Detection Of Mycobacterium Tuberculosis Using DNA Chip And Multi-component Gold Nanoparticle Probes

Tuberculosis (TB) is a chronic communicable disease caused by the Mycobacterium tuberculosis that is commonly infected through the respiratory tract. TB is an increasing and major worldwide problem, which poses a huge threat to human health and has become the greatest public health and social problem in most countries around the world. Therefore, a high sensitivity, efficacy and specificity method for the detection of Mycobacterium tuberculosis is urgently required.In recent years, the application of gold nanoparticles (AuNPs) as oligonucleotide lables instead of fluorescence dyes and isotopes in DNA detection assays was very common. Many experiments revealed that multi-component gold nanoparticle probes were compatible with DNA detection because of their high stability, high labelling density and high hybridization efficiency. Thus, by combination of the nanotechnology and gene chip technique, we established two novel approaches to detect DNA by naked eyes, these are membrane transfer-based colorimetric DNA detection method and silver staining-based crosslinking amplification DNA detection method.Thus, they hold great promise for pathogenic bacteria detection because the methods described here have many desirable advantages including rapid, simple operation and high sensitivity.In the present study, we prepared AuNPs by using the trisodium citrate reduction method. Based on its covalent binding to sulfydryl-modified oligonucleotide and absorption ability of proteins, the multi-component AuNP probes were constructed and further characterized by the transmission electron microscopy (TEM) and ultraviolet absorption spectrometry.In the presence of target DNA, DNA hybridization led to the attachment of multi-component AuNP probes on the chip surface where specific DNA sequences were located in a "sandwich" format, then the invisible signals on the chip could be turned to visible signals by membrane transfer method or silver staining method. Finally, the signals were detected by the naked eye or an ordinary optical scanner to determine the existence of the target DNA.As a result, the AuNP exhibits sphere like shape with the diameter of (13±2) nm, as well as uniform sizes and distributions. The multi-component AuNP probes also have good shapes, distributions and stability. The concentration of AuNP solution is 12.5 nmol/L by calculated from the absorbance at 521 nm. Our results showed that membrane transfer-based colorimetric DNA detection method provided a detection of limit of 1 pmol/L for synthesized target DNA and 0.23 pmol/L for PCR products of Mycobacterium tuberculosis 16S rDNA when the ratio of probes used was 9:1:1 (T10:T40:P2). Specifically, if optimizing the dosage of multi-component AuNP probes and the silver staining time, as litter as 10 fmol/L target DNA could be detected by silver staining-based crosslinking amplification DNA detection method just used naked eyes.Collectively, these two novel methods described here have many desirable advantages including simple operation, high sensitivity, high specificity and no need sophisticated equipment. The multi-component AuNP probes used in the current study not only make ease of hybridization, but aslo transfer the results of chips to visual signals, therefore these two novel methods provide a novel for the detection of Mycobacterium tuberculosis as well as holding great potential applications in the detection of biomolecules.