extraction of dna from human sputum samples for the rapid detection of bacterial pathogens using a microfluidic chip

Diagnóstico de Infecção Bacteriana Respiratória Humana Usando Amplificação Isotérmica

Respiratory tract infections (RTIs) caused by bacterial pathogens primarily contribute to morbidity and mortality worldwide1. It is defined as any upper or lower respiratory symptoms accompanied by fever lasting 2-3 days. While upper respiratory infection is more common than lower respiratory infection, chronic and recurrent respiratory tract infections are also common clinical conditions, posing great risks to individuals and placing a significant burden on healthcare systems2. Common bacterial pathogens of RTIs include Streptococcus pneumoniae3, Haemophilus influenzae4, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Stenotrophomonas maltophilia, among others. These pathogenic bacteria usually colonize the mucosal surfaces of the host&#39;s nasopharynx and upper respiratory tract, causing typical symptoms of RTIs such as sore throat and bronchitis. They cause pneumonia when they spread from the upper respiratory tract to sterile areas of the lower respiratory tract and may spread from person to person through the respiratory tract5. In severe cases, they can also lead to invasive bacterial diseases, especially bacteremic pneumonia, meningitis, and sepsis, which are leading causes of morbidity and mortality in people of all age groups worldwide.
Traditional tests for RTIs involve microbiological culture using throat swabs and sputum respiratory samples6. Additionally, serological tests such as enzyme-linked immunosorbent assay (ELISA) detect antibodies or antigens in serum, while agglutination tests observe the agglutination reaction of antibodies and antigens to detect infection7. Microbial culture is considered the gold standard for diagnosing RTIs, but its low culture positivity rate, poor reliability, and long detection cycle limit diagnostic efficiency8. In reality, rapid and accurate diagnosis of RTIs is crucial for precise eradication of the bacterial pathogen. Quick and effective detection methods can help reduce the transmission rate of pathogens, shorten the duration of infection, and decrease unnecessary antibiotic use9,10. Molecular biology-based methods significantly expedite detection, such as polymerase chain reaction (PCR), which amplifies a target gene&#39;s DNA sequence to detect pathogens. However, traditional PCR necessitates complex temperature cycling equipment, which is cumbersome and time-consuming. Furthermore, every DNA amplification using PCR (except real-time PCR) concludes with electrophoretic separation of the product, which also takes time. Visualization of the product requires dyes, many of which are mutagenic or carcinogenic. Therefore, it is imperative to continuously develop new methods and technologies for diagnosing RTI bacterial pathogens.
Loop-Mediated Isothermal Amplification (LAMP) is a novel and emerging molecular technology initially developed by Notomi et al. in 200011. LAMP can amplify DNA under stable isothermal conditions without complex temperature cycling equipment, which makes it suitable for rapid detection and reduces equipment complexity and cost12. LAMP can detect low concentrations of target DNA with high sensitivity13. It uses multiple specific primers to improve selectivity for target sequences and reduce the possibility of false positives14. LAMP is gradually being widely used in clinical laboratories due to its ease, speed, and intuitive operation, even for detecting RTIs. In this study, we investigated the effectiveness of LAMP in detecting lower RTIs in clinical samples (sputum, bronchial lavage fluid, and alveolar lavage fluid), as shown in Figure 1. It is evident that LAMP offers advantages such as speed, sensitivity, and ease of use over traditional tests in lower RTI detection, making it a promising application.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66677/66677fig01.jpg" /> 
Figure 1: Schematic illustration of the LAMP detection method. <a href="https://www.jove.com/files/ftp_upload/66677/66677fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Autor em destaque: Avançando na detecção rápida de patógenos respiratórios usando chip microfluídico 

Detecção rápida de patógenos bacterianos que causam infecções do trato respiratório inferior por meio de amplificação isotérmica mediada por loop baseada em chip microfluídico

This study focused on the rapid of bacterial pathogen, respiratory by Microfluidic chip technology based on loop mediated isothermal implication and the effectiveness of this approach in the management and the control respiratory in the hospitals and the communities. The main challenge of this method is the risk of with its high sensitivity. Which stricter sample handling and the standard operational protocols, to reduce the risk of false positive results.Additionally, laboratory personnel need to master the relevant skills to effectively apply this method in primary hospitals. Compared to traditional PCR techniques, the Microfluidic chip technology, based on loop mediated isothermal implication, has high specificity and the sensitivity. It can quickly detect up to certain high frequency respiratory pathogens and common resistance genes within one novel.The technique we demonstrated in this study has important practical implications for the control and treatment of respiratory infections. Helping to prevent the spread of bacterial pathogens and providing guidance for the appropriate antibiotic treatment. We intended to continue developing novel diagnostic methods with a focus on rapid and accurate detection of microbial pathogens causing infectious diseases.We will also endeavor to work on developing novel-point care testing methods for infectious diseases, that can be easily deployed in resource limited settings like rural China.

Extração de DNA de amostras de escarro humano para a detecção rápida de patógenos bacterianos usando um chip microfluídico

Para começar, limpe a cavidade oral e os dentes com água limpa. Peça aos pacientes com dentaduras para removê-las. Instrua o paciente a tossir com força para expelir o escarro respiratório profundo para um recipiente de escarro.Para extrair o DNA, adicione um volume apropriado de hidróxido de sódio a 10% à amostra com base em sua viscosidade. Vortex a suspensão por 15 segundos e, em seguida, incube-a a 37 graus Celsius por 30 minutos para liquefazer a amostra. Em seguida, pipete um mililitro da amostra liquefeita em um tubo de centrífuga de 1,5 mililitro.Centrifugue a amostra a 15.777 G por cinco minutos a dois a seis graus Celsius. Em seguida, pipetar o sobrenadante para descartá-lo. Agora, adicione um mililitro da solução de lavagem ao tubo e vórtice para levantar o precipitado do fundo do tubo.Depois de centralizar e descartar o sobrenadante como antes, pipetar 100 microlitros de solução de extração de ácido nucleico para o palete. Com uma pipeta, aspire e misture bem o precipitado com a solução. Transfira o líquido e precipite juntos para um tubo de extração de ácido nucleico.Coloque o tubo em um misturador de vórtice em velocidade média por cinco minutos. Em seguida, transfira o tubo para um banho de metal a uma temperatura constante de 100 graus Celsius por cinco minutos. Finalmente, centrifugue as amostras a 15.777 G por cinco minutos a dois a seis graus Celsius antes de continuar experimentando.

extraction-dna-from-human-sputum-samples-for-rapid-detection

Research

JoVE Journal

Immunology and Infection

Extraction of DNA from Human Sputum Samples for the Rapid Detection of Bacterial Pathogens Using a Microfluidic Chip

63 visualizações.  Southern Medical University. Para começar, limpe a cavidade oral e os dentes com água limpa. Peça aos pacientes com dentaduras para removê-las. Instrua o paciente a tossir com força para expelir o escarro respiratório profundo para um recipiente de escarro.Para extrair o DNA, adicione um volume apropriado de hidróxido de sódio a 10% à amostra com base em sua viscosidade. Vortex a suspensão por 15 segundos e, em seguida, incube-a a 37 graus Celsius por 30 minutos para liquefazer a amostra. Em seguida, pip...

Vídeo: Extração de DNA de amostras de escarro humano para a detecção rápida de patógenos bacterianos usando um chip microfluídico

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...