At present, the status of infectious diseases in the world is grim, but most of the infectious pathogens are still unclear. The rapid determination of pathogens is very important for clinicians to guide clinical medication. However, current diagnostic methods are limited in many aspects, such as detection range, time consuming, specificity, and sensitivity. Traditional pathogen detection methods including microbial culture, microscopy, immunological methods of antigen / antibody, identification, and drug susceptibility play an important role in infection control. PCR methods and genotyping methods for detecting the specific nucleic acid sequences of pathogens have been unable to meet the need for a comprehensive detection of the pathogen spectrum.
With the continuous development of gene sequencing technology, the second generation sequencing is also known as the next generation sequencing technology (next generation sequencing, NGS) technology has gradually grown into the mainstream sequencing technology. The technology is developing rapidly, with high throughput, fast speed and low cost. NGS does not require target-specific primers, and can determine the complete DNA sequence of the strain genome in a single sequence determination, which can be used for the identification and typing of all pathogens. Therefore, this technique can play a huge role in the medical microbiology laboratory and infection control [1-2].
01, and an Introduction to NGS
NGS, or high-throughput sequencing, advances in sequence-based genomic research and novel biological applications, is expected to sequence DNA at an unprecedented rate. Compared with traditional sequencing methods, these new non-Sanger-based technologies have the advantages of high throughput, fast sequencing speed, low running cost and high accuracy. The core idea is side-synthesis-by-side sequencing (Sequencing by Synthesis), which determines the sequence of DNA by capturing the markers of newly synthesized ends. It is characterized by the parallel sequence determination of hundreds of thousands to millions of DNA molecules at a time, and the general read length is shorter. While greatly reducing the sequencing cost, it also greatly improves the sequencing efficiency, which is the most widely used sequencing method in the research field of pathogenic microorganisms. NGS does not need the tedious process of cloning, but performs high-throughput parallel PCR and sequencing reactions of genomic DNA fragments connected to universal joints. The large amount of sequencing data obtained can be analyzed by efficient computer bioinformatics analysis technology to obtain complete DNA sequence information [3]. The relatively established NGS platforms mainly include Roche / 454 FLX, Illumina / Solexa Genome Analyzer and Ap-plied Biosystems SOLID system [4].
1. 1 Ｒoche/454 FLX
Roche / 454 FLX is a sequencing technology using the principle of pyrosequencing. T, A, C, G and four bases are recycled into the PTP (Pico Titer Plate) reaction plate in order, and a pyrophosphate is released when the base pairing. Under the synergistic action of bisphosphatase, luciferase, DNA polymerase and ATP sulfatase, the fluorescein is oxidized to oxidized fluorescein and releases a light signal, emitting a single light signal for every one or several bases extended, and the GS FLX system couples the polymerization of each dNTP on the primer to a single fluorescence signal release. By detecting the presence and intensity of fluorescence signal release, the purpose of measuring DNA sequence in real time. The biggest advantage of the 454 technology lies in its large read length and few repeat sequence. The average read length of the current 454 technology can reach 400bp. One of its main disadvantages is low throughput, relatively high cost, unable to accurately measure the length of homopolymer. Suitable for de novo sequencing and metagenomic sequencing, sequence assembly and acquisition of new genes.
1. 2 Illumina/Solexa
The core idea of Illumina / Solexa sequencing is the method of "sequencing" by seqencing-by synthesis (SBS). The correct base complementation is formed during base elongation, and the modified DNA polymerase, adaptor primers, and four dNTP's with base-specific fluorescence tags are simultaneously added to the system. Genomic DNA fragments are bound to the chip (i. e., Flow cell), and these DNA fragments undergo extension and bridge PCR reactions, expanding each DNA fragment a million-fold to form a single sequence Cluster. Each Cluster is a single-molecule cluster with thousands of copies of the identical template. Four special deoxyribonucleotides with fluorescent groups were used for high-throughput, rapid detection of nucleoacid fragments during base elongation. The biggest advantages of Solexa sequencing technology lie in high throughput, low error rate, low cost and wide application range. Its main disadvantage is that short read length brings difficulties to de novo sequencing splicing, which is suitable for RNA sequencing and epigenetic studies.
1.3 The ABI SOLID technique
Instead of employing commonly used DNA polymerases, ABI SOLID sequencing is a magnetic bead-based massively parallel ligation sequencing platform used by ABI, and the substrate for the ligation reaction is a fluorescently labeled 8-base single-stranded DNA probe. The positions 1 to 5 of the 3 ′ end are random bases, and the base pairs formed at positions 1 and 2 can characterize the fluorescence type of the probe, positions 6 to 8 are special synthetic bases that can be paired at will, and the 5 ′ end is a fluorescent group. In ligation reactions, these probes are paired with the single-stranded DNA template strand emitting a different fluorescence signal that reads to the base order. This yields all the base sequence after five rounds of sequencing reactions. SOLID sequencing has the advantages of high throughput, high accuracy, easy to distinguish SNP and sequencing errors. The original sequencing accuracy is as high as 99.94%, which can be said to be the highest accuracy in the current second-generation sequencing technology. Its main disadvantage is also that the shorter read length brings difficulty to de novo sequencing splicing, used in samples with high GC content, genome resequencing and SNP detection.
02, the application of NGS in infectious diseases
In recent years, infectious diseases are emerging around the world. On the one hand, the number of difficult pathogens is increasing, and on the other hand, the spread of infectious diseases is significantly accelerated. The detection of nucleic acid sequences directly from clinical specimens provides new opportunities for pathogen surveillance and discovery. Molecular techniques have been successfully applied to the identification of Borna disease virus, hepatitis C virus, Synocar virus, Kaposi sarcoma-associated herpesvirus (human herpes virus 8), Bartonella, trophoblastic herpes virus, West Nile virus and infectious sources associated with severe acute coronavirus [5-7]. Infectious diseases threaten human health, and therefore put higher requirements for rapid diagnosis of infectious diseases. NGS technology is playing an increasingly important role in the rapid detection and tracing of infectious diseases.
2.1 NGS in neurological infectious diseases
Central nervous system infection is one of the common diseases in neurology, often difficult and severe diseases. The diagnosis of neuroinfectious diseases is based on the detection of pathogenic organisms from the brain parenchyma or cerebrospinal fluid. However, due to the limitation of current clinical laboratory testing methods, more than half of the cases of encephalitis and meningitis cannot be clearly diagnosed. Current clinical commonly used pathogen detection methods including cerebrospinal fluid cytology, pathogen special staining and morphological identification, bacteria and fungi culture, pathogen nucleic acid detection, etc., mainly for specific types of pathogens for selective detection, while the second generation sequencing technology is through metagenomic analysis, achieve unbiased "whole pathogen" screening [8-10]. Wilson MR et al reported that [11] presented a 14-year-old boy with severe combined immunodeficiency with three fever and headache over a period of 4 months, which developed hydrocephalus, epileptic status, and coma. No examination including brain biopsy identified the pathogenic pathogen. Second-generation CSF sequencing identified 475 of the 3063784 sequence corresponding to Leptospira infection (0. 016%). Targeted penicillin treatment was given, and the patient was discharged home after 32 days. Piantadosi et al reported [12] a 61-year-old man admitted with bilateral headache, gait instability, lethargy and confusion. The Powassan virus was detected by rapid metagenomic sequencing in CSF on day 11 of admission. And 4 weeks earlier than the standard serological testing. The results demonstrate the extremely high sensitivity of metagenomic sequencing, which is essential for the monitoring of emerging and understudied pathogens. In 2018, the multi-center encephalitis cooperation group led by Professor Guan Hongzhi of Neurology, Peking Union Medical College Hospital performed cerebrospinal fluid NGS for suspected infectious encephalitis with unknown causes. As a result, two cases were detected with pseudo-rabies virus (pseudorabies virus, PRV) [13], and NGS could detect previously unknown types of viral encephalitis. Multiple high-quality case reports and clinical studies have shown that NGS has the potential to become a diagnostic tool for neuroinfectious diseases, and some scholars [14] have proposed using NGS as a routine method for the etiological diagnosis of encephalitis.
2.2 Application of NGS in respiratory tract infectious diseases
NGS has the unique potential to overcome both diagnostic and testing challenges, and can significantly improve our ability to detect and diagnose pathogen infections. As the cost of NGS methodologies declines, recent advances in genome sequencing and bioinformatics allow the application of this technology in routine diagnostic settings, and NGS provides us with unprecedented opportunities when gold standard techniques fail to detect putative pathogens. In recent years, outbreaks of infectious diseases caused by new outbreaks of pathogens have brought severe challenges to public health prevention and control. Rapid and accurate identification of pathogenic pathogens is crucial to the development of epidemic prevention and control strategies. In respiratory infections, NGS has been used for a variety of respiratory specimens, including sputum, throat swabs, alveolar lavage fluid, and demonstrated good diagnostic performance. Fischer et al. collected 24 respiratory tract samples (bronchoalveolar lavage fluid, sputum samples and swabs) and 5 bronchoalveolar lavage fluid [15] from patients with pneumonia. In contrast to real-time quantitative PCR detection of influenza virus sequences, influenza virus sequences were detected in 18 out of 24 samples. The complete influenza virus genome can be obtained from eight samples. Furthermore, in 3 of 24 influenza-positive samples, additional viral pathogens could be detected, and 2 of 24 samples showed a significant increase in the number of individual bacterial species known to cause overlapping infections during influenza virus infection. Thus, NGS analysis of respiratory samples from patients with known or suspected influenza provides valuable information relevant to clinical studies. Fisher et al reported that [16] a German police officer was sent to the emergency room due to ARDS, the patient received mechanical ventilation, all diagnostic tests for pathogens usually causing pneumonia were negative, although antimicrobial therapy was immediately initiated, the patient died 6 days after multiple organ failure. When the second police officer was sent to the same emergency room for ARDS, the doctor extracted RNA from bronchoalveolar lavage (BAL) specimens and sequenced Chlamydia psittacus within 50 h. After 11 days of antimicrobial treatment, patient 2's condition improved and the patient was transferred to the general hospital ward. Therefore, rapid, specific and high-throughput pathogen detection methods are important for the effective diagnosis and timely prevention and control of infectious diseases.
2.3 Application of NGS in organ transplant-related infections
Organ transplantation is an important treatment for patients with terminal organ failure, but drugs used to prevent rejection suppress the immune system, leaving transplant recipients often at a higher risk of infection. NGS is able to identify the causative agent of infected patients. Ye et al reported that [17] a 2-year-old boy, a patient with granulocytic leukemia, developed black rash and high fever. Detection of suspected pathogens using isolated cultures and PCR was negative, and some conventional antimicrobial treatments was ineffective, including vancomycin, meropenem, tobramycin, cefepime, and rifampin. In this case, pediatricians use the help of next-generation sequencing technology to discover the potential pathogen Propionibacterium acnes, thus guiding them to use specific drugs for the pathogen, and the patient quickly improved. Palacios et al reported that [18] had three patients who received visceral organ transplantation from the same donor on the same day and died of febrile illness 4 to 6 weeks after transplant. Information of culture, polymerase chain reaction (PCR) and serological analysis, and oligonucleotide microarray analysis are lacking. High-throughput sequencing produced 103,632 sequences, with 14 meeting a novel arenavirus (Arenavirus Burden). Additional sequence analysis suggested that this new arenavirus is associated with the lymphocytic choriomeningitis virus. Specific PCR assays based on unique sequences confirmed the presence of the virus in the recipient kidney, liver, blood, and cerebrospinal fluid. Immunohistochemical analysis revealed arenavirus antigen in the recipient liver and kidney grafts. Both the IgM and IgG antiviral antibodies were detected in the donor serum. Early diagnosis of post-transplant infection mortality.
2.4 NGS in bloodstream infectious diseases
Early detection and identification of pathogens in bloodstream infections (BSI) is important to initiate or adjust antibiotic therapy as soon as possible. The current gold standard for diagnose BSI infection is blood culture, but it usually takes one to several days for NGS to directly detect the pathogens in blood samples, ensuring a faster diagnostic speed. In the diagnosis of sepsis, NGS shows unique advantages, which complement the deficiency of traditional etiology diagnostic methods and has great clinical application value. A single-center study in the ICU of Peking Union Medical College Hospital included 78 blood samples of patients with sepsis [19] by Long et al. According to the test results of bacteria and fungi, the final culture and sequencing results of 8 specimens were all positive, 3 specimens were positive and sequenced negative, and 9 specimens were negative and sequenced positive. Using culture results as the "gold standard", the sensitivity and specificity of NGS were 72.7% and 89.6%, respectively; using blood culture and NGS, the positive rate increased significantly compared with blood culture alone (23.08% and 12.82%, P =0. 013). In addition, 15 viruses were detected in 14 blood samples by second-generation sequencing, all of which were verified by Sanger sequencing. This indicates that second-generation sequencing combined with blood culture can further improve the positive rate of etiological diagnosis. Abril et al [20] reported a case of septic shock and multiple organ failure in a 60-year-old male that was diagnosed within 24 h by a novel whole-genome next-generation sequencing analysis confirmed as canine Capnocytoph-aga infection by plasma whole genome NGS and 16S rRNA sequencing. This technology shows great promise in identifying difficult pathogens, and it has profound implications for the diagnosis of infectious diseases. It shows that metagenomic technology has higher efficiency than traditional detection methods.
03. Summary and outlook
NGS technology, as a rapidly developing new technology, has broken through the limitations of the traditional detection methods. For patients with pathogens that are difficult to cultivate or unknown, NGS can directly detect pathogen DNA in samples, and the established professional pathogen database can obtain accurate and reliable pathogen genetic information through automated data analysis, which promotes the development of clinical microbiology and infection [21-22]. However, there are still some limitations to be addressed: ① NGS workflow improvements, such as shorter library construction and validation time, and automation of data analysis, require further research.② The reference database used for NGS is not perfect enough, and a large number of sequencing data cannot be effectively matched.③ No regularity study compared with routine testing methods [23-24].
In conclusion, secondary sequencing technology is not only a revolution in pathogen diagnosis, but also a landmark monument for pathogen diagnosis, which is of landmark significance. With the continuous development of gene sequencing technology, sequencing technology is more and more widely used in the field of biology and biomedicine. Under the requirements of increasing quantity and quality of sequencing data, it is believed that gene sequencing technology will continue to develop rapidly [25].
Author:, Wang Sumei 1,2,3, Zhang Jiandong 1,2,3, Liu Shuye 1,2,3
institution:
1. Clinical Laboratory Department of Tianjin Third Central Hospital, Tianjin 300170;
2. Tianjin Key Laboratory of Artificial Cell, Tianjin 300170;
3. Artificial Cell Engineering and Technology Research Center, Ministry of Health, Tianjin 300170
Source: Jilin Medicine, January 2021, Volume 42, Issue 1
See: Jilin Medicine for details
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