2-       Only well trained personnel should carry out this procedure.

3-       Handling of samples and other reagents must be done while wearing protective gloves and other customary protective clothes and eyewear. Follow good laboratory practices.  Usual laboratory precautions should be taken at all times.

 4-       Washing the work place surface with bleach is necessary to clean any reagent contamination of the laboratory bench.

5-       The labeling reagent is highly sensitive and appropriate lighting precautions should be taken when handling it.  Use low attenuated diffuse light, dark room, dark operation box or use under red light. 

 Sample preparation:

Sputum from a suspected patient is liquefied with NALC and NaOH, which is routinely done to prepare samples for culture and AFB stain. The sediment is the target sample for the assay.

 Procedure:

 1-       To lyse samples, place 100 ml sediment in 1.5 ml Eppendorf tube, add 20 ml of 2.5 N NaOH.  Heat the mixture at 100^oC for 10 minutes.

 2-       Add 20 ml of 2.5 N HCl to neutralize the mixture to pH 6.0 to 7.5.

 3-       Centrifuge the Eppendorf tubes at 6,000 x g for 5 minutes (this step is optional, but highly recommended if the lysed sample is cloudy or turbid.

 4-       For labeling, add 3 ml of Lumina to the mixture or supernatant.

 5-       Irradiate the mixture for 15 minutes at 320-365 nm using a UV lamp with a long wavelength UV band.

 6-       For hybridization, transfer 100 l of the labeled sample into a 5 ml test tube.

 7-       Add 100 ml hybridization solution.

 8-       Heat at 100^oC for 5 minutes to denature the sample.

 9-       Add 15 ml prewarmed (at 83^oC) beads to the denatured sample.

 10-   Hybridize for 15 minutes at 83^oC.

 11-   Remove hybridization solution from beads on a magnetic separator.

 12-   Wash the beads with wash buffer, at 65^oC, 3 times, five minutes each time.

 13-   Resuspend beads, after the third wash, in 100 ml water.

 14-   For detection, measure chemiluminescence in a luminometer, using hydrogen peroxide (200 ml, 30mM) and sodium hydroxide (200 ml, 1.0 N)         

 Interpretation of Results:

Before starting the assay for the first time, the laboratory will set up the cutoff limits by measuring some true negative samples.  Specimens with light output values equal to or greater than two standard deviations above the cutoff limit can be taken as positive. Specimens with values close to the two standard deviation limit are considered in the gray zone, which need repeat analysis. For this purpose the labeled sample should not be discarded before interpretation of the data.

 Performance of SLAP^TM TB assay:

In a preclinical study more than 200 samples of sputum were tested at AGTI. The sensitivity and specificity of the assay were 92.9% and 97.4% respectively.