Pneumonia is currently ranked in the fourth place in the cause-specific death rates of Japanese. This disease is known to often complicate underlying diseases such as cancer and affect an extremely large number of individuals. Culture tests, which have heretofore been performed to search for microbes causative of pneumonia (causative bacteria), allegedly fail to serve as testing methods sufficiently contributing to the selection of treatment, because they require at least a few days and may involve a nearly 1-week additional drug sensitivity test on the cultured causative bacteria. In the case of severe pneumonia requiring hospitalization in intensive care unit (ICU), the accurate and rapid determination of causative bacteria is exceedingly important for the selection of treatment thereof. According to reports, appropriate initial treatment reliably increases the survival rate of pneumonia patients. In fact, however, a technique of identifying causative bacteria as a substitute for the culture tests still remains to be established. Under the circumstances, pneumonia must be treated with causative bacteria unidentified, possibly leading to the emergence of resistant bacteria due to reluctant use of antibiotics based on empirical treatment.
Highly frequently occurring bacterial strains account for nearly 50% of all causative bacteria responsible for pneumonia. Main causative bacteria, also including viruses, are allegedly of approximately 20 to 30 types. Some of these bacteria cannot be cultured by a usual approach. Also in some cases, causative bacteria are difficult to determine even by culture. Particularly, for pneumonia requiring treatment with antibiotics appropriately selected depending on bacterial strains/bacterial volumes, it is very important to simultaneously detect plural types of pneumonia causative bacteria and quantitatively analyze detected signals. Although the optimum therapeutic drug differs depending on the type of causative bacteria, the fact is that medical ethics compels treatment to be started before determination of causative bacteria. The development of an approach capable of rapidly and quantitatively detecting particular bacteria from among a plurality of bacterial strains has been awaited in order to solve these problems.
Proposed is, for example, a method for simultaneously detecting four types of respiratory infection causative bacteria using primers respectively derived from a lytA gene encoding Streptococcus pneumoniae autolysin (LytA), a gene encoding Haemophilus influenza 16S rRNA, a gene encoding Streptococcus pyogenes 16S rRNA, and a gene encoding Mycoplasma pneumoniae 16S rRNA or a primer set thereof in combination with primers derived from a gene encoding Legionella pneumophila 16S rRNA and a mip gene encoding the Legionella pneumophila causative factor MIP protein (see e.g., patent document 1).
Also proposed are a primer set and a probe oligonucleotide set specific for ten types of respiratory disease-related bacteria consisting of a primer set comprising first oligonucleotides and second oligonucleotides and specifically amplifying target sequences present in Bordetella pertussis, Chlamydophila pneumoniae, Haemophilus influenza, Mycoplasma pneumoniae, Klebsiella pneumoniae, Legionella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae with nucleic acids separated from a bacterium-containing sample as templates, and probes specifically detecting target nucleic acids present in the bacteria (see e.g., patent documents 2 and 3).
Also proposed are: a nucleic acid primer set capable of simultaneously amplifying target sequences of five or more types of respiratory disease causative viruses, wherein each primer is selected from oligonucleotides comprising a fragment of ten or more consecutive bases; and probe oligonucleotides for detecting one or more of measles virus, enterovirus, rhinovirus, SARS-related coronavirus (SARS-coV), varicella-zoster virus (VZV), adenovirus, human parainfluenza virus 1 (HPIV1), human parainfluenza virus 2 (HPIV2), human parainfluenza virus 3 (HPIV3), influenza virus A (IVA), influenza virus B (IVB), respiratory syncytial virus A (RSVA), and respiratory syncytial virus B (RSVB), comprising one or more oligonucleotides of 10 bp to 100 bp in length selected from the group consisting of oligonucleotides comprising a fragment of ten or more consecutive bases and oligonucleotides complementary thereto. It is disclosed that this approach comprises the steps of: obtaining nucleic acids from a sample; amplifying the nucleic acids using the nucleic acid primer set; and detecting the amplification products. It is also disclosed that: the step of obtaining nucleic acids from a sample comprises the steps of separating RNAs from the sample and obtaining cDNAs from the separated RNAs, wherein the step of obtaining cDNAs is performed using, for example, reverse transcriptase; reverse transcriptase reaction using the reverse transcriptase may be based on RT-PCR; and the amplification step may be performed by PCR (see e.g., patent document 4).
Further proposed is a simple, highly sensitive norovirus detection method for specifically amplifying genes broadly classified into norovirus gene groups genogroup I (GI) and genogroup II (GII) present in trace amounts in a sample, the method consisting of steps including the steps of: performing an NASBA method capable of amplifying nucleic acids at a predetermined temperature on RNAs extracted from the sample to obtain complementary single-stranded nucleic acids; and further amplifying the nucleic acids by an RT-LAMP method capable of amplifying, at a predetermined temperature, the amplification products obtained by the NASBA method (see e.g., patent document 5).
The present inventors have proposed a primer set for use in the detection of plural types of pneumonia causative bacteria, the primer set allowing simultaneous detection of Streptococcus pneumoniae, Haemophilus influenza, Mycoplasma pneumoniae, and Chlamydophila pneumoniae by usual PCR as well as multiplex PCR, real-time PCR, RT-PCR, etc. (see e.g., patent document 6), a method for detecting or quantifying a target RNA, comprising preparing, from a bacterium-specific RNA strand in 16S rRNA, a liquid-phase universal primer in which an RNA polymerase promoter sequence is added via a tag sequence to the 5′ end of a DNA sequence corresponding to a specific sequence of the target RNA (see e.g., patent document 7), and a method for detecting pathogenic microbes, wherein the pathogenic microbes are two or more species selected from one or more bacteria selected from bacteria of the genera Staphylococcus, Streptococcus, Klebsiella, Escherichia, Mycobacterium, Legionella, Vibrio, Bacillus, Neisseria, Campylobacter, Chlamydia, Chlamydophila, Mycoplasma, Listeria, Salmonella, and Yersinia, the method comprising a polymerase chain reaction step of carrying out polymerase chain reaction using at least one type of first primer set having a tag sequence and a nucleotide sequence selectively annealing to a target nucleic acid on the DnaJ gene carried by the pathogenic microbes and at least one type of second primer set having a tag sequence substantially identical to the tag sequence of the first primer set, and a step of detecting amplification products comprising the target nucleic acid (see e.g., patent document 8).
The present inventors have also already developed a method for specifically detecting or quantifying a target nucleic acid in a sample, the method comprising the steps of: amplifying the target nucleic acid arbitrarily extracted from the sample using hapten- or peptide-unbound primers to obtain single-stranded nucleic acid; hybridizing the amplification product with a membrane bound-first oligonucleotide probe complementary to the amplification product and a complementary second oligonucleotide probe labeled with a colored high molecular carrier, followed by a detection; and evaluating the detection image by visual judgment, and have established a method comprising performing NASBA amplification reaction with total RNA extracted from, for example, a cultured strain of methicillin-resistant Staphylococcus aureus (MRSA), as a template and detecting the amplification product using a nucleic acid chromatography strip (see e.g., patent document 9).
There is also a report on a reagent (Swiftgene Norovirus GI/GII “Kainos”) for detecting two genotypes of norovirus genes by the NASBA method and nucleic acid chromatography in combination. Judgment using this reagent is based on broad classification into genetically diverse GI type to which 15 or more genotypes belongs and GII type to which 18 or more genotypes belong, and is less than precise for identifying pneumonia causative bacteria.