Single tube amplification and detection of male date palm using polymerase chain reaction and loop-mediated isothermal amplification technique

Q fever, which is caused by Coxiella burnetii, a small, pleomorphic intracellular bacterium, is the most widespread zoonosis in the world. The chronic form of the disease can lead to disability and death. Rapid diagnosis of Q fever is needed in order that effective treatment can be initiated. The conventional retrospective diagnosis of Q fever, based on serology, is useless for the treatment of afflicted patients. Thus, molecular methods have been created to close the diagnostic gap between the onset of the disease and the presence of specific antibodies in serum. A polymerase chain reaction is a suitable and reliable method with high sensitivity and specificity, but it requires expensive equipment and post-amplification protocol. Loop-mediated isothermal amplification (LAMP) is an isothermal technique, conducted at constant temperature that can amplify a negligible amount of DNA to more than 109 copies within one hour, using special primers and polymerase. We have tested the sensitivity and specificity of LAMP in the detection of C. burnetii. The mean positive rate of LAMP and polymerase chain reaction in patients was 100% and 74%, respectively. LAMP reacted negatively with non-C. burnetii pathogens and non-infected blood samples. We conclude that LAMP is a sensitive and specific technique for the detection of C. burnetii and has advantages over serological methods and PCR that make it attractive for diagnosing Q fever in countries around the world.

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Loop-Mediated Isothermal Amplification (LAMP) as a Promising Point-of-Care Diagnostic Strategy in Avian Virus Research

Animals ◽

10.3390/ani12010076 ◽

2021 ◽

Vol 12 (1) ◽

pp. 76

Author(s):

Faiz Padzil ◽

Abdul Razak Mariatulqabtiah ◽

Wen Siang Tan ◽

Kok Lian Ho ◽

Nurulfiza Mat Isa ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Nucleic Acid ◽

Point Of Care ◽

Rolling Circle Amplification ◽

Isothermal Amplification ◽

Screening Methods ◽

Diagnostic Strategy ◽

Chain Reaction ◽

Loop Mediated Isothermal Amplification ◽

Polymerase Chain

Over the years, development of molecular diagnostics has evolved significantly in the detection of pathogens within humans and their surroundings. Researchers have discovered new species and strains of viruses, while mitigating the viral infections that occur, owing to the accessibility of nucleic acid screening methods such as polymerase chain reaction (PCR), qualitative (real-time) polymerase chain reaction (qPCR) and reverse-transcription qPCR (RT-qPCR). While such molecular detection methods are widely utilized as the benchmark, the invention of isothermal amplifications has also emerged as a reliable tool to improvise on-field diagnosis without dependence on thermocyclers. Among the established isothermal amplification technologies are loop-mediated isothermal amplification (LAMP), recombinant polymerase amplification (RPA), strand displacement activity (SDA), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA) and rolling circle amplification (RCA). This review highlights the past research on and future prospects of LAMP, its principles and applications as a promising point-of-care diagnostic method against avian viruses.

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Asymmetric polymerase chain reaction and loop-mediated isothermal amplification (AP-LAMP) for ultrasensitive detection of microRNAs

Chinese Chemical Letters ◽

10.1016/j.cclet.2019.05.003 ◽

2020 ◽

Vol 31 (1) ◽

pp. 159-162 ◽

Cited By ~ 2

Author(s):

Ruili Wang ◽

Ling Lan ◽

Li Liu ◽

Liang Cheng

Keyword(s):

Polymerase Chain Reaction ◽

Isothermal Amplification ◽

Ultrasensitive Detection ◽

Chain Reaction ◽

Loop Mediated Isothermal Amplification ◽

Asymmetric Polymerase Chain Reaction ◽

Polymerase Chain

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Comparison of Reverse Transcriptase Loop-Mediated Isothermal Amplification and Reverse Transcriptase Polymerase Chain Reaction for Detection of Prostate Specific Antigen

Basic & Clinical Cancer Research ◽

10.18502/bccr.v11i1.1649 ◽

2019 ◽

Author(s):

Mohammad Amin Almasi ◽

Marya Esmaili

Keyword(s):

Prostate Cancer ◽

Polymerase Chain Reaction ◽

Reverse Transcriptase ◽

Prostate Specific Antigen ◽

Specific Antigen ◽

Isothermal Amplification ◽

Chain Reaction ◽

Loop Mediated Isothermal Amplification ◽

Polymerase Chain ◽

Positive Controls

Background: Research shows that prostate cancer ranks second among the top five most common cancers in men. It has been confirmed that when circulating Prostate Specific Antigen (PSA) transcripts are successfully detected, prostate cancer cells can be diagnosed at an early stage. A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was developed and compared to reverse transcriptase polymerase chain reaction (RT-PCR) assay for detection of PSA. Methods: 47 patients, including 30 patients with prostate cancer, 15 with Benign Prostate Hyperplasia (BPH) and 2 healthy subjects as negative controls were included in this study. The prostate cancer cell lines (PC3 and LNCaP) of two patients were included in the study as positive controls. Next, RNA was extracted from fresh samples and a first strand cDNA synthesis kit was applied for the synthesis of cDNA. The human prostate specific antigen gene was used to design specific primers. Results: The results indicated that the control subjects and participants suffering from BPH were not positive. 13 out of 15 (86.6%) patients suffering from localized cancer were PSA positive. PSA positive results were observed among all 15 metastatic patients and positive controls (100%). RT-LAMP is an advantageous method because it is highly sensitive (1000-fold), quite cheap, user-friendly, and safe; in addition, it can be quickly performed by visual detection using GineFinderTM dye in a water bath. Conclusion: RT-LAMP technique can be simply and reliably applied with the aid of basic instruments, and its results can be visually inspected in laboratory studies.

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Comparison of a TaqMan real-time polymerase chain reaction assay with a loop-mediated isothermal amplification assay for detection of Gallid herpesvirus 1

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638711427578 ◽

2011 ◽

Vol 24 (1) ◽

pp. 138-141 ◽

Cited By ~ 11

Author(s):

Shan-Chia Ou ◽

Joseph J. Giambrone ◽

Kenneth S. Macklin

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Real Time Pcr ◽

Lamp Assay ◽

Isothermal Amplification ◽

Chain Reaction ◽

Loop Mediated Isothermal Amplification ◽

Gallid Herpesvirus ◽

Herpesvirus 1 ◽

Polymerase Chain

A TaqMan real-time polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) assay were developed to detect Gallid herpesvirus 1 (GaHV-1, formerly Infectious laryngotracheitis virus). The standard curve of real-time PCR was established, and the sensitivity reached 10 copies/μl. In the current study, the conversion between viral titer and GaHV-1 genomic copy number was constructed. Six primers for LAMP assay amplified target gene at 65°C within 45 min, and the detection limit was 60 copies/μl. The 6 primers were highly specific, sensitive, and reproducible for detection of GaHV-1. Although the sensitivity of LAMP was lower than that of real-time PCR, LAMP was faster, less expensive, and did not require a thermocycler. The LAMP assay would be a viable alternative assay in diagnostic laboratories that do not employ real-time PCR technology.

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