bacterial detection of human sputum samples from the lower respiratory tract using a microfluidic chip

İzotermal amplifikasyon kullanılarak insan solunum yolu bakteriyel enfeksiyonu teşhisi

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>

Yazar Spotlight: Mikroakışkan Çip Kullanarak Solunum Patojenlerinin Hızlı Tespitini Geliştirme 

Mikroakışkan çip bazlı döngü aracılı izotermal amplifikasyon yoluyla alt solunum yolu enfeksiyonlarına neden olan bakteriyel patojenlerin hızlı tespiti

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.

Alt Solunum Yollarından İnsan Balgam Örneklerinin Mikroakışkan Çip Kullanılarak Bakteriyel Tespiti

Başlamak için, çözülmüş bir izotermal amplifikasyon reaktifini hafifçe sallayın. Hepsini tüpün dibinde toplamak için reaktifi santrifüjleyin. 20 mikrolitre izotermal amplifikasyon reaktifini hazırlanmış 200 mikrolitrelik bir santrifüj tüpüne pipetleyin.Tüpü kapağı ile örtün ve numune hazırlama alanına aktarın. Şimdi her tüpe 34.5 mikrolitre hedef nükleik asit numunesi ekleyin. İyice karıştırmak için hafifçe çalkalayın.Tüplerin altındaki tüm numuneleri toplamak için tüpleri kısa bir süre santrifüjleyin. Ardından, numune numarasını bir çip ambalaj etiketine yazın, mikroakışkan çipi ambalaj etiketi yukarı bakacak şekilde açın. Çipi giriş ve çıkış portları yukarı bakacak şekilde yerleştirin.Ardından, hazırlanan amplifikasyon reaksiyon sisteminden 50 mikrolitre çekmek için bir pipet kullanın ve giriş portundan çipin ana kanalına ekleyin. Ana kanal dolduğunda eklemeyi durdurun. Giriş ve çıkış portlarının etrafındaki fazla sıvıyı tüy bırakmayan mendille silin.Ardından, önceden ısıtıldıktan sonra nükleik asit analizörü üzerindeki açık tepsi düğmesine tıklayın. Çipi ön tarafı yukarı bakacak şekilde tepsiye yerleştirin ve sabitlemek için yerleştirme küçük silindirinin çipin orta boşluğundan çıktığından emin olun. Çipi nükleik asit analizörüne yerleştirmek için tepsiyi kapat düğmesine basın.Algılama arayüzündeki numune bilgileri alanına numune test bilgilerini girin. Ardından, numune algılamayı başlatmak için algılamayı başlat düğmesine basın. Birinci numunenin Klebsiella pneumoniae ile enfekte olduğu, ikinci ve yedinci numunelerin ise polimikrobiyal enfeksiyonlar gösterdiği bulundu.Pozitif bir gösterge, bir S şekli sergileyen bir amplifikasyon eğrisi ile temsil edilirken, yatay bir çizgi negatif bir göstergeyi temsil ediyordu.

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Unit 1

Bacterial Detection of Human Sputum Samples from the Lower Respiratory Tract Using a Microfluidic Chip

Lower Respiratory Tract Infections

SCIENTISTS IN ACTION

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 ...

İmmünoloji ve Enfeksiyon

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

Mikroakışkan Çip Kullanılarak Bakteriyel Patojenlerin Hızlı Tespiti için İnsan Balgam Örneklerinden DNA Ekstraksiyonu