Detection of Mycoplasma pneumoniae using a highly sensitive rapid diagnostic method with silver amplification technology

To prevent an increase in the frequency of antimicrobial resistance (AMR), the specific and rapid diagnosis of causative pathogens is important, as the results can be used to initiate appropriate antibiotics treatment. Recently, the highly sensitive rapid immunochromatographic assay of silver amplification technology was developed for the detection of Mycoplasma pneumoniae. We investigated the sensitivity and specificity of the silver amplification immunochromatographic assay in adolescent and adult patients. A total of 767 patients with respiratory tract infection (RTI) and 605 with pneumonia were assessed by the silver amplification assay and real-time polymerase chain reaction (PCR). M. pneumoniae was identified by PCR in 95 patients with RTI and in 30 patients with community-acquired pneumonia (CAP), but it was not identified in patients with nursing- and healthcare-associated pneumonia. Eighteen of the 95 RTI patients and 7 of the 30 CAP patients with PCR-positive M. pneumoniae were found to be infected with macrolide-resistant M. pneumoniae. When PCR was used as the control test, the sensitivity, specificity, and overall agreement with the silver amplification assay were 90.5%, 99.0%, and 97.9%, respectively, in RTI patients and 90.0%, 99.1%, and 98.7%, respectively, in pneumonia patients. Our results show that the silver amplification assay has excellent sensitivity and specificity compared with PCR despite being a rapid diagnostic method. The silver amplification assay may be helpful for initiating appropriate antibiotic treatment and preventing AMR.     

Application of recombinase polymerase amplification method for rapid detection of infectious laryngotracheitis virus

Infectious laryngotracheitis is a significant respiratory disease of chickens that causes huge economic losses due to high morbidity and mortality and reduced egg production. A real-time recombinase polymerase amplification (RPA) assay was developed to accurately detect ILTV. The specific probe and primer sets were carefully designed and screened. The real-time RPA assay was carried out at 39 °C for 30 min, and results were obtained within 15 min. The results of the specificity assay showed no fluorescence signals with other avian-related viruses. The sensitivity of the assay was 1 × 10² copies/μL. The low CV value showed that the assay was reproducible. A total of 115 clinical samples were tested using the real-time RPA assay and the real-time PCR assay in parallel; the coincidence rates of the two detection methods were 100%. The results indicated that the real-time RPA assay is a specific, sensitive, rapid, and useful tool for epidemiological studies and clinical diagnosis, especially in the field and in resource-poor areas.     

Rapid analysis of Escherichia coli O157:H7 using isothermal recombinase polymerase amplification combined with triple-labeled nucleotide probes

Rapid analytical methods are urgently needed to evaluate Escherichia coli (E. coli) O157:H7 in food. In this work, a novel recombinase polymerase amplification (RPA)-based lateral flow dipstick (LFD) method was developed to detect E. coli. Briefly, suitable primers and probes were designed and screened. Then, RPA reaction parameters, including volume, time, and temperature, were optimized. The specificity and sensitivity of RPA-LFD were analyzed, and a contaminated milk sample was used to test the detection performance of the proposed method. The optimal RPA reaction conditions included a minimum volume of 10 μL, incubation time of 10 min, temperature range of 39–42 °C, the primer pair EOF4/EOR3, and the probe EOProb. RPA-LFD was highly sensitive, it could detect as little as 1 fg of the genomic DNA of E. coli O157:H7, and 19 nontarget DNA of foodborne bacteria did not yield amplification products. Finally, the limit of detection of RPA-LFD for E. coli O157:H7 in artificially contaminated raw milk was 4.4 CFU/mL. In summary, the RPA-LFD assay developed in this study is an effective tool for the rapid investigation of E. coli O157:H7 contamination in raw milk samples.     

Pyrosequencing-based validation of a simple cell-suspension polymerase chain reaction assay for Campylobacter with application of high-processivity polymerase and novel internal amplification controls for rapid and specific detection

Although Campylobacter is an important food-borne human pathogen, there remains a lack of molecular diagnostic assays that are simple to use, cost-effective, and provide rapid results in research, clinical, or regulatory laboratories. Of the numerous Campylobacter assays that do exist, to our knowledge none has been empirically tested for specificity using high-throughput sequencing. Here we demonstrate the power of next-generation sequencing to determine the specificity of a widely cited Campylobacter-specific polymerase chain reaction (PCR) assay and describe a rapid method for direct cell suspension PCR to quickly and easily screen samples for Campylobacter. We present a specific protocol which eliminates the need for time-consuming and expensive genomic DNA extractions and, using a high-processivity polymerase, demonstrate conclusive screening of samples in <1 h. Pyrosequencing results show the assay to be extremely (>99%) sensitive, and spike-back experiments demonstrated a detection threshold of <10² CFU mL⁻¹. Additionally, we present 2 newly designed broad-range bacterial primer sets targeting the 23S rRNA gene that have wide applicability as internal amplification controls. Empirical testing of putative taxon-specific assays using high-throughput sequencing is an important validation step that is now financially feasible for research, regulatory, or clinical applications.     

Application of a loop-mediated isothermal amplification method for the detection of pathogenic Leptospira

Leptospirosis is an emerging infectious disease, which is considered to be the most widespread zoonotic disease in the world. There are more than 230 known serovars in the genus Leptospira. A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of pathogenic Leptospira spp. was developed and evaluated through amplification of the lipL41 gene coding for the outer membrane protein LipL41. The LAMP assay did not rely on the isolation and culture of leptospires, and no cross-reactivity was observed with other bacterial species. A SYBR Green I-based LAMP assay was also carried out for the real-time detection of DNA amplification. The lower detection limit of the LAMP assay was approximately 100 copies, which was the same as the polymerase chain reaction (PCR) and real-time PCR assays. The accuracy of the LAMP reaction was confirmed by restriction endonuclease analysis of the amplified product. The LAMP assay is easy to perform and inexpensive, and so may be applied in the rapid and specific diagnosis of Leptospira.     

Rapid diagnosis of largemouth bass ranavirus in fish samples using the loop-mediated isothermal amplification method

Largemouth bass ranavirus (LMBV) has been recognized as the causative pathogen responsible for infectious skin ulcerative syndrome in cultured largemouth bass in China. A fast and simple LMBV detection method is urgently needed. Here, a loop-mediated isothermal amplification (LAMP) assay was established for the detection of this virus using primers targeting the major capsid protein gene of LMBV. The amplification conditions were optimized; the assay was specific for the diagnosis of LMBV, as there was no cross-reactivity with other four Iridoviridae viruses (large yellow croaker iridovirus, Singapore grouper iridovirus, tiger frog virus, and soft-shelled turtle iridovirus), grass carp reovirus, white spot syndrome virus, or healthy largemouth bass. The sensitivity of the LAMP assay was found to be 8.55 × 10¹ copies/μL of LMBV DNA, which was 10-fold higher than that of the conventional PCR. Application of the LAMP assay was evaluated using 10 clinical samples, and the results indicated the reliability of the test as a rapid, field diagnostic tool for LMBV detection. Thus, the simplicity and nearly instrument-free LAMP method provides an alternative for rapid and sensitive detection of LMBV and has great potential for early diagnosis of LMBV infection in the farm.     

Rapid and visual detection of Group B streptococcus using recombinase polymerase amplification combined with lateral flow strips

Conventional culture method for detecting Group B streptococcus (GBS), a common pathogen of neonatal meningitis and sepsis, is time-consuming and unsensitive. Even though real-time fluorescence PCR-based molecular method is more accurate, it need special instrument and elaborate protocol. Here, we established a novel molecular method combining recombinase polymerase amplification with lateral flow strips for detecting GBS. The cAMP factor (cfb) gene is a highly specific and sensitive biomarker to identify GBS and is detectable by using 100 genomic copies as the amplification template. Clinical performance of this assay was evaluated by testing 130 samples, in comparison with culture method and real-time fluorescence PCR, and the results achieved 100% accuracy, which were the same with those of real-time fluorescence PCR, and were better than those of culture method with false-negative detection. This study provides a rapid and visual method, with clinical potential, for the detection of GBS infection of patients.     

A PCR-DNA probe assay specific forBacteroides forsythus

Bacteroides forsythusis a fastidious anaerobic Gram-negative organism associated with active periodontal disease. The ability of random amplified polymorphic DNA (RAPD) fingerprinting to generate species-specific markers was exploited towards the construction of a polymerase chain reaction (PCR)-DNA probe assay specific forB. forsythus. The strategy included the four following steps: (1) construction of a first generation DNA probe based on a 507-bp RAPD species-specific marker; (2) cloning and sequencing the 507-bp RAPD marker; (3) design of the primer pair Bf 392-1/Bf 392-2 flanking a 392-bp specific internal sequence; and (4) synthesis of quantities of a 392-bp second generation DNA probe by PCR amplification. The PCR-DNA probe assay includes a PCR amplification of a 392-bp specific sequence in the genomic DNA ofB. forsythusstrains followed by hybridization with the 392-bp digoxigenin-labelled second generation probe. We observed strong, specific hybridization with the amplified DNAs from 11 stains ofB. forsythusand no cross-hybridization with the PCR products from 22 foreign species. The PCR-DNA probe assay must be seen as a highly specific and sensitive method for the detection ofB. forsythusin mixed infections.     

Development and Evaluation of PCR Assay Based on Outer Membrane Protein 22 Gene for Genus Specific Diagnosis of Brucella spp

Brucellosis is a worldwide zoonosis and a significant cause of loss of health in human and animals. Conventionally, definitive diagnosis is done by isolation of brucellae which is time consuming, technically demanding and may be hazardous. The present study was aimed to develop PCR assay based on evolutionarily conserved outer membrane protein 22 gene for rapid diagnosis of Brucella spp. A set of primer was designed after multiple alignment of outer membrane protein 22 gene sequences by clustalW. PCR amplification resulted in single specific amplicon of 639 bp in all the Brucella strains tested. The primers were found to be highly sensitive and specific. PCR assay developed may be used for clinical diagnosis of Brucella infection.     

Fast, simple and highly sensitive double-rounded polymerase chain reaction assay to detect medically relevant fungi in dermatological specimens

BACKGROUND: Early detection of fungal infection is essential for beginning of prompt and specific therapy. In this study we describe a rapid and sensitive procedure to detect, by polymerase chain reaction (PCR) assays, a wide range of medically important opportunistic and pathogenic fungi in dermal specimens from dermatomycoses-affected patients. MATERIALS AND METHODS: Three primer pairs, amplifying fragments of the highly conserved gene coding for small ribosomal RNA (18S rDNA) and the adjacent internal transcribed spacer (ITS) rDNA, previously published by others, were probed on DNA from pure cultures of medically relevant human and animal fungal species. In order to evaluate the specificity of the assay, amplifications of control DNAs from other eukaryotes and prokaryotes were also carried out, and they gave negative results. RESULTS: These primer sets, in single amplification or double-rounded PCR assays, allowed specific amplification when applied to a wide number of fungal DNA from human and animal tissue specimens, including dermatophytes (genera Trichophyton, Microsporum), several yeast species (Candida, Saccharomyces, Cryptococcus, Malassezia) and moulds (Aspergillus, Penicillium). The PCR assay was able to detect as little as 10 pg of fungal DNA, corresponding to approximately 25 fungal genomes per sample specimen. A small-scale DNA extraction method was also developed. This simple, time-saving and sensitive procedure was successfully applied to 40 human and veterinary specimens, and the diagnosis was confirmed with cultural techniques, being shown to work even in the presence of other lesions or contaminating organisms. CONCLUSIONS: This method allows early recognition of fungal pathogen cells in clinical samples as an alternative tool to conventional detection techniques.     

The utility of cerebrospinal fluid for the molecular diagnosis of toxoplasmic encephalitis

The aim of this study was to assess the efficacy of nested polymerase chain reaction (PCR) and the loop-mediated isothermal amplification (LAMP) assay, which were developed to detect and identify toxoplasma parasites in human cerebrospinal fluid (CSF). Nested PCR was performed using primers generated by Dr. L.D. Sibley to target the 18S rDNA instead of the conventionally used primers which target the B1 gene. We also designed Toxoplasma gondii–specific LAMP primers targeting both genes. In vitro detection sensitivity was evaluated using 10-fold serially diluted genomic DNA purified from RH tachyzoites, and clinical sensitivity and specificity were evaluated using clinical CSF samples from 16 patients with toxoplasmic encephalitis (TE) and from 12 patients with other diseases. The 18S rDNA nested PCR showed the highest detection sensitivity limit with a minimum of 1.0 × 10⁻⁸ ng/μL. However, sensitivity and specificity of nested PCR with clinical specimens were 50% and 100%, respectively. The sensitivity of molecular diagnosis of TE is not sufficient; therefore, patients clinically suspected of having TE should be treated promptly. Our molecular diagnostic tool would restrictively facilitate a definitive diagnosis of TE at an early stage in approximately 50% of patients.     

Rapid and sensitive detection of Penaeus monodon nucleopolyhedrovirus (PemoNPV) by loop-mediated isothermal amplification combined with a lateral-flow dipstick

Several methods such as traditional PCR or nested-PCR, immuno assay and histopathology have been developed for detection of Penaeus monodon nucleopolyhedrovirus (PemoNPV) formerly called monodon baculovirus (MBV). However, these methods have various disadvantages including low sensitivity, long assay time, use of toxic substances or unsuitability for field diagnosis. Loop-mediated isothermal amplification of target nucleotide sequences under isothermal conditions, combined with amplicon detection by chromatographic lateral-flow dipsticks allows for more efficient, field friendly detection within 75 min (not including DNA preparation time). In this study, the LAMP amplicon was biotinylated via an inner LAMP primer designed from a BamHI fragment B, a hypothetical protein gene of PemoNPV under isothermal condition at 63 °C for 1 h. Next, the LAMP product was hybridized at 63 °C for 5 min with an optimal FITC-labeled probe that was designed specifically for the LAMP amplicons. The FITC-labeled biotinylated LAMP product picked up gold-labeled, anti-FITC near the LFD origin and the whole, triple-labeled complex was captured by an immobilized biotin-binding protein to yield a red nano-gold stripe at the LFD test line. With a DNA template extracted from PemoNPV-infected shrimp, the LAMP–LFD detection limit was 0.1 pg, whereas one-step PCR and nested-PCR followed with gel electrophoresis was 1 pg. The LAMP–LFD method gave negative test results with buffer and DNA from shrimp infected with other common shrimp DNA viruses including, Penaeus monodon densovirus (PmDNV) formerly called hepatopancreatic parvovirus (HPV), white spot syndrome virus (WSSV) and Penaeus stylirostris densovirus (PstDNV) formerly called infectious hypodermal and hematopoietic necrosis virus (IHHNV). The test platform can be adapted easily for rapid detection of other shrimp viruses, since the LAMP–LFD combination system was a highly sensitive, specific, convenient, and does not require sophisticated instruments.     

Specific detection of Angiostrongylus cantonensis in the snail Achatina fulica using a loop-mediated isothermal amplification (LAMP) assay

Angiostrongylus cantonensis, a rat lungworm, can cause eosinophilic meningitis and angiostrongyliasis in humans following ingestion of contaminated foods or intermediate/paratenic hosts with infective larvae. The snail Achatina fulica is one of the important intermediate hosts of A. cantonensis and is commonly eaten by humans in some countries. In the present study, we developed a loop-mediated isothermal amplification (LAMP) method for the specific detection of A. cantonensis in Ac. fulica. Primers for LAMP were designed based on the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA (rDNA) of A. cantonensis. Specificity tests showed that only the products of A. cantonensis were detected when DNA samples of A. cantonensis and the heterologous control samples Anisakis simplex s.s, Trichuris trichiura, Toxocara canis, Trichinella spiralis and Ascaris lumbricoides were amplified by LAMP. Sensitivity evaluation indicated that the LAMP assay is 10 times more sensitive than the conventional polymerase chain reaction (PCR) assay. The established LAMP assay is rapid, inexpensive and easy to be performed. It can be used in clinical applications for rapid and sensitive detection of A. cantonensis in snails, which has implications for the effective control of angiostrongyliasis.     

Absence of human immunodeficiency virus (HIV) proviral sequences in seronegative hemophilic men and sexual partners of HIV-seropositive hemophiliacs

To detect latent infection with human immunodeficiency virus (HIV), specimens of peripheral blood leukocytes from HIV-seronegative hemophiliacs and from sexual partners of HIV-seropositive hemophiliacs were examined by polymerase chain reaction (PCR). The primer pair SK 38/39 derived from the gag region and/or the primer pair SK 68/69 corresponding to a conserved region of the env gene were used. Whereas HIV proviral DNA was detected by PCR in samples from 86 (97%) of 89 HIV-seropositive hemophiliacs, no HIV-DNA was found in blood samples of 198 HIV-seronegative hemophiliacs at risk. Of 40 HIV-seronegative sexual partners of HIV-infected hemophiliacs, none was PCR positive. Thus, PCR is proving to be a sensitive method by which to confirm infection in seropositive hemophiliacs, while the negative results in HIV-seronegative hemophiliacs and HIV-seronegative sexual partners of HIV-seropositive hemophiliacs suggest that a prolonged seronegative period of latent HIV infection is the exception.     

荧光恒温扩增技术检测肠道病毒71型方法的建立与评价

目的利用荧光恒温扩增(SAT)技术建立一种快速可靠的肠道病毒71型(EV71)检测方法。方法通过设计特异性的EV71 RNA扩增引物及优化探针技术,使用M-MLV反转录酶及T7 RNA多聚酶对EV71 RNA进行核酸扩增,同时利用荧光定量聚合酶链反应(PCR)仪进行实时的荧光信号收集和检测。检测复旦大学附属儿科医院儿童手足口病患儿粪便样本199例,以EV71型核酸检测试剂盒作参比方法,DNA测序为第三方验证方法,对研究数据进行Kappa一致性分析。结果 SAT技术共检测到119例EV71阳性样本,其中21例荧光探针法检测为阴性,经测序证实20例样本仍为阳性。SAT与荧光探针法检测结果的Kappa值为0.789。SAT方法的敏感性100%、特异性98.77%。结论本研究建立的EV71型RNA SAT技术具有敏感性高、特异性强、准确、快速可靠等优点,适用于临床对EV71感染的快速诊断。     

Multiplex PCR assays for the detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae with an internal amplification control

A multiplex polymerase chain reaction (PCR) assay that can simultaneously detect 4 major Vibrio spp., Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae, in the presence of an internal amplification control (IAC) was developed. Species-specific PCR primers were designed based on the gyrB gene for V. alginolyticus, the collagenase gene for V. parahaemolyticus, the vvhA gene for V. vulnificus, and the ompW gene for V. cholerae. Additionally, an IAC primer pair was designed in conserved regions of the bacterial 16S rRNA gene that is used to indicate false-negative results. A multiplex PCR method was developed after optimization of the reaction conditions. The specificity of the PCR was validated by using 83 Vibrio strains and 10 other non-Vibrio bacterial species. The detection limit of the PCR was 10 CFU per tube for V. alginolyticus, V. parahaemolyticus, V. vulnificus, and 10⁵ CFU per tube for V. cholerae in mixed conditions. This method was used to identify 69 suspicious Vibrio isolates, and the results were consistent with physiological and biochemical tests. This multiplex PCR method proved to be rapid, sensitive, and specific. The existence of IAC could successfully eliminate false-negative results for the detection of V. alginolyticus, V. parahaemolyticus, V. vulnificus, and V. cholerae.     

类志贺邻单胞菌实时荧光TaqMan PCR快速检测体系的建立

目的建立针对类志贺邻单胞菌的高灵敏、高特异的实时荧光TaqMan聚合酶链式反应(PCR)快速检测体系。方法根据类志贺邻单胞菌23S rRNA基因的一段特异性序列设计引物及TaqMan探针,利用实时荧光PCR检测平台探讨该检测体系的灵敏度;用30种其他肠道致病菌及院内感染中常见的致病菌评价该检测体系的特异性。结果实时荧光TaqMan PCR快速检测体系对类志贺邻单胞菌重组质粒的检测灵敏度为1×102拷贝/反应体系;对类志贺邻单胞菌基因组的检测灵敏度为3×10-2pg/反应体系;该检测体系在检测30种其他肠道致病菌及院内感染中常见的致病菌时未出现特异性扩增,整个反应在2 h内完成。结论本研究建立的实时荧光TaqMan PCR检测体系可作为类志贺邻单胞菌灵敏、特异、快速的检测方法。