Lower respiratory tract infection is a common infectious disease that seriously harms human health. According to statistics of the World Health Organization, in 2008, the global mortality rate of lower respiratory tract infection was 6.1%, ranking third in the top ten cause of death among human beings. Common pathogens of lower respiratory tract infections include bacteria, viruses, mycoplasma, chlamydia and parasites. The results of the study show that accurate and reliable diagnosis of pathogens is an important guarantee for effective control of lower respiratory tract infections, improved cure rates, and reduced bacterial resistance. However, the current etiological detection techniques such as bacterial culture and virus isolation commonly used in clinical practice have the disadvantages of long time consumption and poor sensitivity, which greatly reduces the detection efficiency and makes it difficult to play an effective guiding role in clinical anti-infection treatment.
In recent years, with the rapid development of molecular biology, etiological diagnosis and molecular biology techniques have been closely combined to form etiological molecular diagnostic techniques. The rise of this emerging technology has completely broken the limitations of traditional methods of training and detection, and it has played a role in the diagnosis of etiology with its advantages of rapidness, accuracy, sensitivity, and specificity. It is a diagnosis, treatment, and new type of lower respiratory tract infection. It provides reliable technical support in the detection of pathogens and the study of bacterial resistance mechanisms. The progress of etiological molecular diagnostic techniques for lower respiratory tract infections is summarized below.
First, Nucleic Acid Detection Molecular biology technology was first used in etiological diagnosis starting with nucleic acid detection. Since the development of nucleic acid detection technology, it has undergone roughly the techniques of nucleic acid hybridization, separation, amplification, gene chip, PCR, DNA profiling and DNA sequencing.
1. Nucleic Acid Hybridization: The earliest nucleic acid hybridization technology used biotin, radioisotopes, enzymes, and other labeled probes to hybridize with the nucleic acid fragments of the samples, and the pathogens were identified by detecting specific hybridization signals. The technology is characterized by high specificity and sensitivity, almost 100%, especially the subsequent development of fluorescence in situ hybridization (FISH) technology, played an important role in the early stages of etiological molecular diagnosis. The key to nucleic acid hybridization technology lies in the design of probes. Currently, Legionella probes, Mycoplasma pneumoniae probes, Epstein-Barr virus probes, Respiratory syncytial virus probes, and Pneumocystis spp. probes have been introduced one after another, resulting in lower respiratory tract infections. Diagnosis of typical and atypical pathogens becomes possible. The cDNA probes prepared by Edelstein et al. have the specificity of the genus Legionella. After being modified by the American Gene Probe Company, they have become commercial probe kits and have been adopted by clinical laboratories. The DNA probe prepared by Hyman et al. detected only 0.1 ng of Mycoplasma pneumoniae DNA. Hernandez et al. successfully detected respiratory syncytial virus using nucleic acid hybridization technology. Nucleic acid hybridization technology is the basis of other nucleic acid tests, and it has taken an important step in the development of etiological molecular diagnosis.
2. Polymerase Chain Reaction (PCR): Since the advent of the 1980s, PCR technology has been widely used in various fields and is of epoch-making significance. From the earliest standard PCR to the development of multiplex PCR, nested PCR, RT-PCR, and real-time quantitative PCR, the current PCR technology has been There are more than a dozen types, and their amplification ability and accuracy have gradually increased. Standard PCR is mainly for the amplification of a single gene fragment, multiplex PCR can simultaneously amplify multiple gene fragments, and nested PCR and the widely used real-time quantitative PCR are more advantageous in the detection accuracy.
As we all know, for Streptococcus pneumoniae, Haemophilus influenzae and other bacteria, as well as Legionella pneumophila, Mycoplasma pneumoniae and other atypical pathogens, clinical isolation, training is very difficult, greatly reducing the clinical detection rate, and PCR technology can Make up for this defect. Morozumi et al. used real-time quantitative PCR to detect the lytA gene of Streptococcus pneumoniae, the mip gene of Legionella, and the 16S rRNA of Mycoplasma pneumoniae from the clinical samples of 429 adults and children with pneumonia. The entire detection process took only 2 hours, and it was traditional. Compared with the bacterial culture method, the sensitivity and specificity were greatly improved. In addition to the aforementioned pathogens, Kawazu et al. also detected Aspergillus in respiratory secretions using real-time quantitative PCR technology, which broke through the constraints of Aspergillus culture. Not only against bacteria, PCR technology is no less inferior in virus diagnosis. Beckham et al. used a reverse transcription PCR method to detect viral samples from 194 swab swabs with COPD patients infected with respiratory viruses and compared them with routine isolation and serological detection methods. The results showed that the virus detection rate of the PCR method was 41.8%, while the detection rate of the conventional method was only 23.4%. This result is of great significance for the rapid etiological diagnosis of patients with acute exacerbation of COPD. It can also guide the use of medication and shorten the length of hospital stay.
Domestic research on PCR diagnosis of pathogens is also very rapid. Fang Jian et al. used PCR technology to detect 15 respiratory pathogens simultaneously. During the outbreak of SARS in 2003, researchers used real-time quantitative PCR technology and DNA sequencing technology to lock down the culprit of SARS as a novel coronavirus in just 3 months. In 2009, the new Bunia virus found in China also applied this technology.
In addition, PCR technology has also played an important role in detecting bacterial resistance. The results showed that bacterial resistance-related factors such as New Delhi metallo-β-lactamase 1 (NDM-1), extended-spectrum β-lactamase (ESBL), penicillin-binding protein 2a (PBP2a), and outer membrane pores There are corresponding gene segments in the protein (OprD) and so on. Therefore, these genes can be detected by designing different primers. Diene et al. used real-time quantitative PCR to detect the NDM-1 gene in 128 strains, showing the rapid and sensitive advantages of this technology in the study of carbapenem antibiotic resistance mechanisms. Guan Xizhou et al. used a multiplex PCR technique to discover Klebsiella pneumoniae and its resistant phenotypes that also produce AmpC beta-lactamase (AmpC) and ESBL. The use of PCR technology for the study of drug resistance avoids cumbersome procedures such as bacterial isolation, culture, and drug susceptibility testing. In recent years, with the combination of multiple PCR methods and the emergence of a large number of commercial kits, this technology is increasingly maturing.
3. DNA profiling analysis: In addition to nucleic acid hybridization, PCR, and other techniques for the detection of a specific nucleic acid fragment of a pathogen, there are techniques for detecting whole genome DNA of a pathogen. Currently used techniques include plasmid DNA mapping, chromosomal DNA restriction enzyme analysis, and DNA pulse field gel electrophoresis. These techniques provide new means for pathogen detection and typing by using gel electrophoresis, profiling, and other methods to detect pathogen-wide genomic DNA. At present, the plasmid DNA map and DNA pulse field gel electrophoresis typing techniques have been applied to the staphylococcal classification and the resistance mechanism of methicillin-resistant Staphylococcus aureus (MRSA). Plasmid mapping can be applied to all plasmid pathogens; DNA pulse field gel electrophoresis is often used as a "gold standard" for molecular biological typing methods, but these techniques also have complex DNA map analysis and low resolution. Disadvantages such as poor reproducibility.
4. DNA Sequencing: For pathogen nucleic acids, we must not only understand its overall structure and fragment composition, but also understand the DNA base types, number, and arrangement, which requires the use of DNA sequencing technology. In 1977, Sanger and Maxam determined the DNA sequence using the dideoxy chain termination method and chemical degradation method respectively. Since then, DNA sequencing technology has been applied in the fields of biology, medicine, forensics and pharmacy. As early as 1985, British Lambden used this technology to determine the nucleotide sequence encoded by phosphoproteins of respiratory syncytial virus. In recent years, this technology has been used for the detection of pathogens such as fungi, bacteria, Mycobacterium tuberculosis, and Toxoplasma gondii. Especially in the detection of Mycobacterium tuberculosis compared with nucleic acid hybridization technology costs are significantly reduced. However, due to the high sequencing requirements of the sequencing technology, only a small part of the potentially changing sequences of the bacteria can be detected. Therefore, the technology is currently mainly used in virus detection. For example, in 2007, the First People's Hospital of Wenling of China found that lower respiratory tract infections in children. The new virus-Wu polyoma virus Asian species, and completed the sequencing work. However, because of the strict conditions of DNA sequencing technology, complicated instruments, and high cost, general clinical laboratories are not yet available, and they are mainly used for scientific research.
Second, protein detection For organisms, nucleic acids and proteins are important substances that determine their biological properties. Nucleic acids are the genetic material of organisms, while proteins are the representatives of life activities. Therefore, in addition to targeting nucleic acids, the detection of pathogens is also required to study their protein functions. With the completion of the Human Genome Project, the focus of genomics research has shifted to the study of protein function, and the proteomics that came into being in the post-genomic era has also provided a new platform for the study of respiratory pathogens. The detection techniques for pathogen proteomics are mainly two-dimensional electrophoresis and mass spectrometry or chromatographic analysis. The proteins of the pathogens are transferred to the gel by two-dimensional electrophoresis. After image analysis, specific protein spots are selected, followed by enzymatic hydrolysis and subsequent mass spectrometry. Or chromatographic analysis and data processing to determine the nature of pathogens, the current technology is mainly used for bacterial identification and drug resistance mechanisms. Foreign scholars have reported using proteomics mass spectrometry to detect the proteomics of MRSA and methicillin-sensitive Staphylococcus aureus (MSSA), and analyzed and compared the differences between the two, thus confirming proteomics in the diagnosis of pathogens. The role. In addition, Schaar et al. used two-dimensional electrophoresis and mass spectrometry to discover that the outer membrane vesicles of Moraxella catarrhalis have high biological activity and are the key structures for the transport of bacterial pathogenic factors; Soualhine et al. found that the Streptococcus pneumoniae mutation Phosphate transporters in strains are associated with bacterial resistance.
Third, biochip technology In recent years, biochip technology has been rapid development for the detection of pathogens provides a new technology platform. Biochips currently used for pathogen detection mainly include gene chips, protein chips, and microfluidic chip labs. These technologies rely on their advantages of miniaturization, high throughput and automation to detect, classify, analyze and diagnose microbial pathogens. Genotyping, mutation screening, and genome monitoring are playing an increasingly important role.
The gene chip is the earliest molecular biology chip for pathogens. The special gene information representing various pathogens is printed on a gene chip, and then combined with the sample through reverse transcription to detect the presence or absence of specific pathogen genes and their expression levels in the sample, thereby determining the pathogen of infection. , infection process and host response. At present, due to the completion of genome sequencing of many pathogens, gene chip technology is gradually being applied to the clinic. In addition to gene chips, other biological chip technologies are also becoming more mature. For example, Causse et al. used DNA chips and multiple reverse transcription PCR methods to detect respiratory syncytial virus; in 2003, China successfully detected SARS virus using a self-developed genome-wide chip. . In the same way, protein chips also have broad space for the development of pathogen molecular diagnostics. As a platform, differences in protein expression between pathogens can be detected, especially for respiratory viruses. For example, Yang Lan and others used protein chip technology to detect the pathogens of 1022 children with acute respiratory infections. There was no significant difference between the positive detection rate of virus and the ELISA method, which further validated the high efficiency and convenience of the chip technology in the detection of clinical pathogens. .
Another method worth mentioning is the microfluidic chip technology, also known as the chip lab, which is characterized by high throughput, high efficiency, low consumption, and integration. The advantages of pathogen nucleic acid and protein detection are increasingly prominent. . Song et al. used this technology to not only achieve bacterial sorting, but also achieved counting and identification; Sun et al. used a microchip platform to quickly detect RNA of bird flu viruses. Although microfluidic chip technology has just started, it will have broad application prospects in molecular diagnostics.
IV. Prospects In summary, compared with traditional methods such as bacterial culture, molecular diagnostic technology has the advantages of specificity, sensitivity, rapidity, and convenience, which will surely play an increasingly important role in the diagnosis of etiology of lower respiratory tract. Although these technologies still have deficiencies such as false positives and false negatives, they are complicated to operate, expensive, and even some technologies are still in the initial stage and basic research stage, and the clinical needs to be promoted. However, with the improvement of the economic level and the continuous development of science and technology, the transformation and application of molecular biology techniques in the clinical will gradually become the main means of clinical etiological diagnosis. In the near future, with the help of molecular diagnostic technology, a single drop of blood, a drop of urine, and other body fluids or tissues will be able to detect pathogens in the shortest possible time, enabling rapid, convenient, and efficient etiological molecular diagnosis and providing guidance for clinical treatment. At the same time, it will also greatly reduce medical expenses and reduce the economic burden on society.
In recent years, with the rapid development of molecular biology, etiological diagnosis and molecular biology techniques have been closely combined to form etiological molecular diagnostic techniques. The rise of this emerging technology has completely broken the limitations of traditional methods of training and detection, and it has played a role in the diagnosis of etiology with its advantages of rapidness, accuracy, sensitivity, and specificity. It is a diagnosis, treatment, and new type of lower respiratory tract infection. It provides reliable technical support in the detection of pathogens and the study of bacterial resistance mechanisms. The progress of etiological molecular diagnostic techniques for lower respiratory tract infections is summarized below.
First, Nucleic Acid Detection Molecular biology technology was first used in etiological diagnosis starting with nucleic acid detection. Since the development of nucleic acid detection technology, it has undergone roughly the techniques of nucleic acid hybridization, separation, amplification, gene chip, PCR, DNA profiling and DNA sequencing.
1. Nucleic Acid Hybridization: The earliest nucleic acid hybridization technology used biotin, radioisotopes, enzymes, and other labeled probes to hybridize with the nucleic acid fragments of the samples, and the pathogens were identified by detecting specific hybridization signals. The technology is characterized by high specificity and sensitivity, almost 100%, especially the subsequent development of fluorescence in situ hybridization (FISH) technology, played an important role in the early stages of etiological molecular diagnosis. The key to nucleic acid hybridization technology lies in the design of probes. Currently, Legionella probes, Mycoplasma pneumoniae probes, Epstein-Barr virus probes, Respiratory syncytial virus probes, and Pneumocystis spp. probes have been introduced one after another, resulting in lower respiratory tract infections. Diagnosis of typical and atypical pathogens becomes possible. The cDNA probes prepared by Edelstein et al. have the specificity of the genus Legionella. After being modified by the American Gene Probe Company, they have become commercial probe kits and have been adopted by clinical laboratories. The DNA probe prepared by Hyman et al. detected only 0.1 ng of Mycoplasma pneumoniae DNA. Hernandez et al. successfully detected respiratory syncytial virus using nucleic acid hybridization technology. Nucleic acid hybridization technology is the basis of other nucleic acid tests, and it has taken an important step in the development of etiological molecular diagnosis.
2. Polymerase Chain Reaction (PCR): Since the advent of the 1980s, PCR technology has been widely used in various fields and is of epoch-making significance. From the earliest standard PCR to the development of multiplex PCR, nested PCR, RT-PCR, and real-time quantitative PCR, the current PCR technology has been There are more than a dozen types, and their amplification ability and accuracy have gradually increased. Standard PCR is mainly for the amplification of a single gene fragment, multiplex PCR can simultaneously amplify multiple gene fragments, and nested PCR and the widely used real-time quantitative PCR are more advantageous in the detection accuracy.
As we all know, for Streptococcus pneumoniae, Haemophilus influenzae and other bacteria, as well as Legionella pneumophila, Mycoplasma pneumoniae and other atypical pathogens, clinical isolation, training is very difficult, greatly reducing the clinical detection rate, and PCR technology can Make up for this defect. Morozumi et al. used real-time quantitative PCR to detect the lytA gene of Streptococcus pneumoniae, the mip gene of Legionella, and the 16S rRNA of Mycoplasma pneumoniae from the clinical samples of 429 adults and children with pneumonia. The entire detection process took only 2 hours, and it was traditional. Compared with the bacterial culture method, the sensitivity and specificity were greatly improved. In addition to the aforementioned pathogens, Kawazu et al. also detected Aspergillus in respiratory secretions using real-time quantitative PCR technology, which broke through the constraints of Aspergillus culture. Not only against bacteria, PCR technology is no less inferior in virus diagnosis. Beckham et al. used a reverse transcription PCR method to detect viral samples from 194 swab swabs with COPD patients infected with respiratory viruses and compared them with routine isolation and serological detection methods. The results showed that the virus detection rate of the PCR method was 41.8%, while the detection rate of the conventional method was only 23.4%. This result is of great significance for the rapid etiological diagnosis of patients with acute exacerbation of COPD. It can also guide the use of medication and shorten the length of hospital stay.
Domestic research on PCR diagnosis of pathogens is also very rapid. Fang Jian et al. used PCR technology to detect 15 respiratory pathogens simultaneously. During the outbreak of SARS in 2003, researchers used real-time quantitative PCR technology and DNA sequencing technology to lock down the culprit of SARS as a novel coronavirus in just 3 months. In 2009, the new Bunia virus found in China also applied this technology.
In addition, PCR technology has also played an important role in detecting bacterial resistance. The results showed that bacterial resistance-related factors such as New Delhi metallo-β-lactamase 1 (NDM-1), extended-spectrum β-lactamase (ESBL), penicillin-binding protein 2a (PBP2a), and outer membrane pores There are corresponding gene segments in the protein (OprD) and so on. Therefore, these genes can be detected by designing different primers. Diene et al. used real-time quantitative PCR to detect the NDM-1 gene in 128 strains, showing the rapid and sensitive advantages of this technology in the study of carbapenem antibiotic resistance mechanisms. Guan Xizhou et al. used a multiplex PCR technique to discover Klebsiella pneumoniae and its resistant phenotypes that also produce AmpC beta-lactamase (AmpC) and ESBL. The use of PCR technology for the study of drug resistance avoids cumbersome procedures such as bacterial isolation, culture, and drug susceptibility testing. In recent years, with the combination of multiple PCR methods and the emergence of a large number of commercial kits, this technology is increasingly maturing.
3. DNA profiling analysis: In addition to nucleic acid hybridization, PCR, and other techniques for the detection of a specific nucleic acid fragment of a pathogen, there are techniques for detecting whole genome DNA of a pathogen. Currently used techniques include plasmid DNA mapping, chromosomal DNA restriction enzyme analysis, and DNA pulse field gel electrophoresis. These techniques provide new means for pathogen detection and typing by using gel electrophoresis, profiling, and other methods to detect pathogen-wide genomic DNA. At present, the plasmid DNA map and DNA pulse field gel electrophoresis typing techniques have been applied to the staphylococcal classification and the resistance mechanism of methicillin-resistant Staphylococcus aureus (MRSA). Plasmid mapping can be applied to all plasmid pathogens; DNA pulse field gel electrophoresis is often used as a "gold standard" for molecular biological typing methods, but these techniques also have complex DNA map analysis and low resolution. Disadvantages such as poor reproducibility.
4. DNA Sequencing: For pathogen nucleic acids, we must not only understand its overall structure and fragment composition, but also understand the DNA base types, number, and arrangement, which requires the use of DNA sequencing technology. In 1977, Sanger and Maxam determined the DNA sequence using the dideoxy chain termination method and chemical degradation method respectively. Since then, DNA sequencing technology has been applied in the fields of biology, medicine, forensics and pharmacy. As early as 1985, British Lambden used this technology to determine the nucleotide sequence encoded by phosphoproteins of respiratory syncytial virus. In recent years, this technology has been used for the detection of pathogens such as fungi, bacteria, Mycobacterium tuberculosis, and Toxoplasma gondii. Especially in the detection of Mycobacterium tuberculosis compared with nucleic acid hybridization technology costs are significantly reduced. However, due to the high sequencing requirements of the sequencing technology, only a small part of the potentially changing sequences of the bacteria can be detected. Therefore, the technology is currently mainly used in virus detection. For example, in 2007, the First People's Hospital of Wenling of China found that lower respiratory tract infections in children. The new virus-Wu polyoma virus Asian species, and completed the sequencing work. However, because of the strict conditions of DNA sequencing technology, complicated instruments, and high cost, general clinical laboratories are not yet available, and they are mainly used for scientific research.
Second, protein detection For organisms, nucleic acids and proteins are important substances that determine their biological properties. Nucleic acids are the genetic material of organisms, while proteins are the representatives of life activities. Therefore, in addition to targeting nucleic acids, the detection of pathogens is also required to study their protein functions. With the completion of the Human Genome Project, the focus of genomics research has shifted to the study of protein function, and the proteomics that came into being in the post-genomic era has also provided a new platform for the study of respiratory pathogens. The detection techniques for pathogen proteomics are mainly two-dimensional electrophoresis and mass spectrometry or chromatographic analysis. The proteins of the pathogens are transferred to the gel by two-dimensional electrophoresis. After image analysis, specific protein spots are selected, followed by enzymatic hydrolysis and subsequent mass spectrometry. Or chromatographic analysis and data processing to determine the nature of pathogens, the current technology is mainly used for bacterial identification and drug resistance mechanisms. Foreign scholars have reported using proteomics mass spectrometry to detect the proteomics of MRSA and methicillin-sensitive Staphylococcus aureus (MSSA), and analyzed and compared the differences between the two, thus confirming proteomics in the diagnosis of pathogens. The role. In addition, Schaar et al. used two-dimensional electrophoresis and mass spectrometry to discover that the outer membrane vesicles of Moraxella catarrhalis have high biological activity and are the key structures for the transport of bacterial pathogenic factors; Soualhine et al. found that the Streptococcus pneumoniae mutation Phosphate transporters in strains are associated with bacterial resistance.
Third, biochip technology In recent years, biochip technology has been rapid development for the detection of pathogens provides a new technology platform. Biochips currently used for pathogen detection mainly include gene chips, protein chips, and microfluidic chip labs. These technologies rely on their advantages of miniaturization, high throughput and automation to detect, classify, analyze and diagnose microbial pathogens. Genotyping, mutation screening, and genome monitoring are playing an increasingly important role.
The gene chip is the earliest molecular biology chip for pathogens. The special gene information representing various pathogens is printed on a gene chip, and then combined with the sample through reverse transcription to detect the presence or absence of specific pathogen genes and their expression levels in the sample, thereby determining the pathogen of infection. , infection process and host response. At present, due to the completion of genome sequencing of many pathogens, gene chip technology is gradually being applied to the clinic. In addition to gene chips, other biological chip technologies are also becoming more mature. For example, Causse et al. used DNA chips and multiple reverse transcription PCR methods to detect respiratory syncytial virus; in 2003, China successfully detected SARS virus using a self-developed genome-wide chip. . In the same way, protein chips also have broad space for the development of pathogen molecular diagnostics. As a platform, differences in protein expression between pathogens can be detected, especially for respiratory viruses. For example, Yang Lan and others used protein chip technology to detect the pathogens of 1022 children with acute respiratory infections. There was no significant difference between the positive detection rate of virus and the ELISA method, which further validated the high efficiency and convenience of the chip technology in the detection of clinical pathogens. .
Another method worth mentioning is the microfluidic chip technology, also known as the chip lab, which is characterized by high throughput, high efficiency, low consumption, and integration. The advantages of pathogen nucleic acid and protein detection are increasingly prominent. . Song et al. used this technology to not only achieve bacterial sorting, but also achieved counting and identification; Sun et al. used a microchip platform to quickly detect RNA of bird flu viruses. Although microfluidic chip technology has just started, it will have broad application prospects in molecular diagnostics.
IV. Prospects In summary, compared with traditional methods such as bacterial culture, molecular diagnostic technology has the advantages of specificity, sensitivity, rapidity, and convenience, which will surely play an increasingly important role in the diagnosis of etiology of lower respiratory tract. Although these technologies still have deficiencies such as false positives and false negatives, they are complicated to operate, expensive, and even some technologies are still in the initial stage and basic research stage, and the clinical needs to be promoted. However, with the improvement of the economic level and the continuous development of science and technology, the transformation and application of molecular biology techniques in the clinical will gradually become the main means of clinical etiological diagnosis. In the near future, with the help of molecular diagnostic technology, a single drop of blood, a drop of urine, and other body fluids or tissues will be able to detect pathogens in the shortest possible time, enabling rapid, convenient, and efficient etiological molecular diagnosis and providing guidance for clinical treatment. At the same time, it will also greatly reduce medical expenses and reduce the economic burden on society.
1. Machine Introduction:
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