Amplification Techniques of Recombinase & Polymerase and their Application in Parasite Detection

To study the application principles of recombinase polymerase amplification (RPA) and the specific situations of detecting parasites, the principles of RPA are analyzed to find the optimal temperature conditions, advantages, and disadvantages. Then, the parasites are detected to observe the application characteristics of the RPA method. The results show that RPA is a kind of novel isothermal nucleic acid amplification technology, which is an open detection method. It has high sensitivity and specificity when being operated at 37-42°C, which makes it very suitable for early detection of pathogen infection. Besides, it also has high sensitivity and specificity in parasite detection. Therefore, the RPA technology has better performances and excellent applications in parasite detection, which has a certain significance for the future application of the technology in more fields.

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Trends in Diagnosis for Active Tuberculosis Using Nanomaterials

Current Medicinal Chemistry ◽

10.2174/0929867325666180912105617 ◽

2019 ◽

Vol 26 (11) ◽

pp. 1946-1959 ◽

Cited By ~ 3

Author(s):

Le Minh Tu Phan ◽

Lemma Teshome Tufa ◽

Hwa-Jung Kim ◽

Jaebeom Lee ◽

Tae Jung Park

Keyword(s):

Sensitivity And Specificity ◽

High Efficiency ◽

Point Of Care ◽

High Sensitivity ◽

Signs And Symptoms ◽

Diagnostic Methods ◽

Advantages And Disadvantages ◽

Treatment And Prevention ◽

Clinical Tools ◽

Diagnostic Applications

Background:Tuberculosis (TB), one of the leading causes of death worldwide, is difficult to diagnose based only on signs and symptoms. Methods for TB detection are continuously being researched to design novel effective clinical tools for the diagnosis of TB.Objective:This article reviews the methods to diagnose TB at the latent and active stages and to recognize prospective TB diagnostic methods based on nanomaterials.Methods:The current methods for TB diagnosis were reviewed by evaluating their advantages and disadvantages. Furthermore, the trends in TB detection using nanomaterials were discussed regarding their performance capacity for clinical diagnostic applications.Results:Current methods such as microscopy, culture, and tuberculin skin test are still being employed to diagnose TB, however, a highly sensitive point of care tool without false results is still needed. The utilization of nanomaterials to detect the specific TB biomarkers with high sensitivity and specificity can provide a possible strategy to rapidly diagnose TB. Although it is challenging for nanodiagnostic platforms to be assessed in clinical trials, active TB diagnosis using nanomaterials is highly expected to achieve clinical significance for regular application. In addition, aspects and future directions in developing the high-efficiency tools to diagnose active TB using advanced nanomaterials are expounded.Conclusion:This review suggests that nanomaterials have high potential as rapid, costeffective tools to enhance the diagnostic sensitivity and specificity for the accurate diagnosis, treatment, and prevention of TB. Hence, portable nanobiosensors can be alternative effective tests to be exploited globally after clinical trial execution.

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Clinical evaluation of ID NOW influenza A & B 2, a rapid influenza virus detection kit using isothermal nucleic acid amplification technology - A comparison with currently available tests

Journal of Infection and Chemotherapy ◽

10.1016/j.jiac.2019.08.015 ◽

2020 ◽

Vol 26 (2) ◽

pp. 216-221 ◽

Cited By ~ 2

Author(s):

Keiko Mitamura ◽

Hideaki Shimizu ◽

Masahiko Yamazaki ◽

Masataka Ichikawa ◽

Takashi Abe ◽

...

Keyword(s):

Influenza Virus ◽

Nucleic Acid ◽

Clinical Evaluation ◽

Influenza A ◽

Virus Detection ◽

Nucleic Acid Amplification ◽

Isothermal Nucleic Acid Amplification ◽

Nucleic Acid Amplification Technology ◽

Influenza Virus Detection

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Traditional and new applications of HCR in biosensing and biomedicine

The Analyst ◽

10.1039/d1an01371h ◽

2021 ◽

Author(s):

Rong Zhou ◽

Zhuoer Zeng ◽

Ruowei Sun ◽

Wenfang Liu ◽

Qubo Zhu ◽

...

Keyword(s):

Nucleic Acid ◽

Double Helix ◽

Nucleic Acid Amplification ◽

Hybridization Chain Reaction ◽

Chain Reaction ◽

Single Stranded Dna ◽

Isothermal Nucleic Acid Amplification ◽

Hybridization Event ◽

Nucleic Acid Amplification Technology ◽

New Applications

Hybridization chain reaction is a very popular isothermal nucleic acid amplification technology. A single-stranded DNA initiator triggers an alternate hybridization event between two hairpins forming a double helix polymer. Due...

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Comparison between molecular methods (PCR vs LAMP) to detect Candida albicans in bronchoalveolar lavage samples of suspected tuberculosis patients

Microbiology Research ◽

10.4081/mr.2017.7306 ◽

2018 ◽

Vol 8 (2) ◽

Cited By ~ 1

Author(s):

Meysam Goodarzi ◽

Mohammad Hassan Shahhosseiny ◽

Mansour Bayat ◽

Seyed Jamal Hashemi ◽

Mohammad Ghahri

Keyword(s):

Bronchoalveolar Lavage ◽

Sensitivity And Specificity ◽

Isothermal Condition ◽

High Sensitivity ◽

High Specificity ◽

Nucleic Acid Amplification ◽

Lamp Method ◽

Specificity And Sensitivity ◽

Suspected Tuberculosis ◽

Pcr Test

With the increase of patients suffering from immune deficiency infections also increased pulmonary fungi even in people with defective immune system can cause fatal and lethal candidiasis. The timely diagnosis of pulmonary candidiasis is one of the problems that has been detected. Polymerase chain reaction (PCR) test and Loop mediated isothermal amplification (LAMP) method optimized on the basis of α INT1 gene and then sensitivity and specificity were evaluated. LAMP is a novel nucleic acid amplification technique with high specificity and sensitivity which has been done under isothermal condition. Samples were the bronchoalveolar lavage suspected of tuberculosis (TB) reviews for TB disease negative have been reported. DNA extraction carried out by standard phenol/chloroform method on samples and PCR test and LAMP was done. PCR and LAMP testing was performed on samples and products of 441 bp were amplified and observed with agarose gel electrophoresis. At the end of the LAMP reaction, SYBR Green was used for identifying negative and positive results. Among the 60 quantities sera, only 7 cases were PCR positive but 8 cases were LAMP positive. In comparison, between LAMP and PCR, the LAMP technique in spite of its simplicity, high sensitivity and specificity, could be an appropriate replacement for PCR.

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Development of a two-step nucleic acid amplification test for accurate diagnosis of the Mycobacterium tuberculosis complex

Scientific Reports ◽

10.1038/s41598-021-85160-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Chien-Ru Lin ◽

Hsin-Yao Wang ◽

Ting-Wei Lin ◽

Jang-Jih Lu ◽

Jason Chia-Hsun Hsieh ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Nucleic Acid ◽

Real Time ◽

Sensitivity And Specificity ◽

Real Time Pcr ◽

Disease Transmission ◽

High Sensitivity ◽

Nucleic Acid Amplification ◽

Amplification Efficiency ◽

Pcr Assay

AbstractThe Mycobacterium tuberculosis complex (MTBC) remains one of the top 10 leading causes of death globally. The early diagnosis of MTBC can reduce mortality and mitigate disease transmission. However, current nucleic acid amplification diagnostic test methods are generally time-consuming and show suboptimal diagnostic performance, especially in extrapulmonary MTBC samples or acid-fast stain (AFS)-negative cases. Thus, development of an accurate assay for the diagnosis of MTBC is necessary, particularly under the above mentioned conditions. In this study, a single-tube nested real-time PCR assay (N-RTP) was developed and compared with a newly in-house-developed high-sensitivity real-time PCR assay (HS-RTP) using 134 clinical specimens (including 73 pulmonary and 61 extrapulmonary specimens). The amplification efficiency of HS-RTP and N-RTP was 99.8% and 100.7%, respectively. The sensitivity and specificity of HS-RTP and N-RTP for the diagnosis of MTBC in these specimens were 97.5% (77/79) versus 94.9% (75/79) and 80.0% (44/55) versus 89.1% (49/55), respectively. The sensitivity and specificity of HS-RTP and N-RTP for the diagnosis of MTBC in pulmonary specimens were 96.3% (52/54) versus 96.3% (52/54) and 73.7.0% (14/19) versus 89.5% (17/19), respectively; in extrapulmonary specimens, the sensitivity and specificity of HS-RTP and N-RTP were 100% (25/25) versus 92% (23/25) and 83.3% (30/36) versus 88.9% (32/36), respectively. Among the AFS-negative cases, the sensitivity and specificity of HS-RTP and N-RTP were 97.0% (32/33) versus 90.9% (30/33) and 88.0% (44/50) versus 92.0% (46/50), respectively. Overall, the sensitivity of HS-RTP was higher than that of N-RTP, and the performance was not compromised in extrapulmonary specimens and under AFS-negative conditions. In contrast, the specificity of the N-RTP assay was higher than that of the HS-RTP assay in all types of specimens. In conclusion, the HS-RTP assay would be useful for screening patients suspected of exhibiting an MTBC infection due to its higher sensitivity, while the N-RTP assay could be used for confirmation because of its higher specificity. Our results provide a two-step method (screen to confirm) that simultaneously achieves high sensitivity and specificity in the diagnosis of MTBC.

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Strategies for Isothermal Amplification of Nucleic Acids: are they Ready to be Applied in Point of Care Diagnosis of Mycosis?

Biointerface Research in Applied Chemistry ◽

10.33263/briac113.1055910571 ◽

2020 ◽

Vol 11 (3) ◽

pp. 10559-10571

Keyword(s):

Nucleic Acid ◽

Clinical Laboratory ◽

Multiple Displacement Amplification ◽

Rolling Circle Amplification ◽

High Sensitivity ◽

Isothermal Amplification ◽

Nucleic Acid Amplification ◽

Clinical Samples ◽

Rolling Circle ◽

Isothermal Nucleic Acid Amplification

The early detection of invasive fungal infection (IFD) is significant in order to decrease mortality in susceptible patients. There is, therefore, a need for sensitive and specific fungal species detection assays in a clinical laboratory for early targeted therapy. The isothermal amplification method may be useful for the screening of fungal isolates, especially in resource-poor settings. Therefore, our aim was to review the isothermal nucleic acid amplification methods and their applications in fungal pathogen detection. Out of 50 reported studies, 28, 12, 6, 2, and 2 studies used the isothermal-based assays of a loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), multiple displacement amplification (MDA) and polymerase Spiral Reaction (PSR), respectively. Thirty-two studies used clinical samples, 18 pure culture, and four environmental samples. The diagnostic accuracy of isothermal nucleic acid amplification testing for pathogenic fungal was reported as high (sensitivity 0.89–1.0 and specificity 0.63–1.0) in all studies irrespective of the sample tested. Although the isothermal-based assays showed high sensitivity and specificity in reported studies, it is still poorer than that of PCR assays. However, improving the assay to make it simpler, more effective, and inexpensive compared with newer PCR methods are still needed.

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Global Threshold Using Otsu and Active Contour for Detection of Malaria Parasites in Thick Blood Smear

BIO Web of Conferences ◽

10.1051/bioconf/20214104002 ◽

2021 ◽

Vol 41 ◽

pp. 04002

Author(s):

Amin Siddiq Sumi ◽

Hanung Adi Nugroho ◽

Rudy Hartanto

Keyword(s):

Active Contour ◽

Detection Method ◽

High Sensitivity ◽

Morphological Operations ◽

Thick Blood Smear ◽

Improve Image Quality ◽

Plasmodium Parasite ◽

Blood Smears ◽

Global Threshold ◽

Parasite Detection

Malaria is a disease caused by the plasmodium parasite and has caused many fatalities. In general, identifying malaria parasite infection can be done by visually observing thick and thin blood smears through microscopic devices. Identification of parasites in thick blood preparations has a higher level of difficulty than thin blood preparations. In thick blood preparations, various objects such as artefacts and noise have a structure similar to the structure of parasitic objects. This paper aims to develop a parasite detection method based on image processing in thick blood smears, consisting of two main stages. First is to improve image quality by applying contrast value stretching, converting green channels, and refining each image. Second is to segment the plasmodium parasite using global threshold Otsu and active contour followed by several morphological operations. The proposed method produces a high sensitivity of 98.06% with an average negative false rate of 1.4%. With the sensitivity level obtained, it can be interpreted that most of the parasitic objects have been detected correctly in one blood sample image.

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Ultrasensitive detection of microRNAs using catalytic hairpin assembly coupled with enzymatic repairing amplification

Chemical Communications ◽

10.1039/c6cc07116c ◽

2016 ◽

Vol 52 (93) ◽

pp. 13584-13587 ◽

Cited By ~ 28

Author(s):

Chong-Hua Zhang ◽

Ying Tang ◽

Ying-Ying Sheng ◽

Hui Wang ◽

Zhan Wu ◽

...

Keyword(s):

Nucleic Acid ◽

Nucleic Acid Amplification ◽

Ultrasensitive Detection ◽

Isothermal Nucleic Acid Amplification ◽

Nucleic Acid Amplification Technology ◽

Catalytic Hairpin Assembly ◽

Mirna Detection

A novel isothermal nucleic acid amplification technology is developed by coupling CHA with enzymatic repairing amplification for sensitive and selective miRNA detection.

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