RNA (ribonucleic acid) polymerase based multiplex detection method for NTPs (nucleoside triphosphates) by adopting in-vitro transcription machine, kit and application

Abstract

The invention discloses an RNA (ribonucleic acid) polymerase based multiplex detection method for NTPs (nucleoside triphosphates) by adopting an in-vitro transcription machine, a kit and an application. The method is high in practicability and is the unique detection method capable of detecting four NTPs simultaneously, accurate quantification of the four different NTPs can be realized quite rapidly (one minute) with high selectivity, high sensitivity, high throughput and low cost by simply changing sequences of template strands and coding strands, composition of other excessive NTPs and matched molecular beacons, a reaction system is simple, complicated probe design and synthesis steps are not needed, reaction components are all commercial reagents, all that is needed is to simply mix the components during a reaction, development of the kit is easy, research, application and clinical detection are facilitated greatly, and the method has great scientific significance and business value.

Description

technical field [0001] The invention belongs to the technical field, and relates to a method for rapid multiple detection of NTPs by an in vitro transcription machine based on RNA polymerase, a kit and an application thereof, which are used for ultra-fast, high-selectivity, high-sensitivity and detection of ATP, CTP, UTP and GTP High-throughput detection. Background technique [0002] Nucleoside triphosphates (NTPs, also known as nucleoside triphosphates) are the most basic and important functional molecules in life, consisting of a molecule of ribose, a molecule of base and three molecules of phosphate. According to the base class, NTPs are divided into adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP) and guanosine triphosphate (GTP). As the raw material of nucleic acid molecules, NTPs not only ensure the normal replication, repair, transcription and reverse transcription of nucleic acids, but also play an important role in almost all c...

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Problems solved by technology

Specifically, the current sensing and recognition principles of NTPs are divided into six categories: one is organic small molecule fluorescent probes (X.Li, et al. Angew. Chem. Int. Ed. 2014, 53, 7809), which has the advantage of responding Fast, but the synthesis steps are extremely complicated, and the cytotoxicity, sensitivity (μM) and selectivity are poor; the second is the gene-encoded fluorescent protein or polypeptide (H.Yaginuma, et al.Sci.Rep.2014,4,6522), Although the biocompatibility is good, the expression and purification steps are complicated, and the sensitivity and selectivity are also poor; the third is the aptamer screened in vitro (A.B.Iliuk, etal. Anal. Chem. 2011, 83, 4440), ATP aptamer The essence of the ligand is deoxyribose nucleic acid (deoxyribonucleic acid, DNA), poor selectivity to adenosine diphosphate (adenosine diphosphate, ADP) and adenosine monophosphate (adenosine monophosphate, AMP), and the aptamer of GTP is ribonucleic acid ( ribonucleic acid, RNA), the stability is not good, and the incubation time between the aptamer and the target molecule is too long (more than 30 minutes), but the aptamer of UTP and CTP is rarely reported; the fourth is based on the specific recognition of ATP or GTP DNAzyme (A.Sreedhara, etal.J.Am.Chem.Soc.2004,126,3454; S.A.Mcmanus, et al.J.Am.Chem.Soc.2013,135,7181), but the sensitivity ( μM) is very poor; the fifth is based on ATP-dependent luciferase (P.E.Stanley, etal.Biolumin.Chemilumin.1989,4,289), its activity is easily affected by environmental factors, and the fluorescence emitted by the chemical reaction is unstable. After 30 minutes The light intensity can be reduced by 10%, and the versatility is poor, and CTP, UTP and GTP cannot be detected; the sixth is based on ATP-dependent T4 DNA ligase (L.M.Lu, et al.J.Am.Chem.Soc.2011,133,11686) , the connection time is at least half an hour, and it also cannot be used for CTP, UTP and GTP detection

To sum up, the existing sensing methods are all single-target sensors for detecting ATP or GTP, and can only meet one or two aspects in terms of selectivity, sensitivity, response time and simplicity, and cannot be sensitive to the outside world. Rapid detection of NTP content and correlation changes during stimulation is difficult to meet the needs of future scientific research and clinical analysis for rapid multiple detection of NTPs

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