Method for quantifying number of cells of bacterium in sample

Abstract

The present invention addresses the problem of providing a method whereby it becomes possible to quantify the number of cells of a bacterium in a sample rapidly and with high accuracy by employing PCRmethod. A method which can solve the problem is a method in which the identification and quantification of bacterial cells in a sample are performed through the following steps: (1) a first PCR stepof carrying out PCR method using nucleic acid derived from the sample as a template and also using a universal primer pair for the amplification of 16S rRNA gene in the bacterium to obtain a first amplification product; (2) a second PCR step of carrying out nested PCR method using a primer pair for the amplification of an internal sequence in a sequence occurring in the first amplification productobtained in the first PCR step to produce a second amplification product; and (3) a cell number quantification step of determining the number of cells of the bacterium in the sample from the quantityof the second amplification product obtained in the second PCR step using data for calibration use. In addition to the steps (1) to (3), the following steps (4) and (5) may be additionally carried out: (4) a bacterial species identification step of identifying the species of the bacterium in the sample; and (5) the bacterial cell number correction step of employing the number of cells, which hasbeen obtained in the cell number quantification step, as a provisional number of the cells of the bacterium, and correcting the provisional number of cells on the basis of the number of copies of a 16S rRNA operon of each of the control bacterium and the bacterial species identified in the bacterial species identification step to determine the number of the cells in the sample.

Description

Technical field [0001] The present invention relates to a method for quickly and accurately quantifying the number of bacteria in a test sample, particularly a blood test sample, or quantifying the number of bacteria and identifying bacterial species using the PCR method. Background technique [0002] The traditional examination items used to determine the severity of sepsis are blood culture, endotoxin, procalcitonin, white blood cell count, CRP (C-reactive protein, C-reactive protein), blood pressure, body temperature, respiratory rate, pulse rate, etc. , Recently added Presepsin (soluble CD14 subtype, sCD14-ST). Because blood culture results take time, it is difficult to feed back into early treatment. Since the white blood cell count and CRP are the host's infection defense response, there is a time difference between the test value and the severity of the disease, which often deviates from the actual severity. Procalcitonin is difficult to apply in terms of specificity and...

AI Technical Summary

Problems solved by technology

However, pathogenic bacteria in patient test samples are often small in number, and thus sufficient sensitivity for accurate quantification may not be achieved in normal real-time PCR.

In addition, even if sufficient sensitivity is obtained, there is a problem that contamination is easily detected in bacterial universal PCR (a PCR that detects almost all bacteria), which creates difficulties in determining pathogenic bacteria

As a result, the quantitative examination of pathogenic bacteria in patient samples is very useful for the treatment of infectious diseases, but it has not yet been put into practical use due to technical unsolved problems

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